Clinical Immunology: Principles and Practice [5 ed.] 9780702068966, 9780702070396

Citation preview

CHAPTER  

Clinical Immunology: Principles and Practice FIFTH EDITION

ROBERT R. RICH MD Professor of Medicine and Dean Emeritus, University of Alabama at Birmingham, Birmingham, AL, USA

THOMAS A. FLEISHER MD Executive Vice President, American Academy of Allergy, Asthma and Immunology, Milwaukee, WI; Scientist Emeritus, NIH Clinical Center, National Institutes of Health, Bethesda, MD, USA

WILLIAM T. SHEARER MD, PhD Allergy and Immunology Service, Texas Children’s Hospital, Professor of Pediatrics and Immunology, Section of Allergy and Immunology, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

HARRY W. SCHROEDER, JR. MD, PhD Professor of Medicine, Microbiology, and Genetics, Division of Clinical Immunology and Rheumatology, Director, UAB Program in Immunology, University of Alabama at Birmingham, Birmingham, AL, USA

ANTHONY J. FREW MD, FRCP Professor of Allergy and Respiratory Medicine, Department of Respiratory Medicine, Royal Sussex County Hospital, Brighton, UK

CORNELIA M. WEYAND MD, PhD Professor of Medicine, Stanford University, Stanford, CA, USA

For additional online content visit ExpertConsult.com

i

© 2019, Elsevier Limited. All rights reserved. First edition 1996 Second edition 2001 Third edition 2008 Fourth edition 2013 Fifth edition 2019 The right of Robert R. Rich, Thomas A. Fleisher, William T. Shearer, Harry W. Schroeder Jr., Anthony J. Frew, Cornelia M. Weyand to be identified as authors of this work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Chapter 9, Cytokines and cytokine receptors is in the public domain. Chapter 79, Lymphomas; Elaine S. Jaffe and Stefania Pittaluga contributions are the public domain. Chapter 88, Protein kinase antagonists as therapeutic agents for immunological and inflammatory disorders; John J. O’Shea and Massimo Gadina contributions are in the public domain. Chapter 92, Flow Cytometry is in the public domain.

Notices Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-7020-6896-6 E-ISBN: 978-0-7020-7039-6

Content Strategist: Belinda Kuhn/Kayla Wolfe Content Development Specialist: Joanne Scott Content Coordinator: Joshua Mearns Project Manager: Andrew Riley Design: Amy Buxton Illustration Manager: Amy Faith Heyden Illustrators: Martin Woodward, DNA Illustrations, Inc. Marketing Manager: Kristin Koehler

Printed in China Last digit is the print number: 9 8 7 6 5 4 3 2 1

vi

Preface to the First Edition

P R E FA C E T O T H E F I R S T E D I T I O N Clinical immunology is a discipline with a distinguished history, rooted in the prevention and treatment of infectious diseases in the late nineteenth and early twentieth centuries. The conquest of historical scourges such as smallpox and (substantially) polio and relegation of several other diseases to the category of medical curiosities is often regarded as the most important achievement of medical science of the past fifty years. Nevertheless, the challenges facing immunologists in the efforts to control infectious diseases remain formidable; HIV infection, malaria and tuberculosis are but three examples of diseases of global import that elude control despite major commitments of monetary and intellectual resources. Although firmly grounded in the study and application of defenses to microbial infection, since the 1960s clinical immunology has emerged as a far broader discipline. Dysfunction of the immune system has been increasingly recognized as a pathogenic mechanism that can lead to an array of specific diseases and failure of virtually every organ system. Pardoxically, although the importance of the immune system in disease pathogenesis is generally appreciated, the place of clinical immunology as a practice discipline has been less clear. As most of the noninfectious diseases if the human immune system lead eventually to failure of other organs, it has been organ-specific subspecialists who have usually dealt with their consequences. Recently, however, the outlook has begun to change as new diagnostic tools increasingly allow the theoretical possibility of intervention much earlier in disease processes, often before irreversible target organ destruction occurs. More importantly, this theoretical possibility is increasingly realized as clinical immunologists find themselves in the vanguard of translating molecular medicine from laboratory bench to patient bedside. In many settings, clinical immunologists today function as primary care physicians in the management of patients with inmune-deficiency, allergic, and autoimmune diseases. Indeed many influential voices in the clinical disciplines of allergy and rheumatology support increasing coalescence of these traditional subspecialities around their intellectual core of immunology. In addition to his or her role as a primary care physician, the clinical immunologist is increasingly being looked to as a consultant, as scientific and clinical advances enhance his or her expertise. The immunologist with a ‘generalist’ perspective can be particularly helpful in the application of unifying principles of diagnosis and treatment across the broad spectrum of immunologic diseases. Clinical Immunology: Principles and Practice has emerged from this concept of the clinical immunologist as both primary care physician and expert consultant in the management of patients with immunologic diseases. It opens in full appreciation of the critical role of fundamental immunology in this rapidly evolving clinical discipline. Authors of basic science chapters were asked, however, to cast their subjects in a context of clinical relevance. We believe the result is a well-balanced exposition of basic immunology for the clinician. The initial two sections on basic principles of immunology are followed by two sections that focus in detail on the role of the immune system in defenses against infectious organisms. The approach is two-pronged. It begins first with a systematic survey of immune responses to pathogenic agents followed by a detailed

vi

treatment of immunologic deficiency syndromes. Pathogenic mechanisms of both congenital and acquired immune deficiency diseases are discussed, as are the infectious complications that characterize these diseases. Befitting its importance, the subject of HIV infection and AIDS receives particular attention, with separate chapters on the problem of infection in the immunocompromised host, HIV infection in children, anti-retroviral therapy and current progress in the development of HIV vaccines. The classic allergic diseases are the most common immunologic diseases in the population, ranging from atopic disease to drug allergy to organ-specific allergic disease (e.g., of the lungs, eye and skin). They constitute a foundation for the practice of clinical immunology, particularly for those physicians with a practice orientation defined by formal subspecialty training in allergy and immunology. A major section is consequently devoted to these diseases, with an emphasis on pathophysiology as the basis for rational management. The next two sections deal separately with systemic and organ-specific immunologic diseases. The diseases considered in the first of these sections are generally regarded as the core practice of the clinical immunologist with subdisciplinary emphasis in rheumatology. The second section considers diseases of specific organ failure as consequences of immunologically mediated processes that may involve virtually any organ system. These diseases include as typical examples the demyelinating diseases, insulin-dependent diabetes mellitus, the glomerulonephritides and inflammatory bowel diseases. It is in management of such diseases that the discipline of clinical immunology will have an increasing role as efforts focus on intervention early in the pathogenic process and involve diagnostic and therapeutic tools of ever-increasing sophistication. One of the major clinical areas in which the expertise of a clinical immunologist is most frequently sought is that of allogeneic organ transplantation. A full section is devoted to the issue of transplantation of solid organs, with an introductory chapter on general principles of transplantation and management of transplantation rejection followed by separate chapters dealing with the special problems of transplantation of specific organs or organ systems. Appreciation of both the molecular and clinical features of lymphoid malignancies is important to the clinical immunologist regardless of subspecialty background, notwithstanding the fact that primary responsibility for management of such patients will generally fall to the haematologist/oncologist. A separate section is consequently devoted to the lymphocytic leukemias and lymphomas that constitute the majority of malignancies seen in the context of a clinical immunology practice. The separate issues of immune responses to tumors and immunological strategies to treatment of malignant diseases are subjects of additional chapters. Another important feature is the attention to therapy of immunologic diseases. This theme is constant throughout the chapters on the allergic and immunologic diseases, and because of the importance the editors attach to clinical immunology as a therapeutic discipline, an extensive section is also devoted specifically to this subject. Subsections are devoted to issues of immunologic reconstitution, with three chapters on treatment of immunodeficiences, malignancies and metabolic diseases by

Preface to the First Edition bonemarrow transplantation. Also included is a series of chapters on pharmaceutical agents currently available to clinical immunologists, both as anti-allergic and anti-inflammatory drugs, as well as newer agents with greater specificity for T cell-mediated immune responses. The section concludes with a series of chapters that address established and potential applications of therapeutic agents and approaches that are largely based on the new techniques of molecular medicine. In addition to pharmaceutical agents the section deals in detail with such subjects as apheresis, cytokines, monoclonal antibodies and immunotoxins, gene therapy and new experimental approaches to the treatment of autoimmunity. The book concludes with a section devoted to approaches and specific techniques involved in the diagnosis of immunologic diseases. Use of the diagnostic laboratory in evaluation of complex problems of immunopathogenesis has been a hallmark of the clinical immunologist since inception of the discipline and many clinical immunologists serve as directors of diagnostic immunology laboratories. Critical assessment of the utilization of techniques ranging from lymphocyte cloning to flow cytomeric phenotyping to molecular diagnostics are certain to continue as an important function of the clinical immunologist, particularly in his or her role as expert consultant.

vii

In summary, we have intended to provide the reader with a comprehensive and authoritative treatise on the broad subject of clinical immunology, with particular emphasis on the diagnosis and treatment of immunological diseases. It is anticipated that the book will be used most frequently by the physician specialist practicing clinical immunology, both in his or her role as a primary physician and as a subsequent consultant. It is hoped, however, that the book will also be of considerable utility to the non-immunologist. Many of the diseases discussed authoritatively in the book are diseases commonly encountered by the generalist physician. Indeed, as noted, because clinical immunology involves diseases of virtually all organ systems, competence in the diagnosis and management of immunological diseases is important to virtually all clinicians. The editors would be particularly pleased to see the book among the references readily available to the practicing internist, pediatrician and family physician. Robert R. Rich Thomas A. Fleisher Benjamin D. Schwartz William T. Shearer Warren Strober

viii

Part one  Principles of Immune Response

P R E FA C E T O T H E F I F T H E D I T I O N Each edition of Clinical Immunology: Principles and Practice has documented important changes in the discipline from the preceding one. This fifth edition is emphatically not an exception to that pattern. Indeed, advances in both the Principles and Practice of clinical immunology have been remarkable. The constant theme of every edition has been to emphasize that our discipline touches virtually all organ systems. The diseases that are covered range from too little to too much immunity; and from dysregulated, malignant or replaced immunological systems and functions. Fundamental concepts that are essential to a precise understanding of normal and disordered immune function and disease pathogenesis are again balanced by clinical descriptions, diagnostic approaches and therapeutic options. Several examples of particularly notable advances are worth highlighting: Our increasing appreciation of the importance of the microbiota to normal immune system development and to the pathogenesis of immunologic and inflammatory diseases; dissection of relationships between the innate and adaptive immune systems that has served to further clarify the expression of inflammatory processes and their interaction in defenses against infectious agents; progress in rapid and cost-effective genomics that has led to the definition of numerous new primary immune deficiencies and provided new insights into the genetic aspects of many other immunologic diseases; understanding of immune deficiencies that reflect development of anti-cytokine auto-antibodies; detailed definition of cell signaling pathways and the structure of cell-surface molecules that have contributed enormously to the treatment of cancer and autoimmunity with a virtual explosion in novel therapeutics including check-point inhibitors and other recently developed immunomodulators; availability of many new humanized and human monoclonal antibodies and development of novel therapeutic approaches such as chimeric-antigen-receptor T cells; wide use of T cell excision circle receptor (TREC) assay to diagnose serious immune deficiencies of the newborn; and exploration of in vivo therapeutic editing of pathological mutations. With these new tools the practice of clinical immunology has become more interesting yet more complex, while offering important improvements in patient care. Our goal with this edition is to enhance the interest of practitioners in the many specialties and subspecialties that the discipline impacts and to assist them in understanding this increasing complexity. With the increasing availability of powerful new therapeutic agents, the expert clinical immunologist today may function as a primary care physician or consultant in the management of patients with immune deficiencies, allergic, and autoimmune diseases involving multiple organ systems – a role

viii

reminiscent of that served by infectious disease physicians, particularly during the era of antibiotic proliferation in the last quarter of the previous century. It is consequently our hope that the book will find a place near the desk of most persons whose practice relies on the science or practice of clinical immunology as we have broadly defined it. We further trust that it will be especially useful to trainees and practitioners preparing for certification or recertification in an immunology-related subspecialty. In an effort to assist the latter group and as an aid to continuing education of all readers, we have added to the online version of the text, which all purchasers of the book can readily access, multiple choice questions relevant to every chapter. Additionally, we continue to believe that a comprehensive text on clinical immunology can be a valuable asset for generalists in any specialty, particularly internists, pediatricians and family physicians, who regularly care for patients across the broad spectrum of immunological disorders, offering an opportunity for physicians in all disciplines to upgrade their skills and education, and to benefit from the onward rush of science and practice improvement in modern clinical immunology. The book continues features that have been well received in previous editions. Chapters are generously illustrated and all chapters contain summary Boxes (commonly in bulleted form) that provide Key Concepts and a Box labeled as On the Horizon, in which authors look to research opportunities for important advances over the next 5-10 years. Furthermore, due to the extraordinarily cross-disciplinary nature of clinical immunology, it is our hope that investigators working in one area might find new ideas and opportunities in the On the Horizon Boxes outside their primary area of focus. Other Boxes similarly summarize content with Clinical Relevance, Clinical Pearls, and Therapeutic Principles. As always, we are immensely grateful to the hundreds of physicians and scientists whose contributions are the essence of the book. Finally, we recognize the diligence and commitment of our colleagues at Elsevier who have supported all aspects of the book’s development and production, particularly Ms. Joanne Scott who has worked with both authors and editors from concept to birth to completion of every chapter. Robert R. Rich Thomas A. Fleisher William T. Shearer Harry W. Schroeder, Jr. Anthony J. Frew Cornelia M. Weyand

LIST OF CONTRIBUTORS

ix

LIST OF CONTRIBUTORS The editor(s) would like to acknowledge and offer grateful thanks for the input of all previous editions’ contributors, without whom this new edition would not have been possible. Roshini Sarah Abraham PhD, D(ABMLI) Consultant Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, MN, USA Professor of Medicine, Professor of Laboratory Medicine and Pathology Cristina Albanesi BSc, PhD Senior Investigator Laboratory of Experimental Immunology Fondazione “Luigi Maria Monti” (FLMM)—Istituto Dermopatico dell’Immacolata (IDI)-IRCCS Rome, Italy Ilias Alevizos DMD, MMSc Assistant Clinical Investigator National Institute of Dental and Craniofacial Research National Institutes of Health Bethesda, MD, USA Juan Anguita PhD Ikerbasque Professor CIC bioGUNE Derio, Bizkaia, Spain Brendan Antiochos MD Instructor Division of Rheumatology Johns Hopkins University School of Medicine Baltimore, MD, USA Cynthia Aranow, MD Investigator, Clinical Research Autoimmune and Musculoskeletal Diseases The Feinstein Institute for Medical Research Manhasset, NY, USA John P. Atkinson, MD Department of Medicine Chief, Division of Rheumatology Samuel B. Grant Professor of Medicine Professor of Molecular Microbiology Washington University School of Medicine St. Louis, MO, USA

Howard A. Austin III MD Senior Clinical Investigator National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD, USA Subash Babu MBBS, PhD Scientific Director NIH-NIRT-International Center for Excellence in Research National Institute for Research in Tuberculosis Chennai, India Mark C. Ballow MD Professor, Department of Pediatrics Division of Allergy, Immunology and Pediatric Rheumatology Women and Children’s Hospital of Buffalo State University of New York at Buffalo School of Medicine and Biomedical Sciences Buffalo, NY, USA James E. Balow MD Clinical Director and Chief Kidney Disease Section National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, MD, USA John W. Belmont MD, PhD Professor Department of Molecular and Human Genetics Baylor College of Medicine Houston, TX, USA Claudia Berek PhD Group Leader, B Cell Immunology Deutsches Rheuma-Forschungszentrum Berlin (DRFZ) Berlin, Germany Timothy Beukelman MD, MSCE Associate Professor of Pediatric Rheumatology Division of Rheumatology The University of Alabama at Birmingham Birmingham, AL, USA

Tapan Bhavsar MD, PhD Clinical Fellow in Hematopathology National Cancer Institute/National Institute of Health Bethesda, MD, USA J. Andrew Bird MD Associate Professor Department of Pediatrics Division of Allergy and Immunology University of Texas Southwestern Medical Center in Dallas Dallas, TX, USA Sarah E. Blutt PhD Assistant Professor Department of Molecular Virology and Microbiology and Department of Molecular and Cellular Biology Baylor College of Medicine Houston, TX, USA Mark Boguniewicz MD Professor, Division of Pediatric Allergy-Immunology National Jewish Health Denver, CO, USA Rafael Bonamichi-Santos, MD Division of Clinical Immunology and Allergy Division University of São Paulo São Paulo, SP, Brazil Bertrand Boisson PhD Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute; Paris Descartes University, France; St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch The Rockefeller University New York, NY, USA Elena Borzova MD, PhD Professor of Clinical Allergy Department of Clinical Allergology Russian Medical Academy of Postgraduate Education Moscow, Russian Federation

ix

x

LIST OF CONTRIBUTORS

Prosper N. Boyaka PhD Professor Department of Veterinary Biosciences The Ohio State University College of Veterinary Medicine Columbus, OH, USA Joshua Boyce MD Professor of Medicine and Pediatrics Director, Inflammation and Allergic Disease Research Section Director, Jeff and Penny Vinik Center for Allergic Disease Research Harvard Medical School, Brigham and Women’s Hospital Boston, MA, USA Sarah K. Browne Office of Vaccine Research and Review Center for Biologics Evaluation and Research Food and Drug Administration Silver Spring, MD, USA Wesley Burks MD Curnen Distinguished Professor Executive Dean School of Medicine The University of North Carolina Chapel Hill, NC, USA Jacinta Bustamante MD, PhD Research Associate Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute Paris Descartes University Paris Sorbonne Cité Associate Professor of Cellular Biology Study Center for Primary Immunodeficiencies Necker Hospital, Assistance Publique Hôpitaux de Paris Paris, France St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch The Rockefeller University New York, NY, USA Virginia L. Calder PhD Senior Lecturer in Immunology Department of Molecular Therapy and Genetics UCL Institute of Ophthalmology London, UK

Matthew Campbell, MD, MS Assistant Professor Department of Genitourinary Medical Oncology Division of Cancer Medicine The University of Texas MD Anderson Cancer Center Houston, TX, USA Adela Rambi G. Cardones, MD Assistant Professor Department of Dermatology Duke University School of Medicine Durham, NC Jean-Laurent Casanova MD, PhD Director St Giles Laboratory of Human Genetics of Infectious Diseases Rockefeller Branch, The Rockefeller University Howard Hughes Medical Institute New York, NY, USA; Co-Director Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute Paris Descartes University Paris Sorbonne Cité; Professor of Pediatrics Pediatric Hematology-Immunology Unit Necker Hospital, Assistance Publique Hôpitaux de Paris Paris, France Mariana Castells MD, PhD Director, Drug Hypersensitivity and Desensitization Center Director, Allergy Immunology Training Program Associate Director Mastocytosis Center Brigham and Women’s Hospital Harvard Medical School Boston, MA, USA Lisa A. Cavacini PhD Associate Professor Department of Medicine University of Massachusetts Medical School MassBiologics Boston, MA, USA Edwin S.L. Chan MD, FRCPC Assistant Professor Department of Medicine New York University School of Medicine New York, NY, USA

David D. Chaplin, MD, PhD Professor of Microbiology and Medicine University of Alabama at Birmingham Birmingham, AL W. Winn Chatham MD Professor of Medicine Louis W. Heck Clinical Scholar Clinical Director, Division of Clinical Immunology and Rheumatology University of Alabama at Birmingham Birmingham, AL, USA Edward S. Chen, MD Assistant Professor Division of Pulmonary and Critical Care Medicine Johns Hopkins University School of Medicine Baltimore, MD USA Javier Chinen MD, PhD Assistant Professor Departments of Pediatrics Baylor College of Medicine Houston, TX, USA Lisa Christopher-Stine MD, MPH Assistant Professor of Medicine Director John Hopkins Myositis Center Johns Hopkins University Bloomberg School of Public Health Baltimore, MD, USA Michael Ciancanelli PhD Research Associate St Giles Laboratory of Human Genetics of Infectious Diseases Rockefeller Branch The Rockefeller University New York, NY, USA Andrew P. Cope BSc, PhD, MBBS, FRCP, FHEA Head, Academic Department of Rheumatology Centre for Molecular and Cellular Biology of Inflammation Division of Immunology, Infection and Inflammatory Disease King’s College School of Medicine King’s College London London, UK Arthritis Research UK Professor of Rheumatology David B. Corry MD Professor Departments of Medicine, Pathology and Immunology Baylor College of Medicine Houston, TX, USA

LIST OF CONTRIBUTORS Filippo Crea MD, FESC, FACC Full Professor Department of Cardiology Catholic University of Sacred Heart Rome, Italy Randy Q. Cron MD, PhD Professor of Pediatrics and Medicine Children’s Hospital of Alabama University of Alabama at Birmingham Birmingham, AL, USA Jennifer M. Cuellar-Rodriguez MD Staff Clinician Laboratory of Clinical Infectious Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA Marinos C. Dalakas MD, FAAN Professor of Neurology Director, Neuromuscular Division Thomas Jefferson University Philadelphia PA and Chief, Neuroimmunology Unit, Department of Pathophysiology National and Kapodistrian University of Athens Medical School Athens, Greece Sara M. Dann PhD Assistant Professor Departments of Internal Medicine, and Microbiology and Immunology University of Texas Medical Branch Galveston, TX, USA Betty Diamond MD Professor Department of Microbiology and Immunology and Medicine (AECOM) The Feinstein Institute for Medical Research Director Laboratory of Autoimmune Diseases and Musculoskeletal Disorders Head, Center for Autoimmune Diseases and Musculoskeletal Disorders Manhasset, NY, USA Terry W. Du Clos MD, PhD Professor of Medicine School of Medicine University of New Mexico; Head of Rheumatology VA Medical Center Albuquerque, NM, USA

Stéphanie Dupuis-Boisson PhD Research Associate Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute Paris Descartes University Paris Sorbonne Cité, Paris, France; St Giles Laboratory of Human Genetics of Infectious Diseases Rockefeller Branch The Rockefeller University New York, NY, USA Todd N. Eagar PhD Assistant Professor Department of Pathology and Genomic Medicine Houston Methodist Hospital Houston, TX, USA Craig A. Elmets MD Director, UAB Skin Diseases Research Center University of Alabama at Birmingham Birmingham, AL, USA Professor and Chair, Department of Dermatology Doruk Erkan MD Associate Physician-Scientist Barbara Volcker Center for Women and Rheumatic Disease New York, NY, USA; Associate Professor of Medicine Weill Cornell Medical College Associate Attending Rheumatologist, Hospital for Special Surgery New York, NY, USA Laura Fanning, MD Instructor of Medicine Harvard Medical School, Brigham and Women’s Hospital Boston, MA, USA Erol Fikrig MD Section Chief, Division of Infectious Diseases Yale University Investigator, Howard Hughes Medical Institute Professor of Epidemiology (Microbial Diseases) and Microbial Pathogenesis Waldemar Von Zedtwitz Professor of Medicine (Infectious Diseases) New Haven, CT, USA Davide Flego, PhD Catholic University of the Sacred Heart UNICATT Institute of Cardiology Milan, Italy

xi

Thomas A. Fleisher MD Executive Vice President American Academy of Allergy, Asthma and Immunology Milwaukee, WI, USA Scientist Emeritus NIH Clinical Center National Institutes of Health Bethesda, MD, USA Luz Fonacier MD Section of Allergy and Immunology NYU Winthrop Hospital Mineola, NY, USA Andrew P. Fontenot MD Henry N. Claman Professor of Medicine Division Head, Allergy and Clinical Immunology Department of Medicine University of Colorado Anschutz Medical Campus Aurora, CO, USA Alexandra F. Freeman MD Staff Clinician Laboratory of Clinical Infectious Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA Anthony J. Frew MD, FRCP Professor of Allergy and Respiratory Medicine Department of Respiratory Medicine Royal Sussex County Hospital Brighton, UK Kohtaro Fujihashi DDS, PhD Professor, Department of Pediatric Dentistry Immunobiology Vaccine Center The Institute for Oral Health Research, School of Dentistry The University of Alabama at Birmingham Birmingham, AL, USA Massimo Gadina PhD Director Office of Science and Technology National Institute of Arthritis Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, MD, USA Moshe E. Gatt MD Resident, Department of Hematology Hadassah-Hebrew University Medical Center Jerusalem, Israel

xii

LIST OF CONTRIBUTORS

M. Eric Gershwin MD Chief, Division of Rheumatology Allergy and Clinical Immunology University of California Davis Health System Distinguished Professor of Medicine, Jack and Donald Chi Professor of Medicine Davis, CA, USA Susan L. Gillespie MD, PhD Associate Professor of Pediatrics Baylor College of Medicine Baylor International Pediatric AIDS Initiative (BIPAI) Texas Children’s Health Center for International Adoption Houston, TX, USA Jörg J. Goronzy MD, PhD Professor of Medicine Stanford University School of Medicine Stanford, CA, USA Sangeeta Goswami, MD, PhD Clinical Specialist Research Instructor, Department of Genitourinary Medical Oncology Research, Division of Cancer Medicine The University of Texas MD Anderson Cancer Center Houston, TX, USA Clive E.H. Grattan MD, FRCP Consultant Dermatologist Dermatology Centre Norfolk and Norwich University Hospital Norwich, UK Neil S. Greenspan MD, PhD Professor of Pathology Case Western Reserve University Cleveland, OH, USA Sarthak Gupta, MD Systemic Autoimmunity Branch National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, MD, USA Claire E. Gustafson, PhD Division of Immunology and Rheumatology Department of Medicine Stanford University Stanford, CA, USA Russell P. Hall III MD J. Lamar Callaway Professor and Chair Department of Dermatology Duke University School of Medicine Durham, NC, USA

Robert G. Hamilton PhD, D(ABMLI) Professor of Medicine and Pathology Johns Hopkins University School of Medicine and Director Johns Hopkins Dermatology, Allergy and Clinical Immunology Reference Laboratory Johns Hopkins University School of Medicine Baltimore, MD, USA Laurie E. Harrington, PhD Associate Professor Department of Cell, Developmental and Integrative Biology University of Alabama at Birmingham Birmingham, AL, USA Leonard C. Harrison MD DSc, DMedSci (hon. causa), FRACP, FRCPA, FAHMS Professor, NHMRC Senior Principal Research Fellow Population Health and Immunity Division The Walter and Eliza Hall Institute of Medical Research Victoria, Australia Sarfaraz A. Hasni MD Lawrence Schulman Clinical Research Scholar National Institute of Arthritis, Musculoskeletal and Skin Diseases National Institutes for Health Bethesda, MD, USA Arthur Helbling MD Associate Professor of Allergology and Clinical Immunology Division of Allergology University Clinic for Rheumatology, Immunology and Allergology (RIA) Inselspital Bern, Switzerland Joanna Hester PhD Kidney Research UK Senior Fellow Nuffield Department of Surgical Sciences University of Oxford Oxford, UK Steven M. Holland MD Chief, Immunopathogenesis Section; Tenured Investigator National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA Chief, Laboratory of Clinical Infectious Diseases

Dennis Hourcade, PhD Professor of Medicine Washington University School of Medicine St. Louis, MO, USA Nicholas D. Huntington PhD Molecular Immunology Division The Walter and Eliza Hall Institute of Medical Research; Department of Medical Biology University of Melbourne Parkville, Victoria, Australia Tracy Hwangpo MD, PhD Instructor Division of Clinical Immunology and Rheumatology, Department of Medicine University of Alabama at Birmingham School of Medicine Birmingham, AL, USA John B. Imboden MD Alice Betts Endowed Chair for Research in Arthritis Professor of Medicine University of California San Francisco, CA, USA Fadi Issa D.Phil. BM BCh, Academic Clinical Lecturer Nuffield Department of Surgical Sciences, University of Oxford Oxford, UK Shai Izraeli MD Associate Professor of Pediatrics Department of Pediatric Hemato-Oncology Edmond and Lily Safra Children Hospital Sheba Medical Center and University of Tel-Aviv School of Medicine Tel-Aviv, Israel Elaine S. Jaffe MD Head, Hematopathology Laboratory of Pathology Center for Cancer Research National Cancer Institute Bethesda, MD, USA Sirpa Jalkanen MD, PhD Academy Professor Center of Excellence University of Turku Turku, Finland Stacie Jones MD Division of Allergy/Immunology, Department of PediatricsUniversity of Arkansas for Medical Sciences and Arkansas Children’s Hospital Little Rock, AR, USA

LIST OF CONTRIBUTORS Emmanuelle Jouanguy PhD Research Associate Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute Paris Descartes University Paris Sorbonne Cité, Paris, France; St Giles Laboratory of Human Genetics of Infectious Diseases Rockefeller Branch The Rockefeller University New York, NY, USA Sarah Kabbani, MD Emory Vaccinology Training Grant Fellow Division of Infectious Diseases, Department of Medicine Emory University School of Medicine Atlanta, GA, USA Stefan H.E. Kaufmann PhD DR. DR H.C Director and Professor of Microbiology and Immunology Max-Planck-Institute for Infection-Biology Charité Universitätsmedizin Berlin Berlin, Germany Farrah Kheradmand MD Professor of Medicine, Pathology and Immunology Department of Medicine Baylor College of Medicine Houston, TX, USA Donald B. Kohn MD Departments of M.I.M.G. and Pediatrics University of California Los Angeles, CA, USA Robert Korngold PhD Chairman and Senior Scientist Department of Research Hackensack University Medical Center Hackensack, NJ, USA Anna Kovalszki MD Assistant Professor University of Michigan Medical School Division of Allergy and Clinical Immunology Department of Medicine University of Michigan Ann Arbor, MI, USA Douglas B. Kuhns PhD Head Neutrophil Monitoring Laboratory Clinical Services Program SAIC-Frederick, Inc. NCI Frederick Frederick, MD, USA

Hrishikesh Kulkarni, MD Pulmonary and Critical Care Fellow Washington University School of Medicine St. Louis, MO, USA Caroline Y. Kuo MD Allergy and Immunology UCLA Medical Center Santa Monica, CA, USA Arash Lahouti M.D Postdoctoral Research Fellow Division of Rheumatology Johns Hopkins University School of Medicine Baltimore, MD, USA C. Ola Landgren MD, PhD Myeloma Service Department of Medicine Memorial Sloan-Kettering Cancer Center New York, CA, USA Arian Laurence PhD Postdoctoral Fellow Molecular Immunology and Inflammation Branch National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, MD, USA Joyce S. Lee MD Assistant Professor of Medicine Department of Medicine University of Colorado Anschutz Medical Campus Aurora, CO, USA Catherine Lemière MD, MSc CIUSS du Nord de l’île de Montréal Université de Montréal Montréal, Canada Donald Y. M. Leung, MD, PhD Division of Pediatric AllergyImmunology National Jewish Health Denver, CO, USA Arnold I. Levinson MD Emeritus Professor of Medicine Pulmonary, Allergy and Critical Care Division Perelman School of Medicine University of Pennsylvania Philadelphia, PA, USA

xiii

Ofer Levy MD, PhD Director, Precision Vaccines Program & Staff Physician Division of Infectious Diseases, Department of Medicine Boston Children’s Hospital; Associate Professor of Pediatrics Harvard Medical School Boston, MA, USA Dorothy E. Lewis PhD Professor of Internal Medicine Division of Infectious Diseases University of Texas Health Sciences Center Houston, TX, USA Phoebe Lin MD, PhD Casey Eye Institute Oregon Health and Science University Portland, OR, USA Andreas Linkermann, FASN Division of Nephrology Department of Internal Medicine III University Hospital Carl Gustav Carus at the Technische Universität Dresden Dresden, Germany Giovanna Liuzzo MD, PhD Aggregate Professor Department of Cardiology Catholic University of Sacred Heart Rome Italy Michael D. Lockshin MD, MACR Co-Director, Mary Kirkland Center for Lupus Research Hospital for Special Surgery Attending Physician, Hospital for Special Surgery Professor of Medicine and Obstetrics-Gynecology Joan and Sanford Weill College of Medicine of Cornell University Director, Barbara Volcker Center for Women and Rheumatic Disease New York, NY, USA Allison K. Lord PhD Department of Medicine Division of Infectious Diseases Massachusetts General Hospital Boston, MA, USA Jay N. Lozier, MD, PhD, Senior Staff Clinician Department of Laboratory Medicine National Institutes of Health Clinical Center Bethesda, MD, USA

xiv

LIST OF CONTRIBUTORS

Amber Luong MD, PhD Assistant Professor of Otorhinolaryngology - Head and Neck, Surgery and Immunology and Autoimmune Diseases The University of Texas Health Science Center at Houston Houston, TX, USA

Douglas R. McDonald MD, PhD Assistant Professor of Immunology Departments of Pediatrics and Immunology Children’s Hospital, Boston and Harvard Medical School Boston, MA, USA

Raashid Luqmani DM, FRCP, FRCPE Professor of Rheumatology/Consultant Rheumatologist Rheumatology Department, NDORMS University of Oxford Oxford, UK

Peter C. Melby MD Professor of Medicine Departments of Internal Medicine, Microbiology and Immunology, and Pathology University of Texas Medical Branch Galveston, Texas, USA

Meggan Mackay, MD Associate Investigator Clinical Research Autoimmune and Musculoskeletal Diseases The Feinstein Institute for Medical Research Manhasset, NY, USA

Stephen D. Miller PhD Professor Department of Microbiology-Immunology Northwestern University Medical School Chicago, IL, USA

Jonathan S. Maltzman, MD, PhD Associate Professor of Medicine Division of Nephrology Stanford University School of Medicine Palo Alto, CA, USA

Anna L. Mitchell MB, BS, BMednsci, MRCP (UK) Clinical Research Training Fellow Institute of Genetic Medicine, International Centre for Life Newcastle University Newcastle-upon-Tyne, UK

Peter J. Mannon MD, MPH Professor of Medicine Mucosal HIV and Immunobiology Center University of Alabama at Birmingham Birmingham, AL, US; Director, Gastroenterology/Hepatology Clinical Research Program Michael P. Manns MD Professor and Chairman Department of Gastroenterology Hepatology and Endocrinology Hannover Medical School Hannover, Germany James G. Martin MD DSc McGill University Health Centre Research Institute and McGill University Craig L. Maynard, PhD Assistant Professor Department of Pathology University of Alabama at Birmingham Birmingham, AL USA Samual McCash, MD Clinical Chemistry Service Department of Laboratory Medicine Memorial Sloan Kettering Cancer Center New York, NY, USA

Amirah Mohd-Zaki UCL Institute of Ophthalmology London, UK Carolyn Mold PhD Professor Department of Molecular Genetics and Microbiology University of New Mexico School of Medicine Albuquerque, NM, USA David R. Moller MD Professor of Medicine Johns Hopkins University School of Medicine Baltimore, MD, USA Dimitrios S. Monos PhD Director, Immunogenetics Laboratory The Philadelphia Children’s Hospital of Philadelphia Professor of Pathology and Lab Medicine Perelman School of Medicine, University of Pennsylvania Philadelphia, PA, USA

Scott N. Mueller, PhD Associate Professor Department of Microbiology and Immunology Peter Doherty Institute for Infection and Immunity The University of Melbourne, at Melbourne; Australian Research Council Queen Elizabeth II Research Fellow, Department of Microbiology and Immunology, University of Melbourne Parkville, VIC, Australia Catharina M. Mulders-Manders MD PhD Resident internal medicine Radboudumc Expertise Center for Immunodeficiency and Autoinflammation Department of Internal Medicine Radboud University Medical Centre Nijmegen, The Netherlands. Mark J. Mulligan, MD, FIDSA Distinguished Professor of Medicine Executive Director The Hope Clinic of the Emory Vaccine Center Division of Infectious Diseases, Department of Medicine Emory University School of Medicine Atlanta, GA, USA Ulrich R. Müller MD Professor, Consultant Spital Ziegler, Spital Netz Bern Bern, Switzerland Pashna N. Munshi, MD John Theurer Cancer Center Hackensack University Medical Center Hackensack, NJ, USA; Lombardi Comprehensive Cancer Center MedStar Georgetown University Hospital Washington, DC, USA Kazunori Murata PhD, DABCC Clinical Chemist Memorial Sloan Kettering Cancer Center New York, NY, USA Philip M. Murphy MD Chief, Laboratory of Molecular Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA Nicolás Navasa CIC bioGUNE Derio, Bizkaia, Spain

LIST OF CONTRIBUTORS Pierre Noel MD Professor of Medicine Division of Hematology-Oncology Mayo College of Medicine Scottsdale, AZ, USA Luigi D. Notarangelo MD Jeffrey Modell Chair of Pediatric Immunology Research Division of Immunology Children’s Hospital Boston Professor of Pediatrics and Pathology Harvard Medical School Boston, MA, USA Robert L. Nussbaum MD, FACP, FACMG Volunteer Clinical Faculty UCSF Chief Medical Officer, Invitae Corporation Thomas B. Nutman MD Head, Helminth Immunology Section Clinical Parasitology Section Laboratory of Parasitic Diseases National Institutes of Health Bethesda, MD, USA Stephen L. Nutt PhD Professor Molecular Immunology The Walter and Eliza Hall Institute Melbourne, VIC, Australia João B. Oliveira MD, PhD Head Human Disorders of Lymphocyte Homeostasis Unit National Institutes of Health Assistant Chief, Immunology Service Department of Laboratory Medicine at National Institutes of Health Bethesda, MD, USA Thomas L. Ortel MD, PhD Chief, Division of Hematology Professor of Medicine and Pathology Medical Director, Clinical Coagulation Laboratory Duke University Medical Center Durham, NC, USA John J. O’Shea MD Scientific Director Molecular Immunology and Inflammation Branch National Institute of Arthritis and Musculoskeletal and Skin Diseases Bethesda, MD, USA

Sung-Yun Pai MD Assistant Professor of Pediatrics Division of Pediatric Hematology-Oncology Children’s Hospital Boston Department of Pediatric Oncology Dana-Farber Cancer Institute Harvard Medical School Boston, MA, USA Lavannya Pandit MD Assistant Professor of Medicine Department of Medicine Baylor College of Medicine Houston, TX, USA Mary E. Paul MD Associate Professor Department of Pediatrics Texas Children’s Hospital Houston, TX, USA Simon H.S. Pearce MD, FRCP Professor of Endocrinology Institute of Genetic Medicine, International Centre for Life Newcastle University Newcastle-upon-Tyne, UK Daniela Pedicino, MD Department of Cardiovascular and Thoracic Sciences Catholic University of the Sacred Heart Rome, Italy Erik J. Peterson MD Center for Immunology Department of Medicine University of Minnesota Minneapolis, MN, USA Capucine Picard MD, PhD Research Associate Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute Paris Descartes University Paris Sorbonne Cité, Paris, France; Professor of Immunology Director Study Center for Primary Immunodeficiencies Necker Hospital, Assistance Publique Hôpitaux de Paris Paris, France Stefania Pittaluga MD, PhD Staff Physician Laboratory of Pathology National Cancer Institute National Institutes of Health Hematopathology Section Bethesda, MD, USA

xv

Debra Long Priel, MS Associate Scientist Neutrophil Monitoring Lab Clinical Services Program Applied Developmental Research Directorate Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research, Frederick, MD, USA Jennifer Puck, MD Department of Allergy and Immunology UCSF Pediatric San Francisco, CA, USA Anne Puel PhD Research Associate Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute Paris Descartes University Paris Sorbonne Cité, Paris, France St Giles Laboratory of Human Genetics of Infectious Diseases Rockefeller Branch The Rockefeller University New York, NY, USA Andreas Radbruch PhD Scientific Director Deutsches Rheuma-Forschungszentrum Berlin (DRFZ) Leibniz Institute Berlin, Germany Stephen T. Reece PhD Senior Scientist Department of Medicine University of Cambridge School of Clinical Medicine Cambridge, United Kingdom John D. Reveille MD Professor of Medicine Division of Rheumatology Department of Medicine University of Texas Health Science Center at Houston Houston, TX, USA Robert R. Rich MD Professor of Medicine and Dean Emeritus University of Alabama at Birmingham Birmingham, AL, USA Chaim M. Roifman MD, FRCPC Professor Department of Paediatrics and Immunology The Hospital for Sick Children Toronto, ON, Canada

xvi

LIST OF CONTRIBUTORS

Antony Rosen MD Director Division of Rheumatology Johns Hopkins University School of Medicine Baltimore, MD, USA James T. Rosenbaum MD Casey Eye Institute, Oregon Health and Science University Dever’s Eye Institute Portland, OR, USA Sergio D. Rosenzweig, MD, PhD Chief, Immunology Service Department of Laboratory Medicine NIH Clinical Center National Institutes of Health Bethesda, MD, USA Barry T. Rouse DVM, PhD, DSc Distinguished Professor Department of Pathobiology University of Tennessee Knoxville, TN, USA Scott D. Rowley MD Chief, Blood and Marrow Transplantation John Theurer Cancer Center Hackensack University Medical Center Hackensack, NJ, USA Shimon Sakaguchi. MD, PhD Distinguished Professor, Experimental Immunology Immunology Frontier Research Center (IFReC) Osaka University Suita, Osaka, Japan Marko Salmi MD, PhD Department of Molecular Medicine Department of Medical Biochemistry and Genetics University of Turku Turku, Finland Andrea J. Sant, PhD Professor of Microbiology and Immunology University of Rochester Medical School David H. Smith Center for Vaccine Biology and Immunology Rochester, NY, USA Sarah W. Satola, PhD Assistant Professor of Medicine Division of Infectious Diseases Department of Medicine Emory University School of Medicine Atlanta, GA, USA

Valerie Saw, MD FRCOphth PhD Consultant Ophthalmic Surgeon and Clinical Lecturer UCL Institute of Ophthalmology London, UK Marcos C. Schechter, MD Infectious Diseases Fellow Division of Infectious Diseases Department of Medicine Emory University School of Medicine Atlanta, GA, USA Harry W. Schroeder, Jr, MD, PhD Director UAB Program in Immunology University of Alabama at Birmingham Birmingham, AL, USA; Professor of Medicine, Microbiology, and Genetics Division of Clinical Immunology and Rheumatology Benjamin M. Segal, M.D. Holtom-Garrett Family Professor of Neurology and Director of the Multiple Sclerosis Center Department of Neurology University of Michigan Medical School Ann Arbor, MI, USA Carlo Selmi MD Department of Medicine and Hepatobiliary Immunopathology Unit IRCCS Istituto Clinico Humanitas Department of Translational Medicine University of Milan, Milan, Italy; Assistant Professor of Medicine Division of Rheumatology, Allergy and Clinical Immunology University of California at Davis Davis, CA, USA Sushma Shankar BM, BCh, Ba Physiol Sci (Hons), MRCS Academic Clinical Fellow Department of Surgery John Radcliffe Hospital Oxford, UK Anu Sharma Postdoctoral Researcher Texas MD Anderson Cancer Center Houston, TX, USA

Padmanee Sharma Genitourinary Medical Oncology Department of Dermatology and Cutaneous Surgery The University of Miami Miller School of Medicine Texas MD Anderson Cancer Center Houston, TX, USA William T. Shearer MD, PhD Allergy and Immunology Service Texas Children’s Hospital; Professor of Pediatrics and Immunology Section of Allergy and Immunology, Department of Pediatrics Baylor College of Medicine Houston, TX, USA Richard M. Siegel MD, PhD Chief, Immunoregulation Section Autoimmunity Branch NIAMS National Institutes of Health Bethesda, MD, USA Anna Simon MD, PhD Associate Professor of Immunodeficiency and Autoinflammation Department of General Internal Medicine N4i Centre for Immunodeficiency and Autoinflammation (NCIA) Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands Gideon P. Smith MD, PhD Director Connective Tissue Diseases Department of Dermatology Massachusetts General Hospital of Harvard University Boston, MA, USA David S. Stephens MD Vice President for Research Robert W. Woodruff Health Sciences Center Emory University Atlanta, GA, USA Professor of Medicine, Microbiology & Immunology, and Epidemiology Emory University School of Medicine Atlanta, GA, USA Robin Stephens PhD Associate Professor Departments of Internal Medicine, and Microbiology and Immunology University of Texas Medical Branch Galveston, TX, USA

LIST OF CONTRIBUTORS Alex Straumann MD Chairman, Swiss EoE Clinic and EoE Research Network Olten, Switzerland Leyla Y. Teos PhD Molecular Biologist National Institute of Dental and Craniofacial Research National Institutes of Health Bethesda, MD, USA Laura Timares PhD Associate Professor of Dermatology Department of Dermatology University of Alabama at Birmingham School of Medicine Birmingham, AL, USA Wulf Tonnus Division of Nephrology Department of Internal Medicine III University Hospital Carl Gustav Carus at the Technische Universität Dresden Dresden, Germany Raul M. Torres PhD Professor of Immunology and Microbiology University of Colorado School of Medicine Aurora, CO, USA Gülbü Uzel MD Staff Clinician Laboratory of Clinical Infectious Diseases, NIAID, NIH Allergy & Immunology - Clinical & Laboratory Immunology and Pediatric Rheumatology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA Jeroen C.H. van der Hilst MD Internal Medicine Resident Department of General Internal Medicine Radboud University Nijmegen Medical Center Nijmegen, The Netherlands Jos W.M. van der Meer MD, PhD Professor of Medicine, Head Department of General Internal Medicine Nijmegen Institute for Infection, Inflammation and Immunity N4i Centre for Immunodeficiency and Autoinflammation (NCIA) Radboud University Nijmegen Medical Centre Nijmegen, The Netherlands

John Varga MD John and Nancy Hughes Professor of Medicine Division of Rheumatology Northwestern University Feinberg School of Medicine Chicago, IL, USA Jatin M. Vyas MD PhD Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital; Department of Medicine, Harvard Medical School Boston, MA, USA Meryl Waldman MD Staff Clinician Kidney Disease Section National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, MD, USA Peter Weiser MD Associate Professor of Pediatric Rheumatology Division of Rheumatology Department of Pediatrics The University of Alabama at Birmingham Birmingham, AL, USA Peter F. Weller MD William Bosworth Castle Professor of Medicine Harvard Medical School Beth Israel Deaconess Medical Center Allergy and Inflammation Division Boston, MA, USA Cornelia M. Weyand MD, PhD Professor of Medicine Stanford University Stanford, CA, USA Fredrick M. Wigley MD Professor of Medicine Department of Medicine Division of Rheumatology Johns Hopkins University School of Medicine Baltimore, MD, USA

xvii

Robert J. Winchester MD Professor of Medicine College of Physicians and Surgeons Columbia University New York, NY, USA James B. Wing, PhD Assistant Professor, Experimental Immunology Immunology Frontier Research Center (IFReC) Osaka University Suita, Osaka, Japan Kathryn J. Wood DPhil Professor of Immunology Nuffield Department of Surgical Sciences University of Oxford John Radcliffe, Hospital Oxford, UK Xiaobo Wu, MD Assistant Professor of Medicine Washington University School of Medicine St. Louis, MO, USA Hui Xu MD, PhD Professor of Dermatology University of Alabama at Birmingham Birmingham, AL, USA Cassian Yee, MD Professor, Department of Melanoma Medical Oncology Division of Cancer Medicine Texas MD Anderson Cancer Center Houston, TX, USA Shen-Ying Zhang MD, PhD Research Associate Laboratory of Human Genetics of Infectious Diseases Necker Branch, Imagine Institute; Paris Descartes University, Paris Sorbonne Cité, Paris, France; St. Giles Laboratory of Human Genetics of Infectious Diseases Rockefeller Branch The Rockefeller University Research Associate St Giles Laboratory of Human Genetics of Infectious Diseases New York, NY, USA

xviii

Part one  Principles of Immune Response

D E D I C AT I O N

To: Susan Rich, Cathryn Rich and Darren Selement, Phoebe and Kenneth Rich, Lynn and Kenneth Todorov Robert R. Rich With thanks and gratitude to my family and my colleagues. Thomas A. Fleisher Lynn Des Prez and Christine, Mark, Christopher, Martin, John, Jesse and Melissa Shearer William T. Shearer Dixie Lee Schroeder; Harry W Schroeder III MD PhD, Maria Isabel, and Anabel Schroeder; Elena, Jeff and Liam Beck; Jeannette Schroeder and Antoni Bernard Harry W. Schroeder, Jr To my family for their support and encouragement and to my students for their interest in immunology and allergy Anthony J. Frew Jörg Goronzy and Dominic and Isabel Weyand Goronzy Cornelia M. Weyand

xviii

1  The Human Immune Response Robert R. Rich, David D. Chaplin

Clinical immunology is a medical subspecialty largely focused on a specific physiological process, inflammation, which is essential to good health, particularly in defense against pathogenic organisms, recovery from injury, and containment of neoplasms. But inflammation, mediated by the cells and soluble products of the immune system, is also a powerful contributor to the pathogenesis of diseases that affect virtually every organ system. A consequent challenge for clinical immunologists, both clinicians and basic scientists, is to reduce a dizzying array of disease descriptions to systematic understanding of pathogenic mechanisms, facilitating translation of fundamental concepts and new discoveries into more effective disease prevention or treatment. This introductory chapter is directed to nonimmunologist clinicians and researchers. It is structured as an introduction to the interacting elements of the human immune system and their disordered functions in diseases. The subtleties, including immunological or molecular genetic jargon unavoidably used, are described in detail in the chapters that follow.

THE HOST–MICROBE INTERACTION The vertebrate immune system is a product of eons of evolutionary relationships between rapidly evolving microbial organisms and their much less rapidly reproducing, and hence less adaptable, hosts.1 In general, the relationship is mutually beneficial, each providing nutrients and other materials essential to the well-being of their partner—the host and its microbiome (Chapter 14). Occasionally, however, a normally beneficial relationship becomes pathological, as pathogenic microbes overwhelm the microbiome, invading host tissues and resulting in host morbidity or even death. Because the vertebrate host cannot win a battle with microbial invaders by rapid mutation and selection, the immune system employs a strategy of complexity and redundancy, which involves both the individual organism and its collective population. Reflecting plasticity of the response, specific defenses differ, depending on the nature of the infectious agent and its point of entry and distribution within the body. Regardless of the defense mechanism, an intended outcome is destruction or neutralization of the invading organism. A secondary consequence, however, can be collateral damage to host cells. These cells can be targeted for damage because they are sites of microbial residence and replication, or they can be damaged as “innocent bystanders.” Depending on the site and severity of the host’s defensive response, it may be accompanied by local and/or systemic symptoms and signs of inflammation, which may lead to long-lasting tissue dysfunction as a result of tissue remodeling and partial repair.

Adaptive and Innate Immunity Immune responses are traditionally classified as adaptive (also termed acquired or specific) and innate (or nonspecific) (Table 1.1). The adaptive immune system, present uniquely in species of the phylum Chordata, is specialized for development of an inflammatory response based on recognition of specific “foreign” macromolecules that are predominantly, but not exclusively, proteins, peptides, and carbohydrates. The vast majority of chordate species are vertebrates, and this book addresses adaptive immunity of that subphylum. Its primary effectors are antibodies, B lymphocytes, T lymphocytes, and antigen-presenting cells (APCs). T and B lymphocytes express surface antigen receptors that are clonally specific as a consequence of receptor–gene rearrangements. Expansion of clones of lymphocytes specific for any particular antigen is induced by antigen encounter and consequent activation and proliferation, thereby constituting the basis of immunological memory. Innate immune responses are phylogenetically far more ancient, being widely represented in multicellular phyla.2 Rather than being based on exquisitely specific recognition of a diverse array of macromolecules (i.e., antigens), they are focused on recognition of common molecular signatures of microbial organisms that are not present in vertebrates.3 Among these structures, which are termed pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), are bacterial cell wall constituents, such as mannose-rich oligosaccharides, lipopolysaccharides, peptidoglycans, and several nucleic acid variants, including double-stranded RNA and unmethylated CpG DNA. For both innate and adaptive immunity responses, defense effector mechanisms can require either direct cell-to-cell contact or the activity of cytokines and chemokines, which are hormonelike soluble molecules that act in the cellular microenvironment (cell-mediated immunity). Most immune responses include participation of both modes of response.2-4 The elements of innate immunity are diverse (Chapter 3). They include physical barriers to pathogen invasion (e.g., skin, mucous membranes, cilia, and mucus), as well as an array of cellular and soluble factors that can be activated by secreted or cell surface products of the pathogen, including PAMPs. Recognition of PAMPs by cells in innate immunity, which also commonly function as APCs to the lymphocytes of adaptive immunity, is via cell membrane or cytoplasmic receptors known as pattern recognition receptors (PRRs). PRRs can be either membrane bound or cytoplasmic. Membrane-bound PRRs include Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). Humans express 10 distinct TLRs, which recognize (among others) specific bacterial glycolipids, lipopolysaccharide; viral single-stranded

3

4

Part one  Principles of Immune Response

TABLE 1.1  Features of Innate and

Adaptive Immune Systems

Distinguishing Features Adaptive Immunity Innate Immunity Germ line-encoded receptors targeting pathogen molecular patterns Does not require immunization Limited memory Includes physical barriers to pathogen

Clonally variable receptors generated somatically by rearrangement of gene elements Consequence of B- and/or T-cell activation Immunological memory well developed Antibody and cytotoxic T cells

Common Features Cytokines and chemokines Complement cascade Phagocytic cells Natural killer (NK) cells “Natural” antibodies

RNA; and bacterial and viral unmethylated CpG DNA. CLR are particularly important in antifungal innate immunity but also have important roles in defenses against bacteria, viruses, and parasites. They comprise a large family that commonly recognizes microbe-specific carbohydrate ligands or structurally similar lectin-like domains. Cytoplasmic PRRs include RIG-1–like receptors (RLRs) and nucleotide oligomerization domain (NOD)like receptors (NLRs). RLRs are involved in recognition of viruses through interaction with intracytoplasmic viral double-stranded RNA (dsRNA), and NLRs recognize bacterial peptidoglycan motifs.4 Cells of the innate immune system are commonly triggered through activation of the NF-κB transcription factor via the MyD88 signaling pathway, thereby inducing an inflammatory response using mechanisms that are broadly shared with those of the adaptive immune system. These include activation of various types of innate lymphoid cells (e.g., natural killer [NK] cells), which are characterized by absence of clonally expressed receptors for specific antigen (see below), activation of granulocytes and other phagocytes, the secretion of inflammatory cytokines and chemokines, and interactions of the many participants in the complement cascade. Additionally, activation of cells of innate immunity that also act as APCs for the adaptive immune system results in upregulation of membrane molecules (e.g., CD80, CD86) that provide the second signal, along with the T-cell receptor (TCR) for antigen, necessary for induction of antigen-specific T cells.5 Finally, because recognition of pathogens by the innate immune system relies on germline encoded, nonrearranged receptors held in common by the specific cell type, innate immunity is more rapidly responsive. It can initiate in minutes to hours and generally precedes development of a primary adaptive immune response by at least several days.

CELLS OF THE IMMUNE SYSTEM The major cellular constituents of both innate and adaptive immunity originate in bone marrow, where they differentiate from multipotent hematopoietic stem cells (HSCs) along several pathways to become granulocytes, lymphocytes, and APCs (Chapter 2).

Granulocytes Polymorphonuclear leukocytes (granulocytes) are classified by light microscopy into four types. By far the most abundant in the peripheral circulation are neutrophils, which are principal effector cells linking the innate and adaptive responses by virtue of their expression of surface receptors for antibody and complement (Chapter 21). They are phagocytic cells that ingest, kill, and degrade microbes and other targets of an immune attack within specialized cytoplasmic vacuoles that contain potent antimicrobial enzymes and oxidative pathways. The phagocytic activity of neutrophils is promoted by their surface display of receptors for antibody molecules (specifically the Fc portion of immunoglobulin G [IgG] molecules) (Chapter 15) and activated complement proteins (particularly the C3b component) (Chapter 21). Neutrophils are the predominant cell type in acute inflammatory infiltrates and are the primary effector cells in immune responses to pyogenic bacteria (Chapter 27). Eosinophils (Chapter 24) and basophils (Chapter 23) are the other circulating forms of granulocytes. A close relative of the basophil, but derived from distinct bone marrow precursors, is the tissue mast cell, which does not circulate in blood. Eosinophils, basophils, and mast cells are important in defenses against multicellular pathogens, particularly helminths (Chapter 31). Their defensive functions are not based on phagocytic capabilities but, rather, on their ability to discharge potent biological mediators from their storage granules into the cellular microenvironment. This process, termed degranulation, can be triggered by antigen-specific IgE molecules that bind to basophils and mast cells via high-affinity receptors for the Fc portion of IgE (FcεR) on their surfaces. In addition to providing a mechanism for anthelmintic host defenses and certain antibacterial responses, this is also the principal mechanism involved in acute (IgEmediated) allergic reactions (Chapters 41–49).

Lymphocytes Three broad categories of lymphocytes are identified on the basis of display of particular surface molecules: B cells, T cells, and innate lymphoid cells; each of these categories can be further subdivided according to specific function and display of distinguishing cell surface molecules (Chapter 2). All lymphocytes differentiate from common lymphoid stem cells in bone marrow. B cells create their immunoglobulin receptors in bone marrow and differentiate into antibody-producing cells in the periphery (Chapter 7). T-cell precursors move from bone marrow to the thymus (or, in some cases, to extrathymic tissue compartments), where they complete their differentiation and selection (Chapter 8). T cells and B cells are the heart of immune recognition, a property reflecting their clonally specific cell surface receptors for antigen (Chapter 4). The TCR is a heterodimeric integral membrane molecule expressed exclusively by T lymphocytes. B-cell receptors for antigen (BCRs) are membrane immunoglobulin (mIg) molecules of the same antigenic specificity that the cell and its terminally differentiated progeny, plasma cells, will secrete as soluble antibodies. Memory B cells and nondividing, long-lived plasma cells may account substantially for persistence of antibody responses (including production of autoantibodies) over many years.6 Receptors for “antigen” on the third class of lymphocytes, innate lymphoid cells (ILCs), are not clonally expressed. ILCs are subdivided into three major groups according to the cytokines that they produce (e.g., group 1 ILCs, including NK cells, produce interferon-γ [IFN-γ] and tumor necrosis factor [TNF]).7 ILCs

CHAPTER 1  The Human Immune Response express receptors for PAMPs and, as such, serve as major effectors of innate immunity. They also recognize target cells that might otherwise elude the immune system (Chapters 2, 17). Thus recognition of NK cell targets is based substantially on what their targets lack rather than on what they express. NK cells express receptors of several types for major histocompatibility complex (MHC) class I molecules via killer immunoglobulin-like receptors (KIRs).8 KIRs are expressed on the plasma membrane of NK cells (and some T cells), which interact with class I molecules to alter NK-cell cytotoxic function. Most KIRs express in their intracellular domain a tyrosine-based inhibitory-motif (ITIM) that suppresses NK activity, thereby preventing NK cell activity directed against normal self-cells. In contrast, some KIRs express a tyrosine-based activation motif (ITAM), which amplifies their activity. NK cells will kill target cells unless they receive an inhibitory signal transmitted by an ITIM receptor. Virus-infected cells and tumor cells that attempt to escape T-cell recognition by downregulating their expression of class I molecules become susceptible to NK cell–mediated killing because the NK cells receive an activation signal and/or fail to receive an inhibitory signal through the ITAM- and ITIM-containing MHC class I receptors. The balance between ITIM and ITAM is regulated by the microenvironmental milieu, increasing expression of ITAM in the presence of viral-infected or cancer cells and of ITIM as necessary to maintain self-tolerance and prevent autoimmunity. A high frequency of ITAM-expressing cells has been reported in some patients with autoimmune diseases.9 Although NK cell–mediated innate immunity has been long considered to lack immunological memory, recent studies suggest that NK cells can exhibit memory of previous encounters with microbes or other antigens, the molecular basis of which remains to be fully elucidated.10 NK cells can also participate in antigenspecific immune responses by virtue of their surface display of the activating ITAM receptor CD16, which binds the constant (Fc) region of IgG molecules. This enables them to function as effectors of a cytolytic process termed antibody-dependent cellular cytotoxicity (ADCC), a mechanism exploited clinically with monoclonal antibody (mAb) therapeutic agents.11 In general, pathways leading to differentiation of T cells, B cells, and ILCs are mutually exclusive, representing a permanent lineage commitment. No lymphocytes express both mIg and TCRs. However, a subset of T cells, termed NKT cells, exhibit both NK-like cytotoxicity and αβTCR with limited receptor diversity.

Antigen-Presenting Cells KEY CONCEPTS Features of Antigen-Presenting Cells • Capacity for uptake and partial degradation of protein antigens • Expression of major histocompatibility complex (MHC) molecules for binding antigenic peptides • Chemokine receptors to allow colocalization with T cells • Expression of accessory molecules for interaction with T cells • Receptors for pathogen- or danger-associated molecular patterns • Secretion of cytokines that program T helper (Th) cell responses

A morphologically and functionally diverse group of cells, all of which are derived from bone marrow precursors, is specialized for presentation of antigen to lymphocytes, particularly T cells (Chapter 6). Included among such cells are dendritic

5

cells, monocytes (present in the peripheral circulation), macrophages (solid tissue derivatives of monocytes), cutaneous Langerhans cells (Chapter 19), and constituents of the reticular endothelial system within solid organs. B lymphocytes that specifically capture antigen via their clonally expressed mIg can also function efficiently in antigen presentation to T cells. Cardinal features of APCs include their expression of both class I and class II Major Histocompatibility Complex (MHC; Chapter 5) molecules as well as requisite accessory molecules for T-cell activation (e.g., B7-1, B7-2/CD80, CD86).12 Upon activation, APC elaborate cytokines that induce specific responses in cells to which they are presenting antigen. In addition to processing and presenting antigen, APCs can regulate activation of the immune system via innate cell surface receptors, which contribute to determination of whether the antigen is pathogen associated. APCs differ substantially among themselves with respect to mechanisms of antigen uptake and effector functions. Immature dendritic cells show high phagocytic and pathogen-killing activity but low ability to present antigen and activate T cells.12 Dendritic cells (DCs) that have ingested a pathogen or foreign antigen can be induced to mature by inflammatory stimuli, especially via cells of the innate immune system and by direct activation through receptors for PAMPs or DAMPs.12,13 Monocytes and macrophages are actively phagocytic, particularly for antibody and/or complement-coated (opsonized) antigens that bind to their surface receptors for IgG and C3b. These cells are also important effectors of immune responses, especially in sites of chronic inflammation. Upon further activation by T-cell cytokines, they can kill ingested microorganisms by oxidative pathways similar to those employed by polymorphonuclear leukocytes. The interaction between B cells acting as APCs and T lymphocytes is notable as the cells are involved in a mutually amplifying circuitry of antigen presentation and response.12 The process is initiated by antigen capture through B cell mIg and ingestion by receptor-mediated endocytosis. This is followed by proteolytic antigen degradation and then display to T cells as oligopeptides bound to MHC molecules. Like other APCs, B cells display CD80, which provides a requisite second signal to the antigen-responsive T cell via CD28, its accessory molecule for activation (Fig. 1.1). As a result of T-cell activation, T-cell cytokines that regulate B-cell differentiation and antibody production are produced, and T cells are stimulated to display the surface ligand CD40L (CD154), which can serve as the second signal for B-cell activation through its inducible surface receptor

BASIS OF ADAPTIVE IMMUNITY The essence of adaptive immunity is molecular distinction between self constituents and potential pathogens (for simplicity, self/nonself discrimination, but perhaps more precisely discrimination between molecular species perceived as signaling potential “danger” and those that do not). This discrimination is a major responsibility of both T lymphocytes and cells of the innate immune system. It reflects the selection of thymocytes that have generated specific antigen receptors, which, upon later encounter, can bind nonself antigenic peptides bound to self-MHC molecules. The consequence of this selection process is that foreign proteins are recognized as antigens, whereas self-proteins are tolerated (i.e., are not perceived as antigens). Additionally, the cells of innate immunity contribute importantly, through PAMPs/DAMPs and several mechanisms still being defined, to the essential

6

Part one  Principles of Immune Response VH

VH Cµ Cµ

VL

VL

CL

CL

Cµ

Cµ

Cµ

Cµ

Vα

Vβ

α1

α2

α1

β1

Cµ

Cµ

Cα

Cβ

β2m

α3

α2

β2

migM

αβTCR

Cell membrane

HLA class I

HLA class II

FIG 1.1  Antigen-Binding Molecules. Antigen-binding pockets of immunoglobulin (Ig) and T-cell receptor (TCR) comprise variable (V) segments of two chains translated from transcripts that represent rearranged V(D)J or VJ gene elements. Thin red bars designate two of the complementarity determining regions (CDRs) that form portions of the Ig antigen-binding site. The red ovals with thick red bars designate the regions of very high sequence variability in both Igs and TCRs that are generated by recombination of the 3’-end of the V gene element with the D and J gene elements or with the J gene element. In the Ig molecule this is designated CDR3. Antigen-binding pockets of Ig molecules are formed by the three-dimensional folding of the heavy and light chains that juxtapose the CDRs of one heavy chain and one light chain. Antigen-binding grooves of MHC molecules are formed with contributions from α1 and β1 domains of class II molecules and from α1 and α2 domains of class I molecules. All of these molecules are members of the immunoglobulin superfamily. C, constant-region domain; β2, beta-2 microglobulin; mIgM, membrane immunoglobulin M; HLA, human leukocyte antigen; MHC, major histocompatibility complex.

distinction between commensal (not dangerous) and potentially pathogenic (dangerous) microbes. T lymphocytes generally recognize antigens as a complex of short linear peptides bound to self-MHC molecules on the surfaces of APCs (Chapter 6). The source of these peptides can be either extracellular or intracellular proteins and derived from either self or foreign (e.g., microbial) molecules. With the exception of superantigens (see below), T cells neither bind antigen in native conformation nor recognize free antigen in solution. The vast majority of antigens for T cells are oligopeptides. However, the antigen receptors of NKT cells can recognize lipid and glycolipid antigens that are presented to them by MHC-like CD1 molecules.14 Antigen recognition by T cells differs fundamentally from that by antibodies, which are produced by B lymphocytes and their derivatives. Antibodies are oriented toward recognition of extracellular threats and, unlike T cells, can bind complex macromolecules and can bind them in their native conformation either at cell surfaces or in solution. Moreover, antibodies show less preference for recognition of proteins; antibodies against carbohydrates, nucleic acids, lipids, and simple chemical moieties can be readily produced. Although B cells can also be rendered unresponsive by exposure to self-antigens, particularly during differentiation in bone marrow, this process does not define foreignness within the context of self-MHC recognition.

Clonal Basis of Immunological Memory An essential element of self/nonself discrimination is the clonal nature of antigen recognition. Although the immune system can recognize a vast array of distinct antigens, all of the receptors of a single T cell or B cell (and their clonal progeny) have identical antigen-binding sites and hence a particular specificity (Chapter 4). A direct consequence is the capacity for antigen-driven

immunological memory. This phenomenon derives from the fact that after an initial encounter with antigen clones of lymphocytes that can recognize the antigen proliferate and differentiate into effector cells, most of which ultimately are consumed or undergo apoptosis, and a smaller population of long-lived memory cells. These memory cells constitute a pool of cells larger than the initial naïve responders. They can elicit a greater and more rapid response upon subsequent antigen encounter. These two hallmarks of adaptive immunity, clonal specificity, and immunological memory provide a conceptual foundation for the use of vaccines in prevention of infectious diseases (Chapter 90). Immunological memory involves not only the T cells charged with antigen recognition but also the T cells and B cells that mediate the efferent limb of an inflammatory response. In its attack on foreign targets, the immune system can exhibit exquisite specificity for the inducing antigen, as is seen in the epitopespecific lysis of virus-infected target cells by cytolytic T cells.

ANTIGEN-BINDING MOLECULES KEY CONCEPTS Features of the Immunoglobulin (Ig) Superfamily • Large family of ancestrally related genes (more than 100 members) • Most products involved in immune system function or other cell–cell interactions • Ig superfamily members have one or more domains of ~100 amino acids, each usually translated from a single exon • Each Ig domain consists of a pair of β-pleated sheets usually held together by an intrachain disulfide bond

Three sets of molecules are responsible for the specificity of adaptive immune responses by virtue of their capacity to bind

CHAPTER 1  The Human Immune Response foreign antigen. These molecules are Igs, TCRs, and MHC molecules (see Fig. 1.1) (Chapters 4, 5). All are products of a very large family of ancestrally related genes, the immunoglobulin superfamily, which includes many other molecules essential to induction and regulation of immune responses.15,16 Members of the Ig superfamily exhibit characteristic structural features. The most notable of these is organization into homologous domains of approximately 110 amino acids that are usually encoded by a single exon with an intradomain disulfide bond, characteristically configured as antiparallel strands, forming two opposing β-pleated sheets.

Immunoglobulins and T-Cell Receptors The remarkable specificity of Ig and TCR molecules for antigen is achieved by a mechanism of genetic recombination that is unique to Ig and TCR genes (Chapter 4). The antigen-binding site of both types of molecules comprises a groove formed by contributions from each of two constituent polypeptides. In the case of immunoglobulins, these are a heavy (H) chain and one of two alternative types of light (L) chains, κ or λ. In the case of TCRs, either of two alternative heterodimers can constitute the antigen-binding molecule, one comprised of α and β chains, and the other of γ and δ chains. The polypeptides contributing to both Igs and TCRs can be divided into an antigen-binding aminoterminal variable (V) domain and one or more carboxy-terminal constant (i.e., nonvariable) domains. Ig constant region domains generally include specific sites responsible for the biological effector functions of the antibody molecule (Chapter 15).

KEY CONCEPTS Comparison of T-Cell and B-Cell Receptors for Antigen Similarities • Members of the immunoglobulin (Ig) superfamily • Two polypeptide chains contribute to antigen-binding site • Each chain divided into variable and constant regions • Variable regions constructed by V(D)J rearrangements • Nongenomic N-nucleotide additions at V(D)J junctions • Exhibit allelic exclusion • Negative selection against receptors with self-antigen specificity • Transmembrane signaling involving coreceptor molecules Differences • Ig can be secreted; T-cell receptor (TCR) is not • Ig recognizes conformational antigen (Ag) determinants; TCR recognizes linear determinants • Ig can bind antigen in solution; TCR binds antigen when presented by major histocompatibility complex (MHC) molecule on antigenpresenting cell (APC) • Somatic hypermutation of Ig genes can enhance antigen-binding affinity • Ig genes can undergo isotype switching • Inflammatory effector functions by the Ig constant domains • Positive selection of TCR for self-MHC recognition

The most noteworthy feature of the vertebrate immune system is the process of genetic recombination that generates a virtually limitless array of specific antigen receptors from a rather limited genomic investment. This phenomenon is accomplished by the recombination of genomic segments that encode the variable domains of Ig and TCR polypeptides17 (Chapter 4). The products of these rearranged gene elements provide a specific B or T cell

7

with its unique antigen receptor. The variable domain of the mature receptor is created by the rearrangement of two or three separate gene segments. These are designated V (variable) and J (joining), for IgL chains and TCR α and γ chains, and V, D (diversity) and J, for IgH and TCR β and δ chains. In addition to rearrangement, N-nucleotide addition also contributes substantially to receptor diversity. N-nucleotide addition results in the insertion, at the time of rearrangement, of one or more nongenomic nucleotides at the junctions between V, D, and J segments through the action of terminal deoxynucleotidyl transferase (TdT).17 This permits receptor diversity to extend beyond germline constraints. Analysis of the linear sequences of many Ig V regions domains has shown that they contain three sites of high sequence variability that have been designated complementarity determining regions 1–3 (CDR1–3) to indicate that they are the sites that contact antigen (see Fig. 1.1). DNA rearrangement involved in generating T- and B-cell receptors is controlled by recombinases that are active in early thymocytes and in B precursor cells in bone marrow. The process is sequential and carefully regulated, generally leading to translation of one receptor of unique specificity for any given T or B lymphocyte. This result is achieved through a process termed allelic exclusion, wherein only one member of a pair of allelic genes potentially contributing to an Ig or TCR molecule is rearranged at a time.18 The process of allelic exclusion is not absolute, and a small number of lymphocytes will express dual functional Ig or TCR transcripts and, in some cases, two distinct surface receptors.19 But B cells exclusively rearrange Ig genes, not TCR genes, and vice-versa for T cells. Moreover, after producing a functional heavy chain, B cells sequentially rearrange L chain genes, typically κ before λ. Thus B cells express either κ or λ chains, but not both. Similarly, thymocytes express α and β genes or γ and δ genes, and only rarely T cells with αδ or γβ receptors. There is one feature of V region construction that is essentially reserved to B cells. This is somatic hypermutation (SHM), a process that can continue at discrete times throughout the life of a mature B cell at both the VHDHJH and VLJL gene exons.20 Because these rearranged gene exons encode the binding groove that contains the specific points of contact with antigen, on occasion the random process of SHM will result in cells expressing mIg with increased affinity for the antigen they recognize. Typically, cells with increased affinity for antigen are activated preferentially, particularly at limiting doses of antigen. Thus the average affinity of antibodies produced during the course of an immune response tends to increase, a process termed affinity maturation. TCRs do not show evidence of SHM. This absence may be related to the focus on selection in the thymus involving corecognition of a self-MHC molecule and self-peptides,21 (Chapter 8) rather than the continuous process of antigen-driven selection in the periphery by B cells after SHM. Thymic selection results in deletion by apoptosis of the vast majority of differentiating thymocytes by mechanisms that place stringent boundaries around the viability of a thymocyte with a newly expressed TCR specificity. Once a T cell is fully mature and ready for emigration from the thymus, its TCR is essentially fixed, reducing the likelihood of emergent autoimmune T-cell clones in the periphery.

Receptor Selection The receptor expressed by a developing thymocyte must be capable of binding with low-level affinity to some particular MHC

8

Part one  Principles of Immune Response

self-molecule, either class I or class II, expressed by a resident thymic epithelial cell or APC. Because their receptors are generated by a process of semirandom joining of rearranging exon segments coupled with N-nucleotide additions, most thymocytes fail this test. They are consequently deleted as not being useful to an immune system that requires T cells to recognize antigen that is bound to self-MHC molecules. Thymocytes surviving this hurdle are said to have been “positively selected”21 (Fig. 1.2A). Conversely, a small number of thymocytes bind with an unallowably high affinity for a combination of MHC molecule plus antigenic peptide expressed by a thymic APC. Because the peptides available for MHC binding at this site are derived almost entirely from self-proteins, differentiating thymocytes with such receptors are intrinsically dangerous as potentially autoimmune. This deletion of thymocytes with high-affinity receptors for self-MHC plus (presumptively) self-peptide is termed “negative selection”21 (Fig. 1.2B), a process that may also involve activity of regulatory T cells (Tregs).22,23 Another feature that distinguishes B cells from T cells is that the cell surface antigen receptors of the former can be secreted in large quantities as antibody molecules, the effector functions of which are carried out in solution or at the surfaces of other cells. Secretion is accomplished by alternative splicing of Ig

messenger RNAs (mRNAs) to include or exclude a transmembrane segment that is encoded by the Ig heavy-chain genes.

Immunoglobulin Class Switching In addition to synthesizing both membrane and secreted forms of Igs, B cells also undergo class switching. Antibody molecules are comprised of five major classes (isotypes). In order of abundance in serum, these are IgG, IgM, IgA, IgD, and IgE (Chapters 4 and 15). In humans the IgG class is further subdivided into four subclasses and the IgA class into two subclasses. The class of Ig is determined by the sequence of the constant region of its heavy chain (CH). The H chain constant region gene locus is organized with exons that encode each of the Ig isotypes and subclasses located downstream (3’) of the variable (VH) genes. Thus an antibody-producing cell with a successfully rearranged VHDHJH exon can change the class of antibody molecule that it synthesizes by utilization of different CH genes without changing its unique antibody specificity. This process, termed class switch recombination, is regulated by cytokines and is accomplished through the action of activationinduced cytidine deaminase.24 There is no process comparable with class switch recombination in T cells. The two types of TCRs are products of four

Positive selection

Die

A

Negative selection

Cortical Epi APC

Cortical Epi APC

Med. Epi APC

Med. Epi APC

MHC

MHC

MHC

MHC

TCR

TCR

TCR

TCR

Thymocyte

Thymocyte

Thymocyte

Thymocyte

or

Try again (Vα–Jα)

Migrate to medulla

Die

Emigrate

B

FIG 1.2  Two-Stage Selection of Thymocytes Based on Binding Characteristics of Randomly Generated T-Cell Receptors (TCRs). (A) Positive selection. “Double-positive” (CD4+, CD8+) thymocytes with TCRs capable of low avidity binding to some specific self-MHC molecule (either class I or class II) expressed by thymic cortical epithelial cells are positively selected. This process may involve sequential attempts at α gene rearrangement in order to express an αβ TCR of appropriate self-MHC specificity. If binding is to a class I molecule, the positively selected thymocyte becomes CD8 single-positive, and if to a class II molecule, a CD4 single-positive. Thymocytes that are unsuccessful in achieving a receptor with avidity for either a class I or a class II self-MHC molecule die by apoptosis. The solid diamond represents a self-peptide derived from hydrolysis of an autologous protein present in the thymic microenvironment or synthesized within the thymic epithelium itself. (B) Negative selection. “Single-positive” (CD4+ or CD8+) thymocytes, positively selected in the thymic cortex, that display TCRs with high avidity for the combination of self-MHC plus some self (autologous) peptide present in the thymus are negatively selected (i.e., die) as potentially “autoimmune.” Those few thymocytes that have survived both positive and negative selection emigrate to the periphery as mature T cells.

CHAPTER 1  The Human Immune Response independent sets of V-region and C-region genes. A large majority of peripheral blood T cells express αβ TCRs, with a small fraction expressing γδ TCRs (usually ≤5% in peripheral blood). There is a higher representation of γδ T cells in certain tissues, particularly those lining mucous membranes, where they may be specialized for recognition of heavily glycosylated peptides or nonpeptide antigens that are commonly encountered in these tissue compartments. Thymocytes are committed to the expression of either αβ or γδ TCR, and their differentiated progeny (T cells) never change their TCR type in the periphery.

Major Histocompatibility Complex MHC molecules constitute a third class of antigen-binding molecules. When an MHC class I molecule was initially crystallized, an unknown peptide was found in a binding groove formed by the first two (α1 and α2) domains of the molecule. This binding groove has since been established as a general feature of MHC molecules.25 It is now known that the function of MHC molecules is to present antigen to T cells in the form of oligopeptides that reside within this antigen-binding groove (Chapter 6). The most important difference between the nature of the binding groove of MHC molecules and those of Ig and TCR is that the former does not represent a consequence of gene rearrangement. Rather, all the available MHC molecules in an individual are encoded in a linked array, which in humans is located on chromosome 6 and designated the human leukocyte antigen (HLA) complex. MHC molecules are of two basic types, class I and class II. Class I molecules are found on the surface of almost all somatic cells, whereas cell surface expression of class II genes is restricted primarily to cells specialized for APC function. Class I molecules have a single heavy chain that is an integral membrane protein comprised of three external domains (see Fig. 1.1). The heavy chain is noncovalently associated with β2 microglobulin, a nonpolymorphic, non–membrane-bound, single-domain Ig superfamily molecule that is encoded in humans on chromosome 15, not linked to the MHC. Class II MHC molecules, in contrast, comprise two polypeptide chains, α and β (or A and B), of approximately equal size, each of which consists of two external domains connected to a transmembrane region and cytoplasmic tail. Both chains of class II molecules are anchored on the cell by a transmembrane domain, and both are encoded within the MHC. Class I and class II molecules have a high degree of structural homology, and both fold to form a peptide-binding groove on their exterior face, with contribution from the α1 and α2 domains for class I molecules and from α1 and β1 domains for class II.25 There are three class I loci (HLA-A, -B, and -C) and three class II subregions (HLA-DR, -DQ, and -DP) that are principally involved in antigen presentation to T cells (Chapter 5). The functions of other class I and class II genes within this complex are less clear. Some, at least, appear to be specialized for binding (presentation) of peptide antigens of restricted type, source, or function (e.g., HLA-E),26 and others (e.g., HLA-DM and HLA-DO) are clearly involved in antigen processing and loading of antigenic peptides into the binding cleft of the HLA-DR, -DQ, and -DP molecules 27 (Chapter 6). Additionally, members of a family of “nonclassic” class Ib molecules, CD1a-d, which are encoded on chromosome 1, outside the MHC, are specialized for binding and presentation of lipid and lipid-conjugate antigens to T cells.14,28 The HLA complex represents an exceedingly polymorphic set of genes (Chapter 5). Consequently, most individuals are

9

heterozygous at each major locus. In contrast to TCRs and Igs, the genes of the MHC are codominantly expressed. Thus, at a minimum, an APC can express six class I molecules and six class II molecules (the products of the two alternative alleles of three class I and three class II loci). This number is, in fact, usually an underestimate, as a consequence of additional complexity in the organization of the class II region (Chapter 5).

ANTIGEN PRESENTATION Because MHC genes do not undergo recombination, the number of distinct antigen-binding grooves that they can form is many orders of magnitude less than that for either TCRs or Igs. Oligopeptides that bind to MHC molecules are the products of self or foreign proteins. They are derived by hydrolytic cleavage within APCs and are loaded into MHC molecules before expression at the cell surface (Chapter 6). Indeed, stability of MHC molecules at the cell surface requires the presence of a peptide in the antigen-binding groove; cells mutant for the loading of peptide fragments into MHC molecules fail to express MHC molecules on their cell surfaces.29 Since in the absence of infection most hydrolyzed proteins are of self-origin, the binding groove of most MHC molecules contains a self-peptide. Class I and class II molecules differ from one another in the length of peptides that they bind, usually 8–9 amino acids for class I and 14–22 amino acids for class II.25 Although important exceptions are clearly demonstrable, they also generally differ with respect to the source of peptide. Those peptides binding to class I molecules usually derive from proteins synthesized intracellularly (e.g., autologous proteins, tumor antigens, virus proteins, and proteins from other intracellular microbes), whereas class II molecules commonly bind peptides derived from proteins synthesized extracellularly (e.g., extracellular bacteria, nonreplicating vaccines, toxins/allergens). Endogenous peptides are generated by the immunoproteasome and then are loaded into newly synthesized class I molecules in the endoplasmic reticulum following active transport from the cytosol. Proteolytic breakdown and loading of exogenous peptides into class II molecules, in contrast, occurs in acidic endosomal vacuoles. As a consequence of proteolytic processing and binding into an MHC molecule, T cells see linear peptide epitopes. In contrast, because B-cell antigen recognition requires neither proteolytic processing nor binding into an MHC molecule, B cells recognize native, three-dimensional epitopes. In addition to the recognition of lipids and lipid-conjugates presented by CD1 molecules, there are other exceptions to the generalization that MHC molecules only present (and T cells only recognize) oligopeptides. It has been known for many years that T cells can recognize haptens, presumably covalently or noncovalently complexed with peptides residing in the antigenbinding groove. This phenomenon is familiar to physicians as contact dermatitis to nonpeptide antigens, such as urushiol (from poison ivy) and nickel ion. Additionally, a newly recognized subset of T cells designated mucosal-associated (semi-)invariant T (MAIT) cells recognize vitamin B2 (riboflavin) and vitamin B9 (folate) derivatives bound to MR1, a nonpolymorphic MHC class I–like molecule; these vitamin derivatives are expressed by many strains of bacteria and yeast.30 As MAIT cells constitute ~5% of human T cells and up to 25% of CD8 cells, their binding specificity suggests a role for these cells in host defenses. Additionally, certain human γδ T cells can recognize a variety of nonpeptide phosphoantigens, such as phosphorylated nucleotides, other phosphorylated small molecules, and alkylamines. The role of

10

Part one  Principles of Immune Response

APCs and MHC-like molecules in presentation of phosphoantigens to γδ T cells remains a subject of investigation.31 Another exception to the generalization of T-cell recognition of oligopeptides is represented by a group of proteins termed superantigens (SAgs).31 SAgs, of which the staphylococcal enterotoxin A represents a prototype, are produced by a broad spectrum of microbes, ranging from retroviruses to bacteria. They differ from conventional peptide antigens in their mode of contact both with MHC class II molecules and TCRs (Chapter 6). They do not undergo processing to oligopeptide fragments but, rather, bind as intact proteins to class II molecules and TCRs outside the antigen-binding grooves. Their interaction with TCRs is predominantly determined by variable residues of the TCR Vβ region. Because SAgs bind more or less independently of the TCRs α chain and the other variable segments of the β chain, they are capable of activating much larger numbers of T cells compared with conventional peptide antigens, and hence their name. SAgs cause a wave of T-cell activation, proliferation, and production of proinflammatory molecules that can have profound clinical consequences, leading to development of such diseases as toxic shock syndrome.31

LYMPHOCYTE ADHESION AND TRAFFICKING The capacity to survey continuously the antigenic environment is an essential element of immune function. APCs and lymphocytes must be able to find antigen wherever it occurs. Surveillance is accomplished through an elaborate interdigitated circulatory system of blood and lymphatic vessels that establish connections between the solid organs of the peripheral immune system (e.g., spleen, lymph nodes, and lymphoid structures in mucosal tissues) in which the interactions between immune cells predominantly occur (Chapter 2). The trafficking and distribution of circulating cells of the immune system is largely regulated by interactions between molecules on leukocyte surfaces and ligands on vascular endothelial cells32 (Chapter 11). Leukocyte-specific cellular adhesion molecules can be expressed constitutively or can be induced by cytokines (e.g., as a consequence of an inflammatory process). Several families of molecules are involved in the regulation of lymphocyte trafficking. Particularly important are selectins and integrins, which ensure that mobile cells home to appropriate locations within lymphoid organs and other tissues. Selectins are proteins characterized by a distal carbohydrate-binding (lectin) domain. They bind to a family of mucin-like molecules, the endothelial vascular addressins. Integrins are heterodimers essential for the emigration of leukocytes from blood vessels into tissues. Members of the selectin and integrin families not only are involved in lymphocyte circulation and homing but are also important in interactions between APCs, T cells, and B cells in induction and expression of immune responses. Certain endothelial adhesion molecules, mostly members of the Ig superfamily, are similarly involved in promoting interactions between T cells and APCs, as well as in leukocyte transmigration from the vasculature. Additionally, receptors for chemokines are important determinants of lymphocyte migration, particularly in guiding tissue-selective cell trafficking.33

LYMPHOCYTE ACTIVATION For both B cells and T cells, initial activation is a two-signal event34 (Chapter 12). This generalization is particularly true for

immunologically naïve cells that have not been previously exposed to antigen. The first signal is provided by antigen. Most commonly, antigens for B cells are proteins with distinct sites, termed epitopes, which are bound by membrane Ig. Such epitopes can be linear, defined by a contiguous amino acid sequence or (more frequently) can be conformationally defined by the three-dimensional structure of the antigen. Epitopes can also be simple chemical moieties (haptens) that have been attached, usually covalently, to amino acid side chains (Chapter 6). In addition to proteins, some B cells have receptors with specificity for carbohydrates, and less commonly lipids or nucleic acids. Antigens that stimulate B cells can be either in solution or fixed to a solid matrix (e.g., a cell membrane). As previously noted, the nature of antigens that stimulate T cells is more limited. TCRs do not bind antigen in solution but are usually stimulated only by small molecules, primarily oligopeptides, which reside within the antigen-binding cleft of a self-MHC molecule. The second signal requisite for lymphocyte activation is provided by an accessory molecule expressed on the surface of the APC (e.g., B7/CD80) for stimulation of T cells or on the surface of a helper T cell (e.g., CD40L/CD154) for activation of B lymphocytes. The cell surface receptors for this particular second signal on T cells is CD28 and on B cells is CD40 (Fig. 1.3). Other cell surface ligand-receptor pairs may similarly provide the second signal (Chapters 8, 12). The growth and differentiation of both T cells and B cells additionally requires stimulation with one or more cytokines, which are peptide hormones secreted in small quantities by activated leukocytes and APCs for function in the cellular microenvironment.35 In the absence of a second signal, cells stimulated only by antigen become unresponsive to subsequent antigen stimulation (anergic)36 (Chapter 12). T cell

B cell

mIg + Ag CD40 TCR

Class II + peptide

CD28

Up-regulation mIg + Ag CD40L

CD40

TCR

Class II + peptide

CD28

CD80 (B7) Cytokines

FIG 1.3  Reciprocal Activation Events Involved in Mutual Simulation of T Cells and B Cells. T cells constitutively express T-cell receptors (TCRs) and CD28. B cells constitutively express mIg and major histocompatibility complex (MHC) class II. Antigen bound to mIg is endocytosed and processed to peptide fragments that bind to MHC class II molecules for presentation to TCRs. Activation of B cells by antigen (Ag) upregulates their expression of CD80 that interacts with CD28 to activate T cells. This upregulates CD40L (CD154) on the T cell and induces cytokine synthesis. Costimulation of B cells by antigen, CD40L, and cytokines leads to Ig production.

CHAPTER 1  The Human Immune Response Signal transduction through the antigen receptor is a complex process involving interactions between the specific receptor and molecules coexpressed in the cell membrane.37 For B cells, this is a heterodimer, Igα/Igβ; and for T cells it is a macromolecular complex, CD3, usually comprising γ, δ, εε and ς chains. Within the cell membrane, antigen receptor stimulation induces phosphorylation of Igα/Igβ or CD3 and hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, leading to generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). As a consequence of signal transduction and secondarily of DAG and IP3 generation, tyrosine and serine/ threonine protein kinases are activated. In turn, these kinases catalyze phosphorylation of a number of signal transducing proteins, leading to activation of cytoplasmic transcription factors NF-AT in T cells and NF-κB in both T cells and B cells. These transcription factors then translocate to the nucleus, where they bind to 5’ regulatory regions of genes that are critical to generalized lymphocyte activation37,38 (Chapter 12).

CELL-MEDIATED IMMUNE RESPONSES T-Cell Subsets T lymphocytes expressing an αβ TCR can be divided into two major subpopulations based on the class of MHC molecule that their TCR recognizes and the consequent expression of one of a pair of TCR accessory molecules, CD4 or CD8 (Chapters 4, 8). Binding of CD4 to class II MHC molecules or CD8 to class I MHC molecules on APCs contributes to the overall strength of intercellular molecular interactions. The ratio of CD4 to CD8 cells in peripheral blood is usually about 2 : 1.

CD4 T Cells, Cytokines, and Chemokines The activities of CD4 T lymphocytes, commonly referred to as T helper (Th) cells, are mediated predominantly through the secretion of cytokines (Chapter 9). Cytokine activity can include autostimulation (autocrine function) if the cell producing the cytokine also expresses a surface receptor for it, or stimulation of other cells in the microenvironment of the cytokine-secreting cell (paracrine function) including B cells, APCs, and other T cells. Although it is now recognized that their biological effects are broader than implied by their name, many of the principal cytokines active in the immune system are known as interleukins (ILs), implying that they are produced by a leukocyte to act on other leukocytes. The specific profile of cytokines produced by CD4 T cells allows further functional subdivision (Chapters 16, 18).35,39 CD4 T cells elaborating the “inflammatory” cytokines involved in effector functions of cell-mediated immunity, such as IL-2 and IFN-γ, are designated Th1 cells. Th2 CD4 T cells synthesize cytokines, such as IL-4 and IL-13, which control and regulate antibody responses and activate cells that are involved in host defense against parasites. Differentiation of Th1 versus Th2 subsets is a process substantially controlled by positive feedback loops, being promoted particularly by IL-12 in the case of Th1 cells and IL-4 in the case of Th2 cells. It is important to note that generalizations regarding cytokine activity are usually oversimplifications, reflecting a substantial overlap and multiplicity of functions (Chapter 9). For example, although IL-2 was initially identified as a T-cell growth factor, it also significantly affects B-cell differentiation. The prototypic inflammatory cytokine, IFN-γ, which promotes differentiation of effector function of cytotoxic lymphocytes (CTLs) and macrophages, is also involved

11

in regulation of Ig isotype switching. And IL-4, although known primarily as a B-cell growth and differentiation factor, can also stimulate proliferation of T cells. A distinct subset of cytokines is a large group of highly conserved cytokine-like molecules, smaller than typical cytokines (~7–12 kilodaltons [kDa]), termed chemotactic cytokines, or chemokines33 (Chapter 10). Chemokines are classified on the basis of the number and spacing of specific cysteine residues. They regulate and coordinate trafficking and activation of leukocytes, functioning importantly in host defenses, and also broadly in a variety of nonimmunological processes, including organ development and angiogenesis. They are characterized by binding to seven-transmembrane-domain G protein–coupled receptors. Of particular interest to clinical immunologists, the chemokine receptors CCR5 and CXCR4 together with CD4 are utilized by human immunodeficiency virus (HIV) as coreceptors to gain entry into target cells.40 Cytokines produced by activated T cells can downregulate as well as initiate or amplify immune responses.41 Downregulating cytokines include IL-10 (produced by both T cells and B cells) and transforming growth factor-β (TGF-β). The functions of IL-10 in vivo are thought to include both suppression of proinflammatory cytokine production and enhancement of IgM and IgA synthesis. TGF-β, produced by virtually all cells, expresses a broad array of biological activities, including promotion of wound healing and suppression of both humoral and cellmediated immune responses. In addition to their central role in initiation and regulation of immune responses, CD4 T cells are important effectors of cell-mediated immunity (Chapter 16). Through the elaboration of inflammatory cytokines, particularly IFN-γ, they are essential contributors to the generation of chronic inflammation, characterized histologically by mononuclear cell infiltrates, where their principal role is thought to be the activation of macrophages. Additionally, in some circumstances CD4 T cells can function as cytotoxic effectors, either directly as CTLs (in which case the killing is “restricted” for recognition of antigen-bound self-MHC class II) or through the elaboration of cytotoxic cytokines, such as lymphotoxin and TNF-α. A third subset of Th cells, designated Th17, has been recognized more recently. With differentiation driven particularly by TGF-β and IL-23 and characterized by the production of the proinflammatory cytokine IL-17, Th17 cells are important in the induction and exacerbation of autoimmunity in a variety of disease models, as well as in host defenses against a broad spectrum of extracellular bacteria, fungi and other pathogens.42 Research continues to identify additional examples of CD4 T cells, which may become recognized as distinct subsets, whose function is governed by other predominantly expressed cytokines to achieve specialized effector responses. One final category of CD4 cells, Tregs, plays a crucial role suppressing the functions of other lymphocytes. Tregs can differentiate either in the thymus (tTregs) or in the periphery (pTregs). A third category of Tregs are induced in vitro (iTregs)43 (Chapter 18). These are commonly characterized by surface expression of CD4 and CD25 and by nuclear expression of the transcription factor Foxp3. Peripheral activation of CD25+ Tregs is via the TCRs; the cells are IL-2 dependent and apparently require cell-to-cell contact for suppressive function. They can suppress functions of both CD4 and CD8 T cells, as well as B cells, NK cells, and NKT cells. In contrast to activation, suppressor effects are independent of the antigen specificity of the target

12

Part one  Principles of Immune Response

cells. Other Tregs are noted for production of inhibitory cytokines, including IL-10- and TGF-β–secreting Th3 cells, and IL-10– producing Tr1 cells.44,45

CD8 T Cells The best understood function of CD8 T cells is that of CTL effectors.46 CTLs are particularly important in host defenses against virus-infected cells, where they can kill target cells expressing viral peptides bound to self-MHC class I molecules (Chapter 17). This process is highly specific and requires direct apposition of CTLs and target cell membranes. Bystander cells, expressing MHC molecules that have bound peptides that the CD8 T cell does not recognize, are not affected. The killing is unidirectional; the CTL itself is not harmed, and after transmission of a “lethal hit,” it can detach from one target to seek another. Killing occurs via two mechanisms: a death-receptor-induced apoptotic mechanism and a mechanism involving insertion of perforins into the target cell membrane to create a pore through which granzymes and other cytotoxic enzymes can be transferred from the CTL into the target cell. CTL activity is enhanced by IFN-γ. As CTL function is dependent on target cell surface display of MHC class I molecules, a principle mechanism of immune evasion by viruses and tumors is elaboration of factors that downregulate class I expression (Chapter 25). However, this increases susceptibility of such cells to cytolysis by NK cells that are activated to attack cells expressing low levels of class I MHC molecules.

ANTIBODY-MEDIATED IMMUNE RESPONSES The structure of antibodies permits a virtually limitless binding specificity of its antigen-binding groove. Antigen binding can then be translated into biological effector functions based on properties of the larger nonvariable (constant) region of its heavy chains (Fc fragment) (Chapter 15). Moreover, in response to cytokines in the cellular microenvironment, through the mechanism of isotype switching an antibody-producing cell can alter the exons that are used to encode its heavy-chain constant region and thereby the biological effects of its secreted product without affecting its antigen-binding specificity. With functional heterogeneity determined by isotype, the antibody molecules provide a broad-based and efficient defense system against extracellular microbes or foreign macromolecules (e.g., toxins and venoms) (Chapters 15, 27, 90). Each antibody class contributes differently to an integrated defense system.47 IgM is the predominant class formed on initial contact with antigen (primary immune response). As a monomeric structure comprises two light (κ or λ) and two heavy (µ) chains, it is initially expressed as a membrane bound antigen receptor on the surface of B lymphocytes. The avidity of serum IgM for antigen binding is increased by its organization into a pentamer of five of the monomeric subunits held together by a joining (J) chain. IgM is essentially confined to the intravascular compartment. As a multivalent antigen binder that can efficiently activate (“fix”) complement, it is an important contributor to immune responses early after the initial encounter with antigen. The synthesis of IgM, compared with other isotypes, is much less dependent on the activity of T lymphocytes. IgG is the most abundant immunoglobulin in serum and the principal antibody class of a secondary (anamnestic or memory) immune response. IgG molecules are heterodimeric monomers with two light (κ or λ) and two heavy (γ) chains joined by interchain disulfide bridges. Because of its abundance, its capacity

KEY CONCEPTS Biological Properties of Immunoglobulin (Ig) Classes • IgM • Principal Ig of the primary immune response • Generally restricted to the vascular compartment • Antigen receptor (monomer) for most naïve B cells • Fixes complement potently • IgG • Principal Ig of secondary immune responses • Binds to Fcγ receptors on neutrophils, monocytes/macrophages, natural killer (NK) cells • Four subclasses with different effector functions • Fixes complement (except IgG4 subclass) • IgA • Principal Ig of mucosal immunity • Two subclasses • IgD • Antigen receptor for mature B cells • Typically coexpressed with membrane IgM • IgE • Binds to Fcε receptors on mast cells and basophils • Antibody of immediate hypersensitivity • Important in defenses against helminths

to fix complement, and the expression on phagocytes of Fcγ receptors, IgG is the most important antibody for systemic secondary immune responses. IgG is the only isotype that is actively transported across the placenta. These transported maternal IgG antibodies provide the neonate with an important level of antibody protection during the early months, when its own antigen-driven antibody responses are first developing (Chapter 38). IgA is the principal antibody in the body’s secretions (Chapter 20). It is found in serum in monomeric form of two light and two heavy (α) chains or as a dimer joined by J chain. In secretions, it is usually present in dimeric form and is actively secreted across mucous membranes by attachment of a specialized secretory component (SC) that is recognized by the polyIg receptor on mucosal epithelial cells. Dimeric IgA is found in high concentration in tears, saliva, and secretions of the respiratory, gastrointestinal, and genitourinary systems; it is relatively resistant to enzymatic digestion. It is particularly abundant in colostrum, where its concentration may be >50 times that in serum, providing passive immunity to the gastrointestinal system of a nursing neonate. IgA does not fix complement by the antibody-dependent pathway and hence does not promote phagocytosis. Its role in host defenses lies in preventing a breach of the mucous membrane surface by microbes or their toxic products. IgD and IgE are present in serum at concentrations much lower than that of IgG. The biological role of IgD remains controversial. B cells can express both membrane IgM and IgD by alternative splicing of the Ig heavy chain gene, or can secrete only IgD via an apparently atypical form of class switch recombination.48 These mechanisms do not require T-cell help. Although IgE is the least abundant isotype in serum, it has dramatic biological effects because it is responsible for immediatetype hypersensitivity reactions, including systemic anaphylaxis (Chapter 42). Such reactions reflect expression of high-affinity receptors for Fcε on the surfaces of mast cells and basophils. Cross-linking of IgE molecules on such cells by antigen induces their degranulation, with the immediate release of preformed potent biological mediators and de novo synthesis and secretion

CHAPTER 1  The Human Immune Response of additional proinflammatory molecules. The protective role of IgE is in host defenses against parasitical infestation, particularly with helminths (Chapter 31).

Complement and Immune Complexes As noted, the biological functions of IgG and IgM are importantly reflections of their capacities to activate the complement system. Through a cascade of sequential substrate–enzyme interactions, the 11 principal components of the antibody-dependent complement cascade (C1q, C1r, C1s, and C2–C9) cause many of the principal consequences of an antigen-antibody interaction (Chapter 21). These include the establishment of pores in a target cell membrane by the terminal components (C5–C9) leading to osmotic lysis; the production of factors (principally C5a) with chemotactic activity for phagocytic myeloid cells; opsonization by C3b, promoting phagocytosis; and the ability to induce degranulation of mast cells (C3a, C4a, and C5a). There are three distinct pathways to complement activation.49 The pathway mediated by the binding of the first component (specifically C1q) to IgG or IgM has been termed the “classical” pathway (CP). The lectin pathway is similar to the CP but is activated by selected carbohydrate-binding proteins, the mannose (or mannan)-binding lectin (MBL), and ficolins, which recognize certain carbohydrate repeating structures on microorganisms. MBL and ficolins are plasma proteins that are homologous to C1q and contribute to innate immunity through their capacity to induce antibody- and C1q-independent activation of the CP. Finally, a large number of substances, including certain bacterial, fungal, and viral products, can directly activate the cascade through a distinct series of proteins also leading to activation of the central C3 component. Although bypassing C1, C4, and C2, this distinct pathway can achieve all the biological consequences of C3–C9 activation. Non–antibody-induced activation of C3 is referred to as the “alternative” pathway (AP) or “properdin” pathway. Additionally, the central components of the cascade (e.g., C5a) can be directly produced by the action of serine proteases of the coagulation system.49 Reflecting these separate pathways to activation and the fact that many types of leukocytes express receptors for activated complement components, the complement system is a major contributor to the efferent limbs of both innate and acquired immune systems. In addition to their roles in pathogen/antigen elimination, constituents of the complement system, together with antigen– antibody (immune) complexes, act at leukocyte surfaces to regulate immune functions. For example, interaction of immune complexes via FcγR on B cells decreases their responsiveness to stimulation. In contrast, complement activation on B-cell surfaces coligates their receptors with B-cell receptors for antigen, rendering the cells more readily activated and resistant to apoptosis. Essential for the proper function of the complement system is a series of downregulatory mechanisms that prevent unwanted activation of the system and that extinguish its activity when no longer needed. The regulatory pathways are mediated by a combination of both soluble complement-binding and digesting molecules and cell surface binding proteins.

APOPTOSIS AND IMMUNE HOMEOSTASIS An immune response is commonly first viewed in a “positive” sense—that is, lymphocytes are activated, proliferate, differentiate, and carry out effector functions. It is equally important, however,

13

that this positive response be tightly regulated by mechanisms that operate to turn off the response and to eliminate cells no longer required.50,51 Under physiological circumstances, once an immune response fades, commonly as a consequence of antigen depletion, two pathways to terminal lymphocyte differentiation become available: apoptosis or differentiation into memory cells. Memory cells are, of course, a key to the effectiveness of the adaptive immune system, since a second activating encounter with antigen (e.g., pathogen) is both more rapid and more productive. Isotype-switched high-affinity antibodies are rapidly produced, and/or clones of CTL effector cells proliferate. But the majority of lymphocytes in an active response are not required for maintenance of immunological memory, and the necessity for homeostasis leads to apoptosis of cells no longer required. Apoptosis (or Regulated Cell Death; RCD) is a unique process of cellular death, widely conserved phylogenetically, and distinguished from death by necrosis by cellular shrinking, DNA fragmentation, and breakdown of cells into “apoptotic bodies” containing nuclear fragments and intact organelles that can be eliminated by phagocytosis without release into the extracellular space of the majority of intracellular, especially nuclear, components. Necrosis can be genetically determined (Regulated Necrosis; RN) or unregulated, reflecting some accidental or otherwise inevitable process (Chapter 13). Apoptosis depends on the activation of cysteinyl proteases, termed caspases, which cleave proteins that regulate DNA repair and the establishment/ maintenance of cellular architecture. In the absence of these apoptotic mechanisms, massive proliferation of cells in lymphoid tissues results and is seen clinically as autoimmune lymphoproliferative syndrome (ALPS), which is characterized by lymphocytosis with lymphadenopathy and splenomegaly as well as autoimmunity and hypergammaglobulinemia.52

MECHANISMS OF IMMUNOLOGICAL DISEASES Immunological diseases can be classified on the basis of our understanding of normal immune physiology and its perturbations in disease states (Table 1.2). One type of immunological disease results from failure or deficiency of a component of the immune system leading to failure of normal immune function (Chapters 32–40). Such disorders are usually identified by increased susceptibility to infection (Chapter 37). Failure of host defense can be congenital (e.g., X-linked agammaglobulinemia; Chapter 34) or acquired (e.g., acquired immunodeficiency syndrome [AIDS]; Chapter 39). It can be global (e.g., severe combined immunodeficiency [SCID]; Chapter 35) or, quite specific, involving only a single component of the immune system (e.g., selective IgA deficiency; Chapter 34). A second type of immunological disease is malignant transformation of immunological cells (Chapters 77–80). Manifestations of leukocyte malignancies are protean, most commonly TABLE 1.2  Mechanisms of Immunological

Diseases

1. Functional deficiency of key immune system components a. Congenital b. Acquired 2. Malignant transformation of immune system cells 3. Immunological dysregulation 4. Autoimmunity 5. Untoward consequences of physiological immune function

14

Part one  Principles of Immune Response

reflecting the secondary consequences of solid organ or bone marrow infiltration or replacement of normal cells by tumor cells, with resulting anemia and immunological deficiency. The remaining types of immunopathogenesis are more specific to the immune system. Dysregulation of an essentially intact immune system constitutes a third general type of immune disorder. Features of an optimal immune response include antigen recognition and elimination, with little adverse effect on the host. Both initiation and termination of the response, however, involve regulatory interactions that can go awry when the host is challenged by antigens of a particular structure or presented in a particular fashion. Diseases of immune dysregulation can result from genetic and environmental factors that act together to produce a pathological immune response, such as acute allergic diseases (Chapters 41–49). Some forms of allergic disease are thought to be a consequence of insufficient exposure to nonpathogenic microbes and other potential allergens in early childhood, resulting in an increased susceptibility to allergy, atopy, and asthma once the immune system has matured. The so-called “hygiene hypothesis” suggests that mucosal tissuecolonizing organisms play key roles in the initial establishment of immune homeostasis.53 The importance of establishing immune homeostasis early in life is also supported by studies demonstrating reduction in the likelihood of food allergy associated with feeding of the allergenic foods to infants at high risk for allergy54-56 (Chapter 45). A fourth type of immunological disorder is the result of failure of a key feature of normal immune recognition, the molecular discrimination between self and nonself. Ambiguity in this discrimination can lead to autoimmune tissue damage (Chapters 50–76). Although such damage can be mediated by either antibodies or T cells, the common association of specific autoimmune diseases with inheritance of particular HLA alleles (Chapter 5) suggests that the pathogenesis of autoimmune diseases usually represents a failure of regulation of the anti-self inflammatory response by T cells. The immunologic attack on self-tissues can be general, leading to systemic autoimmunity, such as systemic lupus erythematosus; or it can be localized, as in organ-specific autoimmune diseases. In the latter instances, the immune system attacks specific types of cells and usually particular cell surface molecules. In most cases, pathology is a consequence of target tissue destruction (e.g., multiple sclerosis, rheumatoid arthritis, or insulin-dependent diabetes mellitus). However, depending on the antigenic specificity of the abnormal immune response, autoimmunity can lead to receptor blockade (e.g., myasthenia gravis or insulin-resistant diabetes) or hormone receptor stimulation (e.g., Graves disease). It is thought by many immunologists that ambiguity in self/nonself discrimination is commonly triggered by an unresolved encounter with an infectious organism or other environmental agent that shares some structural features with self-tissue structures, although this remains a subject of controversy57,58 (Chapter 50). Insight into mechanisms whereby specific HLA alleles predispose to development of autoimmunity and others may be protective are suggested by studies in HLA-transgenic mice, which suggest that alleles that predispose animals to a particular autoimmune disease may reflect a T cell phenotype associated with secretion of pro-inflammatory cytokines. In contrast protective alleles were associated with elaboration of trolerogenic cytokines by regulatory T cells.59 A fifth form of immunological disease occurs as a result of physiological, rather than pathological, immune functions.

Inflammatory lesions in such diseases are the result of the normal function of the immune system. A typical example is contact dermatitis to such potent skin sensitizers as urushiol, the causative agent of poison ivy dermatitis (Chapter 44). These diseases can also have an iatrogenic etiology that can range from mild and self-limited (e.g., delayed hypersensitivity skin test reactions) to life-threatening (e.g., graft-versus-host disease, organ graft rejection).

HOST IMMUNE DEFENSES SUMMARIZED The first response upon initial contact with an invading pathogen depends on components of the innate immune system (Chapter 3). This response begins with recognition of PAMPs expressed by cells of the pathogen. These include lipoproteins, lipopolysaccharide, unmethylated CpG-DNA, and bacterial flagellin, among others. PAMPs bind to PRRs on or within effector cells of the host’s innate immune system, including DCs, granulocytes, and ILCs.3 The best characterized PRRs are the TLRs, first recognized as determinants of embryonic patterning in Drosophila and subsequently appreciated as components of host defenses in both insects and vertebrates. TLR subfamilies can be distinguished by expression either on the cell surface or in intracellular compartments. A second major family of PRRs comprises NLRs, which detect intracellular microbial products. Binding of TLRs or NLRs by PAMP ligands triggers intracellular signaling pathways via multiple “adapters,” leading to a vigorous inflammatory response. The innate immune response also includes the capacity of NK lymphocytes to identify and destroy, by direct cytotoxic mechanisms, cells lacking surface expression of MHC class I molecules, which marks them as potentially pathogenic.4 Additionally, an innate immune response involves elements of the humoral immune system that function independently of antibody, especially the activation of the complement cascade through the lectin pathway and the AP, with consequent opsonization of particles and microbes to promote their phagocytosis and destruction. The nature of the adaptive immune response to any particular pathogenic agent is determined largely by the context in which the pathogen is encountered. Regardless, effectiveness depends on the four principal properties of adaptive immunity: (i) a virtually unlimited capacity to bind macromolecules, particularly proteins, with exquisite specificity, reflecting generation of antigen-binding receptors by genetic recombination and, in the case of B cells, somatic hypermutation; (ii) the capacity for self/ nonself discrimination, consequences of a rigorous process involving positive and negative selection during thymocyte differentiation, as well as negative selection during B-cell differentiation; (iii) the property of immunological memory, reflecting antigen-driven clonal proliferation of T cells and B cells, which results in increasingly rapid and effective responses on second and subsequent encounters with a particular antigen or pathogen; and (iv) mechanisms for pathogen destruction, including direct cellular cytotoxicity, release of inflammatory cytokines, opsonization with antibody and complement, and neutralization in solution by antigen precipitation or conformational alteration plus phagocytosis and intracellular digestion. Although most acquired immune responses include multiple defense mechanisms, several generalizations may be conceptually useful. T cell–mediated (and NK cell–mediated) effector functions are particularly important in defenses against pathogens encountered intracellularly or at cell surfaces, such as intracellular viruses,

CHAPTER 1  The Human Immune Response

15

intracellular bacteria, and tumor cells. These responses involve the production of inflammatory cytokines by CD4 Th1 cells, as well as the direct cytolytic activity of CD8 CTLs. In contrast, host defenses to most antigens encountered primarily in the extracellular milieu are largely dependent on humoral mechanisms (antibody and complement) for antigen neutralization, precipitation, or opsonization and subsequent destruction by phagocytes. Targets of antibody-mediated immunity include extracellular virus particles, bacteria, and toxins (or other foreign proteins). It is worth reiterating, however, that induction of an effective antibody response (including isotype switching) and development of immunological memory (resulting from B-cell clonal expansion and B memory cell differentiation) require antigen activation not only of specific B cells but also CD4 T cells, particularly of the Th2 type. Additionally, antibacterial and antifungal responses that involve prominent responses by neutrophils require CD4 T cells of the Th17 type.

KEY CONCEPTS Characteristic Infections Associated With Immune Deficiency Syndromes Deficiencies of T Cell–Mediated Immunity • Mucocutaneous fungal infections, especially Candida albicans • Systemic (deep) fungal infections • Systemic infection with attenuated viruses (e.g., live viral vaccines) • Infection with viruses of usually low pathogenicity (e.g., cytomegalovirus) • Pneumocystis jiroveci pneumonia Antibody Deficiencies • Infections by encapsulated bacteria (e.g., Streptococcus spp., Haemophilus influenza) • Recurrent pneumonia, bronchitis, sinusitis, otitis media • Giardia lamblia enteritis Phagocyte Deficiencies Infection by gram-positive bacteria (e.g., staphylococci, • streptococci) • Gram-negative sepsis • Systemic fungal infections (e.g., Candida spp., Aspergillus spp.) Adhesion Molecule Deficiencies • Infections with pyogenic bacteria (especially staphylococci) • Cutaneous and subcutaneous abscesses Complement Component Deficiencies • C3 deficiency: Infections with encapsulated bacteria • Deficiency of terminal components: infections with gram-negative bacteria, especially Neisseria spp.

Finally, clinical “experiments of nature” have proven particularly instructive in our efforts to understand the role of specific components of the immune system in overall host defenses60 (Chapter 37). The importance of T cell–mediated immunity in host defenses to intracellular parasites, fungi (Fig. 1.4), and viruses is emphasized by the remarkable susceptibility of patients with T cell–deficiency to pathogenic organisms, such as Pneumocystis jiroveci and Candida albicans, and by the fact that utilizing attenuated live virus vaccines in such patients can lead to devastating disseminated infections. Indeed, the relationship between susceptibility to particular potential pathogens and specific immunological deficiencies is nicely illustrated by recent dem-

FIG 1.4  Leg of a 16-year-old patient with chronic mucocutaneous candidiasis as a consequence of a congenital T-cell deficiency associated with hypoparathyroidism and adrenal insufficiency.

onstrations that the pathogenesis of various familial forms of chronic mucocutaneous candidiasis reflects deficiency of IL-17–mediated immunity.61 However, patients with defects in antibody synthesis or phagocytic cell function are characteristically afflicted with recurrent infections with pyogenic bacteria, particularly grampositive organisms. And patients with inherited defects in synthesis of terminal complement components have increased susceptibility to infection with species of Neisseria. In recent years, immunology has entered the lay lexicon, largely as a result of the HIV pandemic. People throughout the world are now aware of the tragic consequences of immune deficiency. The remarkable progress in understanding this disease, however, depended substantially on earlier studies of relatively rare patients with primary immunodeficiency syndromes, more recent acceleration due to progress in genomic definition of their molecular basis. Similarly, cure of patients with primary immunodeficiencies by cellular reconstitution, particularly bone marrow or stem cell transplantation62 (Chapter 82), presaged recent progress in correction of such diseases by gene replacement therapy63 (Chapter 85). The “present” of clinical immunology is, indeed, bright. But its future potential to impact prevention and treatment of many challenging diseases, including cancer (Chapter 77), through specific analysis of genetic mutations and enhancement or suppression of global or antigen-specific immune responses and check-point inhibition is even more exciting to contemplate.64 A few approaches are broadly hinted at here, and it is hoped that readers will enjoy considering such “perspectives” throughout the book and, given the nature of the immune system, it is also hoped will challenge themselves to transform a particular author’s views to new and different clinical settings. Studies in experimental animals, especially murine studies, have been critical to our understanding of molecular aspects of

16

Part one  Principles of Immune Response ON THE HORIZON

Enhancement of Immune Responses • Use of CRISPR/Cas9 technology in correction of gene defect in monogenic immunodeficiency diseases • Enhancement of molecular-pathway-specific cancer immunotherapy • Prediction of molecular targets and improvements in adjuvants for vaccines Suppression of Immune Responses • Further development of protocols for prevention of childhood allergic diseases by appropriate environmental exposures in infancy • Improved allergen-specific immunotherapy of allergic diseases • Prevention of graft-versus-host disease in allogeneic bone marrow transplantation cases • Induction of antigen-specific immune tolerance in humans • Pharmacological development of specific inhibitors of cytokines, chemokines, and their receptors Immunodiagnostics and Immunopathogenesis • Routine use of genomic analysis for development of personalized medicine applicable to immunological diseases • Development of novel diagnostic tools based on nanotechnology arrays • Application of gastrointestinal microbiome analysis in understanding the pathogenesis of inflammatory bowel diseases, leading to novel therapeutic options • Understanding the role of inflammation in the pathogenesis of leading causes of morbidity and mortality, including myocardial infarction, stroke, and Alzheimer disease

immune system function and have contributed importantly to our appreciation of how aberrations of such functions are involved in the pathogenesis of disease. Insights gained from use of transgenic mice (including murine expression of human genes) and constitutive or conditional gene-knockout mice are essential to a comprehensive view of the immune system at the advancing edge of its clinical application, implying that future progress in clinical immunology will equally depend on detailed analysis in such systems. It has become apparent, however, that there are important differences in aspects of the human and rodent immune systems. Consequently, the carefully studied patient, particularly when coupled with the power of increasingly feasible genome and transcriptome sequencing, will remain the ultimate crucible for understanding human immunity and the roles of the immune system in the pathogenesis of and protection from disease. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Flajnik MF, Kasahara M. Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat Rev Genet 2010;11:47–59. 2. Buchmann K. Evolution of innate immunity: clues from invertebrates via fish to mammals. Front Immunol 2014;5:459. 3. Vivier E, Raulet DH, Moretta A, et al. Innate or adaptive immunity? The example of natural killer cells. Science 2011;331:44–9. 4. Liu J, Cao X. Cellular and molecular regulation in innate inflammatory responses. Cell Mol Immunol 2016;13:711–21. 5. Tukhvatulin AL, Dzharullaeva AS, Shcheblyakov DV, et al. Powerful complex immunoadjuvant based on synergistic effect of combined TLR4 and NOD2 activation significantly enhances magnitude of humoral and cellular adaptive immune responses. PLoS ONE 2016;11:e0155650.

6. Tiburzy B, Kulkarni U, Hauser AE, et al. Plasma cells in immunopathology: concepts and therapeutic strategies. Semin Immunopathol 2014;36:277–88. 7. Juelke K, Romagnani C. Differentiation of human innate lymphoid cells (ILCs). Curr Opin Immunol 2016;38:75–85. 8. Saunders PM, Vivian JP, O’Connor GM, et al. A bird’s eye view of NK cell receptor interactions with their MHC class I ligands. Immunol Rev 2015;267:148–66. 9. Liu H, Li L, Voss C, et al. A comprehensive immunoreceptor phosphotyrosine-based signaling network revealed by reciprocal protein-peptide array screening. Mol Cell Proteomics 2015;14:1846–58. 10. O’Sullivan TE, Sun JC, Lanier LL. Natural killer cell memory. Immunity 2015;43:634–45. 11. Rezvani K, Rouce RH. The application of natural killer cell immunotherapy for the treatment of cancer. Front Immunol 2015;6:578. 12. Den Haan JM, Arens R, van Zelm MC. The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells. Immunol Lett 2014;162:103–12. 13. Hammer GE, Ma A. Molecular control of steady-state dendritic cell maturation and immune homeostasis. Annu Rev Immunol 2013;31:743–91. 14. Rossjohn J, Pellici DG, Patel O, et al. Recognition of CD1d-restricted antigens by natural killer T cells. Nat Rev Immunol 2012;12:845–57. 15. Hirano M, Das S, Guo P, et al. The evolution of adaptive immunity in vertebrates. Adv Immunol 2011;109:125–57. 16. Xu Z, Jin B. A novel interface consisting of homologous immunoglobulin superfamily members with multiple functions. Cell Mol Immunol 2010;7:11–19. 17. Dudley DD, Chaudhuri J, Bassing CH, et al. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences. Adv Immunol 2005;86:43–112. 18. Corcoran AE. Immunoglobulin locus silencing and allelic exclusion. Semin Immunol 2005;17:141–54. 19. Maul RW, Gearhart PJ. AID and somatic hypermutation. Adv Immunol 2010;105:159–91. 20. Mechtcheriakova D, Svoboda M, Meshcheryakova A, et al. Activation-induced cytidine deaminase (AID) linking immunity, chronic inflammation, and cancer. Cancer Immunol Immunother 2012;61:1591–8. 21. von Boehmer H. Selection of the T-cell repertoire: receptor-controlled checkpoints in T-cell development. Adv Immunol 2004;84:201–38. 22. Coutinho A, Caramalho I, Seixas E, et al. Thymic commitment of regulatory T cells is a pathway of TCR-dependent selection that isolates repertoires undergoing positive or negative selection. Curr Top Microbiol Immunol 2005;293:43–71. 23. Simons DM, Picca CC, Oh S, et al. How specificity for self-peptides shapes the development and function of regulatory T cells. J Leukoc Biol 2010;88:1099–107. 24. Hwang JK, Alt FW, Yeap LS. Related mechanisms of antibody somatic hypermutation and class switch recombination. Microbiol Spectr 2015;3:MDNA3-0037-2014. 25. Madden DR. The three-dimensional structure of peptide-MHC complexes. Annu Rev Immunol 1995;13:587–622. 26. Hansen SG, Wu HL, Burwitz BJ, et al. Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E. Science 2016;351:714–20. 27. Mellins ED, Stern LJ. HLA-DM and HLA-DO, key regulators of MHC-II processing and presentation. Curr Opin Immunol 2014;26:115–22. 28. Cohen NR, Garg S, Brenner MB. Antigen presentation by CD1: lipids, T cells, and NKT cells in microbial immunity. Adv Immunol 2009;102:1–94. 29. Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Immunol 2013;31:443–73. 30. Godfrey DI, Uldrich AP, McCluskey J, et al. The burgeoning family of unconventional T cells. Nat Immunol 2015;16:1114–23. 31. Spaulding AR, Salgado-Pabon W, Kohler PL, et al. Staphylococcal and streptococcal superantigen exotoxins. Clin Microbiol Rev 2013;26:422–47. 32. Smith CW. Adhesion molecules and receptors. J Allergy Clin Immunol 2008;121:S375–9.

CHAPTER 1  The Human Immune Response 33. Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol 2014;32:659–702. 34. Esensten JH, Helou YA, Chopra G, et al. CD28 costimulation: from mechanism to therapy. Immunity 2016;44:973–88. 35. Commins SP, Borish L, Steinke JW. Immunologic messenger molecules: cytokines, interferons, and chemokines. J Allergy Clin Immunol 2010;125:S53–72. 36. Chappert P, Schwartz RH. Induction of T cell anergy: integration of environmental cues and infectious tolerance. Curr Opin Immunol 2010;22:552–9. 37. Chisolm DA, Weinmann AS. TCR-signaling events in cellular metabolism and specialization. Front Immunol 2015;6:292. 38. De Obaldia ME, Bhandoola A. Transcriptional regulation of innate and adaptive lymphocyte lineages. Annu Rev Immunol 2015;33: 607–42. 39. Zhu J, Paul WE. Peripheral CD4+ T-cell differentiation regulated by networks of cytokines and transcription factors. Immunol Rev 2010;238:247–62. 40. Ajamkitsumrit B, Dampier W, Martin-Garcia J, et al. Defining differential genetic signatures in CXCR4- and the CCR5-utilizing HIV-1 co-linear sequences. PLoS ONE 2014;9:e107389. 41. Taylor A, Verhagen J, Blaser K, et al. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells. Immunology 2006;117:433–42. 42. Patel DD, Kuchroo VK. Th17 cell pathway in human immunity: lessons from genetics and therapeutic interventions. Immunity 2015;43:1040–51. 43. Abbas AK, Benoist C, Bluestone JA, et al. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol 2013;14:307–8. 44. Campbell DJ. Control of regulatory T cell migration, function, and homeostasis. J Immunol 2015;195:2507–13. 45. Plitas G, Rudensky AY. Regulatory T cells: differentiation and function. Cancer Immunol Res 2016;4:721–5. 46. Tscharke DC, Croft NP, Doherty PC, et al. Sizing up the key determinants of the CD8+ T cell response. Nat Rev Immunol 2015;15:705–16. 47. Schroeder HW Jr, Cavacini L. Structure and function of immunoglobulins. J Allergy Clin Immunol 2010;125:S41–52. 48. Chen K, Cerutti A. The function and regulation of immunoglobulin D. Curr Opin Immunol 2011;23:345–52.

17

49. Bajic G, Degn SE, Thiel S, Andersen GR. Complement activation, regulation, and molecular basis for complement-related diseases. EMBO J 2015;34:2735–57. 50. Zheng l, Lenardo M. Restimulation-induced cell death: new medical and research perspectives. Immunol Rev 2017;277:44–60. 51. Ruland J. Return to homeostasis: downregulation of NF-κB responses. Nat Immunol 2011;12:709–14. 52. Oliveira JB, Bleesing JJ, Dianzani U, et al. Revised diagnostic criteria and classification for the autoimmune lymphoproliferative syndrome (ALPS): report from the 2009 NIH international workshop. Blood 2010;116:e35–40. 53. Gensollen T, Iyer SS, Kasper DL, et al. How colonization by microbiota in early life shapes the immune system. Science 2016;352:539–44. 54. Du Toit G, Roberts G, Sayre PH, et al. Randomized trial of peanut consumption in infants at risk for peanut allergy. N Engl J Med 2015;372:803–13. 55. Perkin MR, Logan K, Marrs T, et al. Enquiring about tolerance (EAT) study: feasibility of an early allergenic food introduction regimen. J Allergy Clin Immunol 2016;137:1477–86. 56. Ierodiakonou D, Garcia-Larsen V, Logan A, et al. Timing of allergenic food introduction to the infant diet and risk of allergic or autoimmune disease. JAMA 2016;316:1181–92. 57. Kivity S, Agmon-Levin N, Blank M, et al. Infections and autoimmunity— friends or foes? Trends Immunol 2009;30:409–14. 58. Steed AL, Stappenbeck TS. Role of viruses and bacteria-virus interactions in autoimmunity. Curr Opin Immunol 2014;31:102–7. 59. Ooi JD, Petersen J, Tan YH, et al. Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells. Nature 2017;545:243–7. 60. Raje N, Dinakar C. Overview of immunodeficiency disorders. Immunol Allergy Clin North Am 2015;35:696–726. 61. Lanternier F, Cypowyj S, Picard C, et al. Primary immunodeficiencies underlying fungal infections. Curr Opin Pediatr 2013;25: 736–47. 62. Booth C, Silva J, Veys P. Stem cell transplantation for the treatment of immunodeficiency in children: current status and hopes for the future. Expert Rev Clin Immunol 2016;12:713–23. 63. Cicalese MP, Aiuti A. Clinical applications of gene therapy for primary immunodeficiencies. Hum Gene Ther 2015;26:210–19. 64. Chen DS, Mellman I. Elements of cancer immunity and the cancerimmune set point. Nature 2017;541:321–30.

CHAPTER 1  The Human Immune Response

17.e1

M U L T I P L E - C H O I C E Q U E S T I ON S 1. Which of the following statements regarding B cells is correct? A. B cells can function as antigen-presenting cells (APCs). B. Activation for antibody production requires antigen presentation by APCs. C. Activation requires association of B-cell receptors for antigen with CD3. D. Expression of major histocompatibility complex (MHC) class II molecules on B cells requires cytokines produced by APCs. 2. Natural killer (NK) cells of the innate immune system: A. Express clonally specific antigen receptors B. Exhibit allelic exclusion of antigen receptors C. Express receptors for immunoglobulin molecules D. Upregulate cytotoxic activity on virus-infected target cells that express increased levels of major histocompatibility complex (MHC) class I molecules 3. Expression of human leukocyte antigen (HLA) class I molecules: A. Exhibits allelic exclusion B. Is associated with activation-induced cytidine deaminase C. Produces an antigen-binding site formed by folding of a single polypeptide chain D. Comprises two polypeptide chains that are both encoded within the HLA gene complex

4. A 2-year-old child is referred for evaluation of massive lymphadenopathy, splenomegaly, and apparent autoimmune anemia of 6 months’ duration without fever or weight loss. You suspect the child’s primary problem is: A. Acute myelogenous leukemia B. Excess production of IL-2 by CD4+ T helper (Th) cells C. Dysregulated proliferation of B cells D. Defective lymphocyte apoptosis 5. A 6-year-old boy is referred for evaluation of a presumptive diagnosis of a genetic deficiency of intercellular adhesion molecules. The referring pediatrician’s basis for this referral was: A. Recurrent viral infections B. Unusual susceptibility to infection with gram-negative bacteria C. Recurrent cutaneous abscesses D. Presence of Pneumocystis jiroveci pneumonia 6. A 35-year old female presents with exophthalmos, tremor, and weight loss. You suspect that the cause of her autoimmune disease is: A. Destruction of target gland hormone-producing cells B. Destruction of target gland stromal cells C. Blocking of target cell hormone receptors D. Stimulation of target cell hormone receptors

2  Organization of the Immune System Dorothy E. Lewis, Sarah E. Blutt

The human immune system consists of organs and movable cells. This design provides central locations for the initial production and differentiation of committed cells from naïve precursors, such as the fetal liver, the bone marrow, and the thymus; and more dispersed sites for the selection and further differentiation of cells into mature effector cells, such as the spleen, lymph nodes, and intestinal Peyer patches. This arrangement also allows regulation of immune responses at locations peripheral to primary lymphoid organs to provide local control of infectious processes. This chapter covers the basic features and the ontogeny of cells involved in the immune response, as well as the essential structure of lymphoid organs and sites of organized immune cells, including skin, the large intestine, and adipose tissue.

IMMUNE CELL DEVELOPMENT

banked, and methods to increase levels of HSC self-renewal, including three-dimensional scaffolding, are under intensive study.6

Tools Essential to an Understanding of Immune Cell Biology Understanding of the categorization and development of hematopoietic cells greatly depends on the use of monoclonal antibodies and flow cytometry to identify stage-specific leukocyte cell surface antigens. Leukocyte differentiation antigen workshops have grouped monoclonal antibodies that recognize the same single molecules on leukocytes by the cluster pattern of cells with which they are identified, hence the term cluster of differentiation (CD) antigen (Table 2.1, Appendix 1).

Ontogeny of the Cells of the Immune System

HEMATOPOIESIS AND LYMPHOPOIESIS

In the first month of embryogenesis, stem cells capable of producing white blood cell progenitors are found in erythropoietic islands that are in the yolk sac.1 The aorta–gonad–mesonephros (AGM), which is adjacent to the liver, produces progenitor cells that develop into hematopoietic stem cells (HSCs). In the sixth week of gestation, or just after the embryonic liver can be identified, progenitor stem cells in the liver begin blood cell production. By the eleventh week, the liver is the major source of hematopoiesis and remains so until the sixth month of gestation.2 HSC-derived progenitor cells can differentiate into granulocytes, erythrocytes, monocytes, megakaryocytes, and lymphocytes.3 Subsequent to skeleton formation, which occurs between the second and fourth months of gestation, white blood cell development starts shifting to bone marrow. This transition is completed by 6 months’ gestation. Cells that differentiate from early stem cells begin to populate the primary lymphoid organs, such as the thymus, by 7 to 8 weeks’ gestation.4 B-cell precursors initiate immunoglobulin (Ig) rearrangements by 7 to 8 weeks’ gestation (Chapter 7), and T-cell precursors that have initiated T-cell receptor (TCR) rearrangement (Chapter 8) can be detected in thymus by 8 weeks’ gestation. In bone marrow, B-cell progenitors congregate in areas adjacent to the endosteum and differentiate in the direction of the central sinus. HSC differentiation is a continuous process and is thus associated with many phenotypic stages. In the bone marrow of aged humans, there is evidence for a myeloid predominance, with a restricted diversity of HSCs.5 Stem cells with different characteristics and limited self-renewal can be induced into peripheral blood via injection of granulocyte– colony-stimulating factor (G-CSF).4 Cord blood cells are being

All mature cells of the hematopoietic and lymphoid lineages are derived from pluripotent stem cells that produce progenitors for lineage specific cells.7 Hematopoietic progenitors mature into cells of the granulocytic, erythroid, monocytic–dendritic, and megakaryocytic lineages. Likewise, lymphoid progenitors mature into B, T, and innate lymphoid cells (ILCs), including natural killer (NK) cells (Fig. 2.1). The site of development differs by cell type. After birth, granulocytes, monocytes, dendritic cells (DCs), erythrocytes, platelets, and B cells develop in bone marrow through the mature B-cell stage (Chapter 7) (Table 2.2). T-cell progenitors leave bone marrow and differentiate into αβ and γδ T cells in the thymus (Chapter 8). Some NK cells develop in the thymus.7 Tissue-specific NK-cell development occurs outside the thymus, including bone marrow, lymph nodes, and the uterus.8

Characteristics of Hematopoietic Stem Cells HSCs are rare in human bone marrow: 1 in 10 000 cells. They occupy distinct niches. One is close to bone and contains osteoblasts (endosteal niche), and the other is associated with the sinusoidal endothelium (vascular niche). Quiescent HSCs can be found near the arterioles in the endosteum. Actively dividing HSCs are located near the sinusoid regions close to the central veins. Different lineages of progenitors have preferred niches for development, and many HSCs are closely associated with perivascular mesenchymal stem cells.3 Long-term human HSCs divide once or twice per year. HSCs have characteristic flow cytometric light-scattering properties (low side scatter, medium forward scatter), no lineage-specific markers (Lin−; e.g., lacking CD2, CD3, CD5, CD7, CD14, CD15,

19

20

Part one  Principles of Immune Response

TABLE 2.1  Important Cell Surface Antigens on Hematopoietic Cells Cell Type

Surface Antigens

Predominant Location

CD34+, Lin−, 90+ CD34+, Lin−, CD38+, CD71+

Bone marrow Blood

CD14, CD35 (CR1), CD64 (FcRγ1)CD68, CD13 CD64,CD35 CD1a, CD207 (Langerin), CD35, CD64 CD21, CD35 FcγRIIb CD80, CD56, Class II CD83 CD40 CD83, CD80, CD86, CD40 CD1a, CD11c CD4, CCR5, CXCR4, CD123

Blood Tissue

CD16 (FcγRIII), CD35 CD88 (C5aR) CD32 (FcγRII) CD23, (FcεRII), CD32 FcεRI

Blood, tissues Blood, tissues Tissues, blood Tissues, blood

T cells

CD7, CD3, CD4, CD8, CD28

B cells

Surface Ig, class II, CD19, CD20, CD22, CD40 CD16, CD56, CD94 CD3, CD56, Vα24 TCR CD4, CD25, Foxp3, GARP CD4, CCR6, IL-17, RORγT CD4, ICOS, PD-1, BCL-6

Thymus, spleen, lymph nodes, mucosa-associated lymphoreticular tissue (MALT), blood Bone marrow, spleen, lymph nodes, MALT, blood

Hematopoietic Stem Cells Bone marrow hematopoietic stem cells (HSCs) Peripheral blood HSCs

Myeloid Cells Monocytes Macrophages Langerhans cells Follicular dendritic cells Interdigitating dendritic cells Myeloid dendritic cells Plasmacytoid dendritic cells (interferon [IFN]-α producing)

Skin B-cell areas, lymph nodes T-cell areas, lymph nodes Mainly tissues

Granulocytes Neutrophils Eosinophils Basophils Mast cells

Lymphocytes

Natural killer T cells (NKT cells)

Regulatory T cells (Tregs) T-helper (Th)17 cells T Follicular helper (Tfh) cells

TABLE 2.2 Normal Distribution of

Hematopoietic Cells in Bone Marrow Cell Type Stem cells Megakaryocytes Monocytes Dendritic cells Lymphocytes Plasma cells Myeloid precursors Granulocytes Red blood cell precursors Plasma cells Myeloid precursors Granulocytes Red blood cell precursors Immature and mature red blood cells

Approximate Proportion (%) 1 1 2 2 15 1 4 50–70 2 1 4 50–70 2 10–20

or CD16), and expressing CD34, CD90, and CD49f.6 As HSCs become active, they lose expression of CD90 and CD49f before diversion to lymphoid or myeloid precursors. Lymphoid precursors express CD10, CD45Ra, whereas myeloid precursors express CD135.

Spleen, lymph nodes, mucosal tissues, blood Blood, tissues Thymus, blood, tissues Intestine, blood, tissues Germinal centers of lymph nodes

Transcription factors are unique for each population. For HSCs, these include SOX8, SOX18, and NFIB. Out of quiescence, HSCs express MYC and IKZF1. Key signaling pathways include Notch and Wnt/β-catenin.3 A long-lived stem cell has the capacity for self-renewal via asynchronous division.9 HSCs circulate in peripheral blood with 10 to 100 times less frequency. Mobilization of “stem cells” to the periphery is induced by G-CSF. Of these induced cells, about 5–20% are true stem cells, although most are Lin−.9 Peripheral blood HSCs are more differentiated than bone marrow HSCs and have less self-renewal properties. Peripheral blood HSCs engraft 2 to 3 days faster than conventional bone marrow HSCs and are important in the reduction of bone marrow transplantation morbidities.

Regulation of Hematopoietic and Lymphopoietic Cell Growth and Differentiation Regulation of stem cell differentiation occurs through interactions with a variety of microenvironmental factors. Cell surface receptors recognize either soluble ligands (e.g., cytokines) released by other cells or surface ligands (e.g., cell interaction molecules) expressed on adjacent cells. These receptors can facilitate differentiation. The differential expression of receptors on the stem cells allows control of proliferation and differentiation along one of the hematopoietic or lymphoid lineages.6

CHAPTER 2  Organization of the Immune System B-1 B cell B cell progenitor

Pre-B cell IL-2

fit 3 Ligand

IL-4 IL-10

Plasma cell

IL-1, IL-13, IL-5, IL-6

CD19, CD76 CD20, CD23

CD5, CD38, CD138

B-2 B cell

Plasma cell

CD19, CD79 CD20, CD23

IL-1, IL-4 IL-13, IL-5 IL-6

CD38, CD138 αβ T cell

T cell progenitor

Lymphoid progenitor SCF IL-7

Pluripotential hematopoietic stem cell IL-1, IL-3 IL-6, IL-11 IL-12, SCF CD34+ or CD34LIN-

γδ T cell IL-7, SCF

IL-7 CD7, CD1

CD34, CD45RA, CD7

CD3, CD4, CD8

Pre-T cell

CD3, CD4, CD8

CD7, CD1

NK cell

IL-6 IL-7 SCF fit 3 ligand

CD3, CD4, CD8 Vα 24i

NK cell progenitor

NK cell IL-15, 1L-12 IL-21

CD16, CD56

CD7, IL-15R

TPO, IL-11, IL-6 GM-CSF, SCF

GM-CSF, SCF, M-CSF, IL-3, IL-1

M-CSF, IL-3 IL-1, IL-6, IL11, TPO

CFU, GEMM

M-CSF, IL-3 SCF

Monoblast

GM-CSF, CFU-GM IL-3

GM-CSF, G-CSF

CD34, CD33

Promonocyte

GM-CSF M-CSF, IL-3

IL-4, TNF-α Monocyte

CD41, CD35, CD64

Myeloblast

CD34, CD33, CD13, CD15 CFU-Eo

Promyelocyte

GM-CSF, G-CSF

CD33, CD13, CD15

Macrophage

GM-CSF M-CSF

GM-CSF, M-CSF, IL-3

CD34, CD33, CD13, HLA-DR GM-CSF, IL-3, SCF

Plasmacytoid dendritic cells CD303; IFNα (DC2)

CD31, CD49f CD42 CD41, CD61 GM-CSF,TNF-α

CD34, CD33, HLA-DR

Myeloid dendritic cells CD83, CD80, CD86 (DC1)

Platelet CD41, CD61, CD42

Megakaryocyte

Myeloid stem cell

GM-CSF, G-CSF

CD68, CD35, CD64

Myelocyte

CD33, CD13, CD15, CD11b

Eosinoophil

GM-CSF, Metamyelocyte G-CSF CD33, CD13, CD15, CD11b GM-CSF

IL-8

Bandform Basophil CD203c

BFU-E SCF, IL-3 IL-4

CD33, CD13, CD15 CD16, CD11b

Mast cell CD117 (c-Kit)

IL-9 GM-CSF, SCF, IL-3, EPO

CD9, CD16, CD23, CD32

GM-CSF, G-CSF Neutrophil

Proerythroblast

CFU-E TPO, EPO CD71, Glycophorin

Erythrocyte EPO

Glycophorin

Glycophorin

FIG 2.1  The differentiation of hematopoietic cells.

CD33, CD13, CD15 CD16, CD11b

21

22

Part one  Principles of Immune Response

Cytokines (Chapter 9) affect the growth and maintenance of pluripotent stem cells, as well as the development and differentiation of specific cell lineages. The effect of the cytokine can depend on whether the cell has previously been or is concurrently being stimulated by other cytokines. The stage of differentiation, as well as the presence or absence of the cytokine’s receptor on the cell surface, also affects the cellular response. Stromal cells located within bone marrow and the thymus regulate hematopoietic and lymphoid cell growth and differentiation by releasing cytokines, such as interleukin 4 (IL-4), -6, -7, and -11; leukemia inhibitory factor (LIF); granulocyte macrophage–colony-stimulating factor (GM-CSF); G-CSF; and stem cell factor (SCF).3 Stromal cells also participate in cell–cell interactions with progenitors that express fibroblast growth factor 1 (FGF-1) and FGF-2, which support HSC expansion. Stromal cells form the intercellular matrix (e.g., fibronectin and collagen), which binds to the selectin and integrin receptors present on hematopoietic and lymphoid progenitors.10 Cytokines That Affect the Growth and Maintenance of Pluripotent and Multipotent Stem Cells Pluripotent stem cells can reconstitute cells of the hematopoietic and lymphoid lineages. Maintenance of pluripotent capacity is mediated through factors that keep HSCs quiescent, including c-kit, N-cadherin, osteopontin, transforming growth factor-β (TGF-β), and Wnt. Factors that have a negative effect on quiescence include Hedgehog and the notch ligands Delta and Jagged.3 Because the stem cell pool is depleted as the progeny differentiate,

a low level of proliferation of stem cells is required to avoid exhaustion. The entry of stem cells into the cell cycle and the subsequent proliferation, as well as commitment to particular lineages, are controlled by cytokines and transcription factors. Data suggest that flt-3 ligand, c-kit ligand, and megakaryocyte growth and development factor all promote long-term stem cell expansion. The combination of c-kit ligand, IL-3, and IL-6 causes more rapid expansion but does not allow long-term extension of precursor cells.11 Several cytokines, alone or in combination, promote stem cell growth (Table 2.3).4 Cytokine combinations are more effective at inducing stem cell growth compared with individual cytokines. IL-1 promotes stem cell growth by inducing bone marrow stromal cells to release additional cytokines and by synergistically stimulating these cells in the presence of other cytokines. IL-3 promotes the growth of hematopoietic progenitors. The effect is significantly enhanced by IL-6, IL-11, G-CSF, and SCF. Stromal cell–derived IL-11 enhances IL-3–induced colony formation in 5-fluorouracil– resistant stem cells. Cytokines secreted by stromal cells (e.g., IL-6, G-CSF, and SCF) exert their effects by shortening the G0 period in stem cells. IL-3 acts on cells after they have left G0. IL-12 is unable to support the growth of primitive hematopoietic stem cells, either by itself or in conjunction with IL-11 or SCF. However, it acts in synergy with IL-3 and IL-11, or IL-3 and SCF, to enhance stem cell survival and growth. In some situations, a cytokine can enhance the growth of hematopoietic and lymphoid cells, whereas in others the same cytokine can inhibit cell growth or enhance differentiation. LIF

TABLE 2.3  Cytokines Important for Hematopoietic Cell Growth and Differentiation Cytokines

Stem Cells

Thymocytes

B Cells

Natural Killer (NK) Cells

Interleukin (IL)-1 IL-2 IL-3 IL-4

Acts on stromal cells

Differentiation Pleomorphic

Proliferation

Proliferation

Promotes (low) Prevents (high) Proliferation/differentiation Maintains potential Proliferation of pro- and pre-B cells Survival Maintains potential

Inhibits IL-2

Proliferation Pleomorphic

IL-5 IL-6 IL-7

Shortens G0

IL-10 IL-11 Oncostatin M IL-12 IL-13

Shortens G0 Survival

IL-15 IL- 21 Stem cell factor (SCF)/c-kit G-CS FLt3 ligand Stromal cell–derived factor (SDF)1-α Leukemia inhibitory factor (LIF) Thrombopoietin Tumor necrosis factor (TNF)-α Transforming growth factor (TGF)-β Macrophage inflammatory protein (MIP)-1α Nerve growth factor (NGF)

Survival Shortens G0 Growth factor

Enhances stimulation Survival/proliferation

Activation proliferation

Atrophy

Proliferation/regeneration Proliferation

Enhances IL-2 Activation

Activation/division of mature B cells Proliferation Proliferation Maintains potential Maintains potential Increases proliferation Chemoattractant

Development/survival Expansion Expansion Expansion

Atrophy

Expansion/regulates self-renewal Proliferation: inhibits granulocytes Inhibits growth enhanced granulocytes Inhibits Proliferation/differentiation

Expansion

23

CHAPTER 2  Organization of the Immune System can enhance the growth and development of bone marrow progenitor cells along multiple lineages in media containing IL-3, IL-6, and GM-CSF. However, in the absence of other cytokines or factors in serum, LIF has little effect on the growth and development of CD34+ progenitors. TGF-β and IL-4 are potent inhibitors of hematopoietic progenitor cell growth; yet they enhance granulocyte development. Tumor necrosis factor-α (TNF-α) inhibits the development of granulocytes, but it can potentiate IL-3 effects on hematopoietic progenitor cell proliferation. Other cytokines have effects on the proliferation and differentiation of multipotent progenitors of hematopoietic and lymphoid cells. GM-CSF and IL-3 promote development of granulocytes, macrophages, DCs, and erythrocytes. IL-6 participates in the development of neutrophils, macrophages, platelets, T cells, and B cells. Thrombopoietin signaling promotes stem cell self-renewal to increase transplantation success.7 Cytokines That Inhibit Hematopoietic Stem Cell Growth Cytokines produced by mature cells can downregulate hematopoietic stem cell growth. Macrophage inflammatory protein-1α (MIP-1α) is an inhibitor of hematopoietic progenitor cell proliferation. Other factors regulate stem cell growth through a variety of mechanisms, including the promotion of terminal differentiation (e.g., interferon-γ [IFN-γ] and TGF-β) or through the induction of apoptosis (e.g., TNF-α). When pathologic conditions exist, these cytokines can have adverse effects on hematopoietic and lymphoid cell development. Cytokines Affecting Development and Differentiation of Specific Cell Lineages Differentiation begins with the commitment of pluripotent stem cells to a specific lineage. Cytokines can have lineage-specific effects that act specifically at late stages of differentiation. Erythropoietin regulates the later stages of erythrocyte differentiation, whereas G-CSF induces granulocyte differentiation and macrophage colony-stimulating factor (M-CSF) promotes macrophage maturation.12 Cytokines that play an important role in the growth and development of specific cell lineages are described below under each cell type.

KEY CONCEPTS

Mature Cells of the Immune System The mature cells of the immune system primarily arise from progenitor cells in bone marrow. They include both nonspecific and antigen-specific effector cells. Antigen-Presenting Cells The central player in both nonspecific and antigen-specific lines of defense is the antigen-presenting cell (Chapter 6). In addition to their nonspecific effector functions, these cells are crucial for the development of specific immune responses. With maturation, these cells enter the blood (Table 2.4) and circulate into the tissues and organs. Antigen-presenting cells (APCs) are found in the solid lymphoid organs and skin (Chapter 19) at a frequency that varies from 0.1–1%. Specialized APCs in B-cell areas of lymph nodes and spleen are termed follicular dendritic cells (FDCs). They trap antigen–antibody complexes important in the generation and maintenance of memory B cells. FDCs do not express major histocompatibility complex (MHC) class II molecules as do other APCs. Instead, they have receptors for immunoglobulin G (IgG) (FcγRI [CD64]) and complement component C3b (CR1 [CD35]), respectively. Monocytes–Macrophages Monocyte–macrophage lineage cells exist in blood (~10% of leukocytes) primarily as monocytes, which are large 10- to 18-µm cells with peanut-shaped, pale purple nuclei as determined by Wright staining (see Table 2.4). The cytoplasm, which is 30–40% of the cell, is light blue and has azurophilic granules that resemble ground glass with intracytoplasmic lysosomes. The cells express MHC class II, CD14 (the receptor for lipopolysaccharide), and distinct Fc receptors (FcRs) for Ig. The latter include FcγRI (or CD64), which has a high affinity for IgG, and FcγRII (or CD32), which is of medium affinity and binds to aggregated IgG. FcγRIII (or CD16) has low affinity for IgG and is associated with

TABLE 2.4 Normal Distribution of White

Blood Cells in the Peripheral Blood of Adults and Children

Cells of the Immune System • Pluripotent stem cells in bone marrow give rise to all lineages of the immune system, platelets, and red blood cells. • Development and regulation of cells of the immune system is associated with programmed appearance of specific cell surface molecules called “cluster of differentiation” (CD) markers and with responsiveness to selective cytokines. • Mature cells of the immune system include antigen-presenting cells (APCs); phagocytic cells, including neutrophils, eosinophils, and basophils; and lymphocytes, including T cells, B cells, and natural killer (NK) cells, as well as other innate lymphoid cells. • APCs include monocytes, macrophages, dendritic cells (DCs), B cells, endothelial cells, epithelial cells, and adipocytes. They can direct the differentiation and function of both innate and acquired immune cells. • Polymorphonuclear (PMN) granulocyte cells are important in the early response to stress, tissue damage, and pathogens. They include neutrophils, eosinophils, and basophils. • Lymphocyte lineages have discrete subpopulations with specialized functions. These include CD4 and CD8 T cells, B-1 and conventional B-2 B cells, and NK and other innate lymphoid cells. CD4 T-helper (Th) subsets include regulatory T cells (Tregs), Th17, and Tfh cells.

APPROXIMATE PERCENTAGE

Adults

Children (0–2 yr)

34–75

400–1000 30–170 2500–7500 1450–3600

ND ND 1000–8500 3400–9000

75–85 27–53 13–23

53–84 32–64 12–30

900–2500 550–1500 300–1000

2500–6200 1300–4300 500–2000

5–15 5–15

06–41 03–18

100–600 200–700

300–3000 170–1100

Cell Type

Adults

Monocytes Dendritic cells Granulocytes Lymphocytes

4–13 0.5–1 35–73 15–52

Children (0–2 yr)

RANGE OF ABSOLUTE COUNTS (NO./µL)

NDa

As % of Lymphocytes T cells CD4 cells CD8 cells B cells Natural killer (NK) cells

a Not determined. Child data adapted from Shearer W, Rosenblatt H, Gelman R, et al. Lymphocyte subsets in healthy children from birth through 18 years of age: the pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003;12:973–80.

24

Part one  Principles of Immune Response

antibody-dependent cellular cytotoxicity (ADCC). It is expressed on macrophages, but not on blood monocytes. Monocytes and macrophages also express CD89, the Fc receptor for IgA.12 Macrophages are more differentiated monocytes that reside in various tissues, including the lungs, liver, and brain.13 Cells of the monocyte–macrophage lineage adhere strongly to glass or plastic surfaces. This process activates them, and this can confound functional studies when they are isolated in this way. Many cells of this lineage phagocytose organisms or tumor cells in vitro. Cell surface receptors, including CD14, Fcγ receptors, and CR1 (CD35), are important in opsonization and phagocytosis. This lineage expresses MHC class II, and some express the lowaffinity receptor for IgE (CD23). Other cell surface molecules include myeloid antigens CD13 (aminopeptidase N) and CD15 (Gal (1–4) or [Fuc (1–3)] GlcNAc) and the adhesion molecules CD68 and CD29 or CD49d (VLA-4). Classic blood monocytes in humans (85%) express high levels of CD14, but no CD16. Nonclassic monocytes express less CD14, but more CD16. This later subset produces more IL-12, TNF-α, and IL-1β. These cells have receptors for various cytokines (e.g., IL-4 and IFN-γ). Activated macrophages are a major source of cytokines, including IFN, IL-1, and TNF, as well as complement proteins and prostaglandins. Macrophages, along with dentritic cells (DCs), are much more plastic in differentiation and function than previously realized. They can be alternatively activated and thereby become suppressive, developing antiinflammatory properties relevant in immune responses to cancer as well in maintaining adipose integrity. Alternative activation is induced by the T-helper cell 2 (Th2) (Chapter 16) cytokines IL-4 and IL-13.14 Monocytes and macrophages arise from colony-forming unit–granulocyte monocyte (CFU-GM) progenitors that differentiate into monoblasts, promonocytes, and then monocytes.12 Mature monocytes leave bone marrow and circulate in the bloodstream until they enter tissues, where they develop into tissue macrophages (alveolar macrophages, Kupffer cells, intestinal gut macrophages, and microglial cells). Tissue macrophages appear to originate from fetal macrophages and seed tissues early in fetal development, where they are maintained by longevity and slow self-renewal.13 Several cytokines participate in the development of monocytes and granulocytes. SCF, IL-3, IL-6, IL-11, and GM-CSF promote development of myeloid lineage cells from CD34+ stem cells, especially at early stages. M-CSF acts at later stages of development and is lineage specific, inducing macrophage maturation.12 Dendritic Cells DCs express high levels of MHC class II molecules and are potent inducers of primary T-cell responses. Except for bone marrow, they are found in virtually all primary and secondary lymphoid tissues and in skin, mucosae, and blood. DCs are abundant in the thymus medulla for selection of thymocytes. DCs are derived from CD34+ MHC class II-negative precursors present in bone marrow, which also give rise to macrophages and granulocytes. GM-CSF and TNF-α are involved in DC development.15 DCs residing in peripheral sites, such as skin, the intestinal lamina propria, lungs, the genitourinary tract, and so on, are typically immature. These cells are more phagocytic with less MHC class I, MHC class II, and costimulatory molecules. These immature DCs take up antigens in tissues for subsequent presentation to T cells, and as they migrate, they mature into efficient APCs.

The predominant APCs of the skin are the Langerhans cells16 found in the epidermis and characterized by rocket-shaped granules called Birbeck granules. Immature tissue DCs in peripheral tissues engulf and process antigen and home to T-cell areas in the draining lymph nodes or spleen.17 Mature DCs can directly present processed antigens to resting T cells to induce proliferation and differentiation, and this is a key functional difference between mature DCs and macrophages. The effector cells produced after this presentation then home to the site of the antigenic assault. TNF-α maintains viability of Langerhans cells in skin and stimulates their migration. In Peyer patches (Chapter 20), immature DCs occur in the dome region underneath follicleassociated epithelium (FAE), where they actively endocytose antigens taken up by M cells. Mature interdigitating DCs are found in T-cell regions, where they can induce Th2 immune responses (Chapter 16). Three types of DC are prominent—two types of “conventional” dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). cDC1s derive from bone marrow are found in lymphoid tissues and express CD1a and CD11c. About 50% peripheral blood DCs are cDC1. cDC2s express CD141, are similar to murine CD8 α/α DCs in function, and are rare in peripheral blood but are common in lymph nodes. pDCs are high producers of IFN-α. They express CD123 and low levels of CD11c, along with BDCA 2 and 4. DCs can be derived from either myeloid or lymphoid lineages. DCs are largely influenced by stimulation with Toll-like ligands found on a variety of stimuli, which then direct the differentiation and function of innate and acquired immune cells. Polymorphonuclear Granulocytes Polymorphonuclear (PMN) granulocytes arise and mature in bone marrow. After release from bone marrow, their life span varies from a few to 5 to 6 days and is regulated by environmental conditions. They constitute 65–75% of the white blood cells in peripheral blood, are 10–20 µm in diameter, and have a multilobed pyknotic nucleus characteristic of cells undergoing apoptosis (see Table 2.4).18 PMNs use diapedesis to gain access to tissues from blood. Granulocytes are early responders to stress, tissue damage, or pathogen invasion. Because of their function in phagocytosis and killing, they possess granules with unique staining characteristics that are used to categorize the cells as neutrophils (Chapter 22), basophils (Chapter 23), or eosinophils (Chapter 24). Neutrophils Most circulating granulocytes are neutrophils (90%). Their granules are azurophilic and contain acid hydrolase, myeloperoxidase, and lysozymes. These granules fuse with ingested organisms to form phagolysosomes, which kill the invading organism. In some cases, there is extracellular release of granules after activation via the Fc receptors. Neutrophils express CD13, CD15, CD16 (FcγRIII), and CD89 (FcαR). In response to bacterial infection, the number of circulating granulocytes typically increases. This often includes the release of immature granulocytes, called band or stab cells, from bone marrow. In a mild infection, both the number and the function of neutrophils are increased. This is associated with a delay in apoptosis. With more severe infection, function may be impaired due to the immaturity of cells. A newly described function of some neutrophils is to release neutrophil extracellular traps, which can capture

CHAPTER 2  Organization of the Immune System microbes extracellularly and then use autophagy to digest them intracellularly.19 Neutrophils mature from CFU-GM progenitor cells and differentiate within a 10- to 14-day period. These progenitors give rise to myeloblasts, promyelocytes, myelocytes, and finally mature neutrophils. SCF, IL-3, IL-6, IL-11, and GM-CSF promote the growth and development of neutrophil precursors. Other cytokines are important for differentiation of CFU-GM progenitors into mature neutrophils.20 G-CSF induces maturation of neutrophil precursors into mature neutrophils. IL-4 enhances neutrophil differentiation induced by G-CSF, while inhibiting the development of macrophages induced by IL-3 and M-CSF. Eosinophils Eosinophils typically comprise 2–5% of the white cells in blood. They exhibit a unique form of diurnal variation. Peak production occurs at night, perhaps because glucocorticoid levels are lower. Eosinophils are capable of phagocytosis followed by killing, although this is not their main function. The granules in eosinophils are much larger than in neutrophils and are actually membrane-bound organelles. The crystalloid core of the granules contains a large amount of the major basic protein (MBP), which can neutralize heparin and is toxic. During degranulation, the granules fuse to the plasma membrane, and their contents are released into the extracellular space. Organisms that are too large to be phagocytosed, such as parasites, can be exposed to cell toxins by this mechanism. The MBP can damage schistosomes in vivo, although damage is minimized because the MBP is confined to a small extracellular space. Eosinophils also release products that counteract the effects of mast cell mediators. Whether eosinophils are absolutely required for helminth control is controversial. Eosinophils mature from a progenitor (CFU-Eo) into an eosinophilic myeloblast, then an eosinophilic promyelocyte, a myelocyte, and finally a mature eosinophil.21 Three cytokines are important in the development of eosinophils: GM-CSF, IL-3, and IL-5. GM-CSF and IL-3 promote eosinophil growth and differentiation. SCF also has an effect on eosinophil function. Eotaxin (CCL11) promotes eosinophilia. IL-5 has more lineagespecific effects on eosinophil differentiation. Although it also affects some subsets of T and B cells, it is important for eosinophil survival and maturation. Eosinophils are involved in the pathophysiology of asthma, with contribution to airways dysfunction and tissue remodeling, and IL-5 is being targeted to correct eosinophilia. Basophils and Mast Cells Basophils represent less than 1% of the cells in the peripheral circulation. They are characterized by large, deep-purple granules. Mast cells are found in proximity to blood vessels and are much larger than peripheral blood basophils. Their granules are less abundant, and the nucleus is more prominent. There are two different types of mast cells—designated mucosal and connective tissue—depending on their location. Mucosal mast cells require T cells for their proliferation, whereas connective tissue mast cells do not. Both types have granules that contain effector molecules. After degranulation, which is effected by cross-linkage of cell surface IgE bound to cells via the high-affinity receptor for IgE, basophils and mast cells release heparin, histamine, and other effector substances to mediate an immediate allergic attack (Chapters 23 and 42).

25

Basophils and mast cells share a number of phenotypic and functional features that suggest derivation from a common precursor. Both basophils and mast cells contain basophilicstaining cytoplasmic granules; both express the high-affinity IgE receptor (FcεRI); and both release a number of similar chemical mediators that participate in immune and inflammatory responses, particularly anaphylaxis. They both have been implicated in allergic inflammation and in fibrosis. However, basophils and mast cells also have some distinct morphological and functional characteristics that suggest that they may be distinct lineages of cells, rather than cells at different stages within the same lineage. In human, transcription factor analysis places basophils closer to eosinophils than mast cells.22 Basophils mature from a progenitor (CFU-BM) into basophilic myeloblasts and then into basophilic promyelocytes, myelocytes, and finally mature basophils. Less is known about the stages of mast cell development, although they are probably derived from the same CFU-BM progenitor as basophils. In humans, SCF induces the most consistent effects on the growth and differentiation of both basophils and mast cells. Both IL-3 and SCF are important for intestinal mast cell differentiation. Il-6 can also increase mast cell numbers. This probably explains why T cells are needed for their development.28 Both IL-4 and IL-9 stimulate mast cell development in mice. However, in humans, only IL-9 acts in synergy with SCF to enhance mast cell growth. Additional cytokines that affect basophil growth include nerve growth factor and GM-CSF or TGF-β, and IL-5 for basophil differentiation. Platelets and Erythrocytes Hematopoietic stem cells give rise to platelets and erythrocytes. Platelets are necessary for blood clot formation and mediate a number of immune functions. Mature red blood cells are necessary for oxygen delivery to tissues.23 Platelets derive from CFU-GEMM progenitors, which differentiate into burst-forming units for megakaryocytes (BFU-MEG). BFU-MEG then differentiate into CFU-MEG, promegakaryoblasts, megakaryoblasts, megakaryocytes, and finally platelets.24 Several cytokines, particularly thrombospondin, IL-1, IL-3, GM-CSF, IL-6, IL-11, and LIF, affect the growth and differentiation of platelets. Erythrocytes also derive from CFU-GEMM progenitors, but their progenitors are burst-forming units for erythrocytes (BFU-E), which, in turn, differentiate into CFU-E, pronormoblasts, basophilic normoblasts, polychromatophilic normoblasts, orthochromic normoblasts, reticulocytes, and finally erythrocytes. Again, several cytokines, notably GM-CSF, SCF, IL-9, thrombospondin, and erythropoietin, regulate erythrocyte development. Lymphocytes Lymphocytes, the central cell type of the specific immune system, represent about 25% of the white blood cells in blood (see Table 2.4). Small lymphocytes range from 7–10 µm in diameter and contain a nucleus that stains dark purple with Wright staining, and a small cytoplasm. Large granular lymphocytes range from 10–12 µm in diameter and contain more cytoplasm and scattered granules. The three types of lymphocytes that circulate in the peripheral blood—T cells, B cells, and ILCs, including NK cells—constitute approximately 80%, 10%, and 10% of the total blood lymphocyte population, respectively (Chapters 7, 8, and 17). In the thymus, most of the lymphocytes (90%) are T cells; however, in the spleen and lymph nodes, only about 30–40%

26

Part one  Principles of Immune Response

are T cells. The preponderant lymphocytes in these locations are B cells (60–70%).25 T Lymphocytes T lymphocytes arise from lymphocyte progenitors in bone marrow committed to the T-cell lineage before moving to the thymus. In the early stages of embryogenesis, T-cell precursors migrate to the thymus in waves.26 Associated with this migration is the developing ability of thymic education elements, epithelial cells, and DCs to select appropriate T cells.27 In the thymus, T cells rearrange their specific antigen receptors (TCRs) (Chapter 4) and then express CD3 along with the TCRs on their surface (Chapter 8). Resting T cells in blood typically range from 7–10 µm in diameter and are agranular, except for the presence of a structure termed Gall body, which is not found in B cells (see Table 2.4). The Gall body is a cluster of primary lysosomes associated with a lipid droplet. A minority of T cells in blood (about 20%) are of the large granular type, that is, they are 10–12 µm in diameter and contain primarily lysosomes that are dispersed in the cytoplasm. Golgi apparati also are found. The preponderant form of the TCR, found on about 95% of circulating T cells, expresses TCRαβ.28 Some CD3+ cells do not express either CD4 or CD8 (double-negative, or DN) and express an alternative TCRγδ. Further differentiation in the thymus occurs from CD3+ cells that express both CD4 and CD8 (double-positive, or DP) to cells expressing either CD4 or CD8 but not both (Chapter 8). These mature cells then circulate in peripheral blood at a ratio of about 2 : 1 (CD4:CD8) and populate lymph nodes, the spleen, and other secondary lymphoid tissues. T-cell progenitors, which are CD7+, arise in bone marrow from a multipotential lymphoid stem cell. After migration to the thymus, the CD7+ progenitors give rise to a population of CD34+, CD3−, CD4−, and CD8− T-cell precursors, which undergo further differentiation into mature T cells. Cytokines produced by thymic epithelial cells (e.g., IL-1 and soluble CD23) promote differentiation into CD2+, CD3+ thymocytes (see Table 2.3). IL-7 induces the proliferation of CD3+ DN (CD4− CD8−) thymocytes, even in the absence of comitogenic stimulation. IL-7 is an absolute requirement for human T-cell development.29 IL-2 and IL-4 demonstrate complex effects on thymocyte development. Both can promote development of prothymocytes, as well antagonizing their development. IL-6 acts as a costimulator of IL-1– or IL-2–induced proliferation of DN thymocytes and can stimulate the proliferation of mature, cortisone-resistant thymocytes alone.

node homing and proliferation, but later stage cells home to the periphery, are effector cells, and do not proliferate.30 Th cells mature in response to foreign antigens. Their function is dependent on the production of cytokine patterns, which characterize them as Th type 1 (Th1), Th2, or Th17.31 The precursor Th cell first differentiates into a Th0 cell, which produces IFN-γ and IL-4. The cytokine environment subsequently determines whether Th1 or Th2 cells predominate. Th1 cells produce primarily IFN-γ, IL-2, and TNF-α and are important in cell-mediated immunity to intracellular pathogens, such as the tubercle bacillus. Th1 cells primarily use T-bet transcription factor. Th2 cells produce predominantly IL-4, -5, -6, -10, and -13, as well as IL-2; they predominate in immediate or allergic type 1 hypersensitivity and primarily use GATA-3 transcription factor. Other populations of CD4 T cells can develop and rely on IL-23 or IL-12 action upon the cells. If T cells are exposed to IFN-γ, they upregulate both IL-12R and IL-23R, which then produce either conventional Th1 cells or another subset, Th17, which produces IL-17 and is important for controlling immune cell activation in the gastrointestinal (GI) tract. Overactive function of this subset has been associated with autoimmunity. The Th17 population preferentially uses RORγt transcription factor. T follicular helper cells are those classically determined to help B cell responses in germinal centers. They are CD4+, ICOS+, and PD-1+; and they express transcription factor BCL-6. It is likely that there are other epigenetically altered T cells that allow diversity of function during an immune response. A minor subpopulation (90%) are T cells, which are either CD8+ or CD4−CD8−. Although the majority of IEL T cells express TCRαβ, a substantial number express TCRγδ. The function of IELs remains incompletely understood, but they can be cytotoxic and also maintain oral tolerance. As part of their effector function, they produce several cytokines, including IL-5 and IFN-γ. Two other T-cell subsets play antagonizing roles in controlling inflammation within the intestinal lamina propria: Tregs and Th17 cells. Tregs are FOXP3+ and function to repress inflammation, whereas Th17 cells mount inflammatory and autoimmune responses through production of IL-17.52 Another T-cell subset, which comprises NKT cells, expresses the characteristics of both T cells and NK cells. NKT cells express perforin and granzymes but recognize antigens through non-MHC–mediated pathways.53 Other cells, including macrophages, DCs, eosinophils, mast cells, and a few neutrophils, are also found in the lamina propria and mediate effector functions. There is an elaborate network of APCs, including DCs and macrophages distributed within the lamina propria and GALT. Two major DC subsets, characterized by CD103+CD11b− and CD103+CD11b+, develop into distinct lineages on the basis of secreted factor requirement. CD103+CD11b+ are predominately localized to the lamina propria and migrate to the mesenteric lymph nodes upon activation. In contrast, CD103+CD11b− populations are localized to the Peyer patches. The GI tract contains the largest number of resident macrophages in the body. These macrophages are similar to macrophages in other tissues and express CD68, lysozyme, ferritin, MHC II, CD11b, CX3CR1, and CD74 but do not migrate to the mesenteric lymph nodes. Respiratory Tract Surrounding the entrance to the throat are three tonsillar groups: palatine tonsils, lingual tonsils, and pharyngeal tonsils or adenoids. Together, these are known as Waldeyer ring. Tonsils reach full development in childhood and involute by puberty. The palatine tonsils, one located on each side of the pharynx, each measures approximately 2.5 × 1.25 cm. Except at the pharyngeal surface, they are surrounded by a poorly organized capsule that is covered with stratified squamous epithelium. Trabeculae subdivide the

tonsil into lobules. Blood vessels and nerves enter through the capsule and extend within trabeculae (Fig. 2.10). The surface of the tonsil is covered by pits, which are the openings of crypts. The crypts extend down into the tissue of the tonsil with branching, increasing surface area. Abundant lymphoid follicles in each lobule contain germinal centers that are predominantly B cells. The lymphoid tissue surrounding the follicles contains T cells, macrophages, DCs, and some B cells. The lingual tonsils consist of 35–100 separate crypts surrounded by lymphoid tissue and are located at the root of the tongue. The pharyngeal tonsils, or adenoids, are accumulations of lymphoid tissue, 2.5–4.0 cm long, located on the median dorsal wall of the nasopharynx. They contain a series of longitudinal folds, but not crypts. The lingual and pharyngeal tonsils also contain lymphoid nodules with germinal centers. The palatine tonsils and adenoids (nasopharyngeal tonsils) comprise the nasopharyngeal–associated lymphoreticular tissues. Inductive immune responses to inhaled antigens within the respiratory tract occur mainly in the bronchus-associated lymphoid tissue (BALT). BALT consists of lymphoid aggregates located within the bronchial wall near bifurcations of the major bronchial branches (Fig. 2.11). These structures are analogous to the GALT present in the GI tract and function to provide T- and B-cell protection against inhaled microbes. BALT is present at birth and rapidly expands when exposed to antigenic stimulation. The specialized epithelium overlying the lymphoid aggregates consists of M cells heavily infiltrated with lymphocytes and with DCs below the epithelium. The main result of BALT immune induction is secretory IgA production.54 The diffuse mucosal tissue of the respiratory tract is minimal. Pools of lymphocytes are present within the lung interstitium, which is 10–20% T cells. Macrophages are present on both the air and the mucosal sides of the lungs and airways.50 Minimal inflammation occurs in the bronchial mucosa as a result of Tregs that inhibit T-cell activation and expansion. Instead, antigen is carried by local macrophages to the regional lymph nodes, where most respiratory effector immune responses originate. Communication occurs between the GI and respiratory mucosae through cell trafficking. Antigen-reactive T and B cells from Peyer patches can populate the bronchial mucosa. This sharing feature has been exploited in the development of oral vaccines against respiratory microbes.55 Genital Tract The male and female reproductive tracts are components of the common mucosal system. The genital tract immune system must maintain a delicate balance between tolerance of germinal center cells, spermatozoa, and the fetus and the recognition of microbes. The female reproductive tract has been studied the most. Its mucosal immune system is influenced by hormones that regulate all aspects of innate and adaptive immunity.56 “Professional” APCs, including macrophages and DCs, are present in the stroma of both the uterus and the vaginal tract, where they have unique phenotypes. Reproductive tract NK cells play a role in host defense, implantation of the embryo, and pregnancy and also express a distinct phenotype. CD8 T cells predominate and, along with B cells and macrophages, form unique lymphoid aggregates. Formation of these nodules depends heavily on hormone regulation. Both secretory IgA and IgG are expressed in genital secretions, and levels vary with the stage of the menstrual cycle. The production and transport of antibody produced in the genital tract

CHAPTER 2  Organization of the Immune System

35

A Capsule

Blood vessels

Germinal center Follicular dendritic cells Lymphatic follicle Mucosal epithelium

C

B

D

FIG 2.10  Human Tonsil. (A) Organization of lymphoid follicles and germinal centers. (B) Tonsillar tissue stained with hematoxylin and eosin. (C) Tonsillar tissue stained with anti-CD3 to demonstrate the distribution of T cells. (D) Tonsillar tissue stained with anti-CD19 to demonstrate the distribution of B cells.

depends on hormonal and local factors, including IL-1b, IL-6, and IL-10, all of which influence the maturation of B cells to plasma cells within the mucosa.56

SKIN Skin also serves as a specialized secondary immune organ (Chapter 19). It contains two layers, the epidermis and the dermis. The epidermis is the outermost layer and contains three distinct cell types: keratinocytes, melanocytes, and Langerhans cells (Fig. 2.12). Keratinocytes are squamous epithelial cells and are the principal cell type. They secrete a number of cytokines and

chemokines, including IL-1, -6, -10, -18, and -33; TGF-β; TNF-α; CXCL9; CXCL10; CXCL11; and CCL20. These can profoundly influence immune cell recruitment and responses. The second cell type is the pigment-producing melanocyte. Melanocytes derive from the neural crest and reside in the basal layer of the epidermis. The third cell type, and the one of particular importance for the immune system, is the Langerhans cell. Langerhans cells are scattered throughout the epidermis within the malpighian, or prickle cell, layer. They are important for both normal and pathologic cutaneous T-cell responses. After encountering antigen in the presence of keratinocyte cytokines, such as TNF-α and IL-6, Langerhans cells migrate from the epidermis to the dermis,

36

Part one  Principles of Immune Response

Bronchiole with mucus

Lymphoid aggregate Blood vessel

A

sebaceous glands. The dermal vasculature includes an extensive network of plexuses with arterioles, capillaries, and venules. Dermal lymphatics are associated with the vascular plexuses. In normal skin, a small number of lymphocytes can be found in perivascular areas. These lymphocytes are mostly T cells with distinctive features, including expression of a memory phenotype (CD45RO) and expression of a cutaneous lymphocyte–associated antigen that binds to the vascular addressing endothelial cell leukocyte adhesion molecule-1 (ELAM-1, or CD62E) present on the endothelium. This latter interaction plays an important role in homing of memory T cells to inflamed regions of skin. The dermis also contains mast cells important for immediate hypersensitivity reactions. Tregs are abundant in the skin. CD8 T cells are common in the epidermis. CD4 T cells are common in dermis infected with shingles.57

Commensal Organisms/Toll-Like Receptors

Lung tissue

Humans live in symbiosis with over 1000 different species of viruses, bacteria, protozoa, and fungi that far outnumber human cells. Collectively termed commensal microbiota, these organisms are essential to the development, maturation, organization, and regulation of the mucosal immune system.58 Many of their immune interactions involve TLR triggering. TLRs activate and prime both innate and specific immune responses. Production of IgA, induction of regulatory T cells, and stimulation of antiinflammatory cytokines are associated with commensal microbiota.59 Thus the types and quantities of microorganisms present at a mucosal surface is an important component of the mucosal immune response.60

ON THE HORIZON • Understanding how stem cells self-renew is a key to exploiting them for gene therapy. • Exploiting innate and acquired immune cell function requires an understanding of the multiple subpopulations of cells and the manner in which they are induced. • Increasing generation of new T cells and B cells later in life might enhance immune function, and thus prolong quality of life. • The role of adipose tissue in hematopoietic stem cell (HSC) development in bone marrow and control of inflammation in obesity is fundamental to controlling the epidemic of obesity. • Exploiting interactions between the mucosal immune system and commensal populations may improve health, prevent inflammation, and lead to less antibiotic use. • Cellular migration via the lymphatics to the brain is unknown; inflammation and cancer spread might be reduced by controlling inflammation of the lymphatics.

B

FIG 2.11  Lymphoid regions in the human lung.

ACKNOWLEDGMENTS enter the afferent lymphatics, and migrate to draining lymph nodes. There they present antigen to T cells to promote primary immune responses. Other types of dermal DCs, including those that are macrophage-like, expressing CD14, and those that are DC-like, expressing CD1c and CD14, have consequences for atopic dermatitis and psoriasis.57 The dermis lies under the epidermis. It contains abundant fibroblasts producing collagen, a principal component of skin. The dermis also contains blood vessels and various epidermal adnexal structures, such as hair follicles, sweat glands, and

We thank Dr. Gregory R. Harriman at BioAdvance for his work on previous editions of this chapter; Dr. Edwina Popek, Pathology Department, Texas Children’s Hospital, Houston, Texas, for providing histopathologic images of lymphoid tissues; Dr. Gregory Stelzer and Wendy Schober for the flow cytometric display; Eleanor Chapman, Anna Wirt, Terry Saulsberry, Yvette Wyckoff, and Pamela Love for help with the manuscript; and Dr. Jerry McGhee for critical review of the first edition. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

37

CHAPTER 2  Organization of the Immune System Stratum corneum

Stratum corneum

Epidermis

Epidermis Langerhans cells

Dermis

Dermis

Blood vessels / Lymphatics Blood vessel

Lymphatics A

C

B

D

FIG 2.12  Lymphoid Regions in Human Skin. (A, C) Organization of the epithelial tissue. (B) Epithelial tissue stained with hematoxylin and eosin. (D) Epithelial tissue stained with anti-CD207 to demonstrate the distribution of Langerhans cells (note brown cells).

REFERENCES 1. Rampon C, Huber P. Multilineage hematopoietic progenitor activity generated autonomously in the mouse yolk sac: analysis using angiogenesis-defective embryos. Int J Dev Biol 2003;47:273–80. 2. Ciriza J, Thompson H, Petrosian R, et al. The migration of hematopoietic progenitors from the fetal liver to the fetal bone marrow: lessons

learned and possible clinical applications. Exp Hematol 2014;41: 411–23. 3. Smith JNP, Calvi LM. Current concepts in bone marrow microenvironmental regulation of hematopoietic stem and progenitor cells. Stem Cells 2013;6:1044–50. 4. Copley MR, Eaves CJ. Developmental changes in hematopoietic stem cell properties. Exp Mol Med 2013;45:e55.

38

Part one  Principles of Immune Response

5. Pang WW, Price EA, Sahoo D, et al. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age. Proc Natl Acad Sci 2011;108(50):20012–17. 6. Doulatov S, Notta F, Laurenti E, et al. Hematopoiesis: a human perspective. Cell Stem Cell 2012;10:120–36. 7. Metcalf D. Hematopoietic cytokines. Blood 2008;111:485–91. 8. Yu J, Freud AG, Caligiuri MA. Location and cellular stages of natural killer cell development. Trends Immunol 2013;34:573–82. 9. Wilson A, Laurenti E, Oser G, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 2008;135:1118–29. 10. Zhang CC, Lodish HF. Cytokines regulating hematopoietic stem cell function. Curr Opin Hematol 2008;15(4):307–11. 11. Kondo M, Wagers MJ, Manz MG, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol 2003;21:759–806. 12. Varol C, Mildner A, Jung S. Macrophages: development and tissue specialization. Annu Rev Immunol 2015;33:643–75. 13. Hoeffel G, Ginhoux F. Ontogeny of tissue-resident macrophages. Front Immunol 2015;6:486. 14. Bouhlel MA, Derudas B, Rigamonti E, et al. PPARγ activation primes human monocytes into alternative M2 macrophages with antiinflammatory properties. Cell Metab 2007;6:137–43. 15. Boltjes A, van Wijk F. Human dendritic cell functional specialization in steady-state and inflammation. Front Immunol 2014;5:131. 16. Mende I, Karsunky H, Weissman IL, et al. Flk2þ myeloid progenitors are the main source of Langerhans’ cells. Blood 2006;107:1383–90. 17. Randolph GJ, Angeli V, Swartz MA. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nature Immunol Rev 2005;5:617–28. 18. Friedman AD. Transcriptional regulation of granulocyte and monocyte development. Oncogene 2002;21:3377–90. 19. Kruger P, Saffarzadeh M, Weber A, et al. Neutrophils: between host defence, immune modulation, and tissue injury. PLoS Pathog 2015;11(3):1–23. 20. Lieber JG, Webb S, Suratt BT, et al. The in vitro production and characterization of neutrophils from embryonic stem cells. Blood 2004;103:852–9. 21. Rosenberg HF, Dyer KD, Foster PS. Eosinophils: changing perspectives in health and disease. Nat Rev Immunol 2013;13:9–22. 22. Chirumbolo S. State-of-the-art review about basophil research in immunology and allergy: is the time right to treat these cells with the respect they deserve? Blood Transfus 2012;10:148–64. 23. Hattangadi SM, Wong P, Zhang L, et al. From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications. Blood 2011;118:6258–68. 24. Woolthuis CM, Park CY. Hematopoietic stem/progenitor cell commitment to the megakaryocyte lineage. Blood 2016;127:1242–8. 25. Blom B, Spits H. Development of human lymphoid cells. Annu Rev Immunol 2006;24:287–320. 26. Haddad R, Guimiot F, Six E, et al. Dynamics of thymus colonizing cells during human development. Immunity 2006;24:217–30. 27. Spits H. Development of alpha beta T cells in the human thymus, thymic involution. Nature Immunol Rev 2002;2:760–72. 28. Gary DHD, Ueno T, Chidgey AP, et al. Controlling the thymic microenvironment. Curr Opin Immunol 2005;17:137–43. 29. Takhama Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol 2006;6:127–36. 30. Mahnke YD, Brodie TM, Sallusto F, et al. The who’s who of T-cell differentiation: human memory T-cell subsets. Euro J Immunol 2013;43:2797–809. 31. Synder-Cappione JE, Tincati C, Eccles-James IG, et al. A comprehensive ex vivo functional analysis of human NKT cells reveals production of MIP1 alpha and MIP-1 beta, a lack of IL17 and a Th1bias in males. PLoS ONE 2010;5:15412–20. 32. Kronenberg M. Towards an understanding of NKT cell biology: progress and paradoxes. Annu Rev Immunol 2005;26:877–900. 33. Jonuleit H, Schmitt E. The regulatory T cell family: distinct subsets and their interrelations. J Immunol 2003;171:6323–7.

34. Nagasawa T. Microenvironmental niches in the bone marrow required for B-cell development. Nature Immunol Rev 2006;6:107–16. 35. Tobón GJ, Izquierdo JH, Cañas CA. B Lymphocytes: development, tolerance, and their role in autoimmunity—focus on systemic lupus erythematosus. Autoimmune Dis 2013;2013:827254. 36. Herzenberg LA, Tung JW. B cell lineages: documented at last. Nat Immunol 2006;7:225–6. 37. Barone F, Vossenkamper A, Boursier L, et al. IgA producing plasma cells originate from germinal centers that are induced by B cell receptor engagement in humans. Gastroenterology 2011;140:947–56. 38. Moretta L, Moretta A. Unravelling natural killer cell function: triggering and inhibitory human NK receptors. EMBO J 2004;23:255–9. 39. Farag SS, Caligiuri MA. Human natural killer cell development and biology. Blood 2006;20:123–37. 40. Fu B, Tian Z, Wei H. Subsets of human natural killer cells and their regulatory effects. Immunology 2013;141:483–9. 41. Artis D, Spits H. The biology of innate lymphoid cells. Nature 2015;517:293–301. 42. Guyden JC, Martinez M, Chilukuri RV, et al. Thymic nurse cells participate in heterotypic internalization and repertoire selection of immature thymocytes: their removal from the thymus of autoimmune animals may be important to disease etiology. Curr Mol Med 2015;15:828–35. 43. Hanabuchi S, Watanabe N, Liu Y-J. TSLP and immune homeostasis. Allergol Intl 2012;61:19–25. 44. Steiniger BS. Human spleen microanatomy: why mice do not suffice. Immunology 2015;145:334–46. 45. Choi I, Lee S, Hong Y-K. The new era of the lymphatic system: no longer secondary to the blood vascular system. Cold Spring Harb Perspect Med 2012;2:a006445. 46. Ferrante AW. The immune cells in adipose tissue. Diabetes Obes Metab 2013;15:34–8. 47. Brandtzaeg P. Mucosal immunity: induction, dissemination and effector functions. Scand J Immunol 2009;70:505–15. 48. Kunizawa J, Nochi T, Hiroshi K. Immunological commonalities and distinctions between airway and digestive immunity. Trends Immunol 2008;29:505–13. 49. Wright P. Inductive/effector mechanisms for humoral immunity at mucosal sites. Am J Reprod Immunol 2011;65:248–52. 50. Newberry RD, Lorenz RG. Organizing a mucosal defense. Immunol Rev 2005;206:6–21. 51. Bienenstock J, McDermott MRM. Bronchus- and nasal-associated lymphoid tissues. Immunol Rev 2005;206:22–31. 52. Grainger JR, Askenase MH, Guimont-Desrochers F, et al. Contextual functions of antigen-presenting cells in the gastrointestinal tract. Immunol Rev 2014;259:75–87. 53.  Montalvillo E, Garrote JA, Bernardo D, et al. Innate lymphoid cells and natural killer T cells in the gastrointestinal tract immune system. Rev Esp Enferm Dig 2014;106:334–45. 54. Kyd JM, Foxwell AR, Cripps AW. Mucosal immunity in the lung and upper airway. Vaccine 2001;19:2527–33. 55. Boyaka PN, Tafaro A, Fischer R, et al. Therapeutic manipulation of the immune system: enhancement of innate and adaptive mucosal immunity. Curr Pharm Des 2003;9:1965–72. 56. Hickey DK, Patel MV, Fahey JV, et al. Innate and adaptive immunity at mucosal surfaces of the female reproductive tract: stratification and integration of immune protection against the transmission of sexually transmitted infections. J Reprod Immunol 2011;88:185–94. 57. Heath WR, Carbone FR. The skin-resident and migratory immune system in steady state and memory: innate lymphocytes, dendritic cells and T cells. Nature Immunol 2013;14:978–85. 58. Spasova DS, Surh CD. Blowing on embers: commensal microbiota and out immune system. Front Immunol 2014;5:318. 59. Hevia A, Delgado S, Sanchez B, et al. Molecular players involved in the interaction between beneficial bacterial and the immune system. Front Microbiol 2015;6:1285. 60. Kurashima Y, Goto Y, Kiyono H. Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation. Euro J Immunol 2013;43:3108–15.

CHAPTER 2  Organization of the Immune System

38.e1

M U L T I P L E - C H O I C E Q U E S T I ON S 1. Characteristics of hematopoietic stem cells include: A. Deleted as progeny develop B. Derive all lineages of blood cells C. Form distinct niches in fetal liver D. Rely on stromal factors to regulate differentiation

3. Mucosal immune sites include (more than one correct answer): A. Vaginal and rectal tissues B. M cells which transport antigens across the lumen C. Inguinal and axial lymph nodes D. Mesenteric lymph nodes

2. Interactions between innate and acquired immune cells involve (more than one correct answer): A. Processing and presentation of antigens B. Stimulation of macrophages by TLR-ligands C. Only tissue based interactions D. Migration of immature dendritic cells to lymph nodes

4. Lymph nodes are (more than one correct answer): A. Located throughout the body B. Delivery sites for phagocytized organisms C. Location of T- and B-cell rearrangements D. Peyer patches in the gastrointestinal tract

3  Innate Immunity Douglas R. McDonald, Ofer Levy

Innate immunity is the first line of host defense against infection. All living organisms are continually exposed to microbes. For example, the human gut is colonized by trillions of commensal bacteria. The innate immune system must accommodate commensal bacteria but be able to be activated by pathogens (e.g., Salmonella or Shigella). Potentially life-threatening infections can result from naturally occurring defects in the innate immune response (Chapter 36). A defining characteristic of innate immunity is its existence before microbial exposure. Innate immune responses are initiated rapidly by microbes and precede the development of adaptive immune responses. The adaptive immune system is characterized by the tremendous diversity of its receptors and its antigen ligands. The innate immune system responds to a more constrained set of antigens that are typically essential and invariant structural components specific to microbes. These microbial components are known as pathogen-associated molecular patterns (PAMPs).1 They include microbial cell wall components and nucleic acids. PAMPs are recognized by pattern recognition receptors (PRRs), and they are highly potent and effective in initiating inflammatory responses. “Trained immunity” is used to describe the phenomenon of enhanced innate immune responses following microbial exposure.2,3 This increase in host resistance to reinfection can provide “cross-protection” against other infectious agents. For example, macrophages and natural killer (NK) cells can expand and contract their cell populations, upregulate genes involved in pathogen recognition and presentation, and secrete cytokines that augment the antimicrobial activity of bystander cells. Thus there is a growing appreciation that the adaptive and innate immune systems have certain similar characteristics.

BARRIERS TO INFECTION Skin and Mucosa The epithelial layers of the skin and the linings of the gastrointestinal (GI), genitourinary (GU), and respiratory tracts provide a mechanical barrier to microbial entry and thus play an essential role in host defense. The stratum corneum of the skin is the first barrier encountered by microbes (Chapter 19). The skin is persistently colonized with numerous microbes. Thus an intact physical barrier is essential to prevent activation of the immune system under nonpathological conditions. Key cellular components of the skin immune barrier include keratinocytes, dendritic cells (DCs), macrophages, T lymphocytes, and mast cells. These cells express a wide variety of pathogen recognition receptors and secrete a broad range of cytokines, chemokines, and antimicrobial proteins and peptides (APPs) that mediate inflammatory

KEY CONCEPTS The Innate Immune System • The innate immune system provides the initial immune response to pathogens. • Although less specific than the adaptive immune system, the innate immune system must differentiate commensal from pathological microbes. • The innate immune system comprises barriers to the environment (e.g., skin, mucosa), antimicrobial peptides and proteins, cells (e.g., neutrophils, macrophages, monocytes), and soluble factors (i.e., cytokines, chemokines, complement). • Pathogen detection is mediated by a variety of germline-encoded pathogen recognition receptors (PRRs) that can recognize invariant microbial structures known as pathogen-associated molecular patterns (PAMPs). • Activation of the innate immune system leads to subsequent activation of the adaptive immune system. • The innate immune system has a form of memory or “trained immunity” such that innate immune activation can modulate innate immune responses to subsequent unrelated stimulus/infection.

responses to infection. Genetic disorders of the skin that compromise skin integrity, such as epidermolysis bullosa (Chapter 63), can result in life-threatening infections. Skin disorders that impair barrier function, such as atopic dermatitis (AD) (Chapter 44) or eczema, are common.4 Filaggrin (FLG) is a key a structural component of the outermost layer of the epidermis. Loss of function variants in filaggrin (R510X, 2282del4) is estimated to be present in up to 50% of patients with AD.4 FLG mutations are a risk factor for the development of early-onset AD and thus for sensitization to food allergens (Chapter 45), allergic rhinitis, and asthma (Chapter 72) (the atopic march). Eczematous skin can lead to reduced expression of APPs and increased susceptibility to cutaneous bacterial (e.g., Staphylococcus, Streptococcus) and viral (e.g., herpes) infections.5 The luminal surfaces of the intestines are sites of continual exposure to massive numbers of microbes. Intestinal epithelial cells (IECs) (Chapter 20) protect against infection by forming a physical barrier through tight junctions and by producing mucus (goblet cells) and APPs. IECs express apical junction complexes, including E-cadherin, ZO-1, claudin, and occludin, which function to form a tight monolayer that prevents penetration by bacteria.6 A breakdown in epithelial gut homeostasis can lead to inflammatory bowel diseases (e.g., Crohn disease, ulcerative colitis) (Chapter 75) and increased susceptibility to bacterial infection.7

39

40

Part one  Principles of Immune Response

Influenza viruses and respiratory syncytial virus replicate in airway epithelial cells, leading to cell death and inflammation. The impaired barrier function of the airways can lead to increased susceptibility to secondary invasive bacterial infections by Streptococcus pneumoniae and other pyogenic bacteria. Inflammatory bowel diseases also result in impaired barrier functions of the small and large intestines, which can be associated with increased translocation of bacteria across gut mucosa, potentially leading to serious infection.

CLINICAL PEARLS Innate Immunity Barriers • Innate immune barriers consist of epithelial layers, including those of skin and the gastrointestinal, respiratory, and genitourinary tracts. • Barrier function is an underappreciated component of the innate immune system. • Defects of barrier function, such as epidermolysis bullosa and atopic dermatitis, increase the risk of infection. • Production of antimicrobial peptides and proteins at barrier sites plays a vital role in preventing invasion by microbes.

Antimicrobial Proteins and Peptides Among the APPs produced by the skin, GI, GU, and respiratory tract epithelia are bactericidal/permeability-increasing protein (BPI), defensins (β-strand peptides connected by disulfide bonds), and cathelicidins (linear α-helical peptides)8 (Table 3.1). Most APPs have a net positive charge, which enhances their affinity for negatively charged microbial cell membranes. Binding of APPs to microbes can increase membrane permeability and target cell death. TABLE 3.1  Epithelial Antimicrobial

Proteins and Peptides (APPs) Antimicrobial Peptide Dermicidin Psoriasin RNase 7 RNase 5/angiogenin Cathelicidin (LL-37) BPI hBD-1 hBD-2 hBD-3 hBD-4 SLPI Elafin Adrenomedullin

MIP-3α/CCL20 Lysozyme

Lactoferrin

Source

Target Organism

Eccrine sweat glands Keratinocytes, sebocytes Keratinocytes Keratinocytes Keratinocytes, sebocytes Epithelia-oral, GI, urogenital tract Keratinocytes, sebocytes Keratinocytes, sebocytes Keratinocytes Keratinocytes Keratinocytes Keratinocytes Keratinocytes, hair follicles, eccrine/ apocrine sweat glands, sebocytes Keratinocytes Keratinocytes, sebocytes, hair bulb cells Milk, saliva, tears, nasal secretions, neutrophils

Broad spectrum G− Broad spectrum C albicans G+, G− G−, (G+, fungi) G− G− Broad spectrum G+, G− Broad spectrum Broad spectrum G+, G−

Broad spectrum G+, G−

Broad spectrum

RNase, ribonuclease; BPI, bactericidal/permeability-increasing protein; CCL, chemokine ligand; G+, gram-positive; G−, gram-negative; GI, gastrointestinal; hBD, human β-defensin; MIP, macrophage inflammatory protein; SLPI, secretory leukocyte peptidase inhibitor.

BPI is a ~55-kilodalton (kDa) cationic and hydrophobic protein with high affinity for the lipid A region of lipopolysaccharide (endotoxin). It is found in neutrophil primary (azurophilic) granules and is also inducible in epithelial cells. BPI inhibits gram-negative bacteria by its endotoxin neutralizing and microbicidal and opsonic properties.9 Neutralization of endotoxin may also help limit inflammatory responses to gram-negative bacteria. Some APPs, such as lysozyme (Lz), have enzymatic activities, which cleaves peptidoglycans found in bacterial cell walls. Other APPs bind to and compete for nutrients, a form of so-called nutritional immunity. Lactoferrin (Lf), for example, binds iron, a nutrient essential to bacterial survival.9 Defensins are classified by the linking pattern of cysteines and their sizes. α-defensins are expressed in neutrophils and Paneth cells of the small intestine, whereas β-defensins are expressed by mucosal surface epithelia, including those of skin, eyes, and the oral, urogenital, and respiratory tracts.10 Defensins have a broad specificity of antimicrobial activities against bacteria, mycobacteria, fungi, parasites, and viruses (Table 3.2). They have also been shown to enhance antigen uptake and processing, and to stimulate the chemotaxis of monocytes, macrophages, and mast cells.10,11 The expression of several of the defensins is constitutive. For others, inflammatory stimuli (bacterial products, proinflammatory cytokines) will increase defensin expression (human neutrophil proteins 1–3 and human β-defensin-2). Given the increasing incidence of antibiotic resistant bacteria, there is great interest in the potential uses of APPs as treatment for bacterial infections and infections with multidrug-resistant organisms.12,13

HUMORAL INNATE IMMUNITY The Acute Phase Response A variety of soluble proteins found in plasma help recognize PAMPs and function as mediators of innate immunity. Tumor

TABLE 3.2 Neutrophil-Derived

Antimicrobial Proteins and Peptides (APPs) Neutrophil APP

Granule Type

Target Organism

Lysozyme Azurocidin Elastase Cathepsin G Proteinase 3 BPI α-defensins (HNP-1 to -4) Cathelicidin (hCAP-18) Lactoferrin SLPI

Azurophil, specific Azurophil, secretory Azurophil Azurophil Azurophil Azurophil Azurophil

G+, G+, G+, G+, G+, G−, G+,

Specific Specific Specific

NGAL Lysozyme Azurocidin Elastase Cathepsin G

Specific Azurophil, specific Azurophil, secretory Azurophil Azurophil

G+, G−, mycobacteria G+, G−, fungi, viruses G+, G−, Aspergillus fumigatus, C. albicans G+, G−, fungi G+, G− G+, G−, C. albicans G+, G− G+, G−

G− G−Candida albicans G− G− G− (G+, fungi) G−, fungi, viruses

BPI, bactericidal/permeability-increasing protein; G+, gram-positive; G−, gram-negative; hCAP, human cathelicidin antimicrobial protein; HNP, human neutrophil peptide; NGAL, neutrophil gelatinase-associated lipocalin; SLPI, secretory leukocyte peptidase inhibitor.

CHAPTER 3  Innate Immunity KEY CONCEPTS Humoral Innate Immunity • Cytokines and chemokines are essential mediators of the innate immune response. • Cytokines are redundant and pleiotropic. To avoid host tissue response, their synthesis is tightly controlled. • Acute phase reactants (i.e., cross-reactive protein [CRP]) are induced by cytokines (interleukin [IL]-6) and play roles in opsonization of microbes. Plasma CRP is used to monitor infections and inflammation. • Defects in the complement system result in invasive bacterial infections, primarily from encapsulated bacteria.

necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induce production of acute phase reactants in hepatocytes, including members of the pentraxin family (e.g., serum amyloid A [SAA], serum amyloid P [SAP], and cross-reactive protein [CRP]). These pentraxins bind to components of the bacterial cell wall.14 TNFα and IL-1β also induce production of IL-6 from mononuclear phagocytes, endothelial cells, and fibroblasts. IL-6 is another potent inducer of acute phase reactants, including CRP and fibrinogen. CRP, SAA, and SAP function as opsonins and can bind phosphorylcholine and phosphatidylethanolamine expressed on bacteria and apoptotic cells, enhancing phagocytosis of bacteria and apoptotic cells by macrophages. Lipopolysaccharide-binding protein (LBP) is an acute phase reactant synthesized by the liver in response to gram-negative bacterial infections. LBP binds to LPS and subsequently forms a complex with CD14, TLR4, and MD-2, which functions as a high-affinity receptor for LPS.15 Mannose-binding lectin (MBL) is a member of the calciumdependent (C-type) lectins (collectins) produced by the liver in response to infection. MBL binds to carbohydrates with terminal mannose and fucose residues that are expressed on microbial cell surfaces.16 MBL can bind to the C1q receptor on macrophages to enhance phagocytosis and can activate the complement system via the lectin pathway (discussed below). Surfactant protein-A and surfactant protein-D are collectins expressed in the lung and can bind a variety of microbes and inhibit their growth.13,17 They also function as opsonins that promote ingestion by alveolar macrophages. Finally, ficolins are plasma proteins capable of binding to several types of bacteria and can activate complement.

CLINICAL PEARLS Complement • Deficiency of early components of the complement cascade create susceptibility to invasive infections with encapsulated bacteria and development of a lupus-like syndrome. • Deficiency of late components of the complement cascade (C5–9) result in susceptibility to meningitis caused by Neisseria meningitis. • Deficiency of C1-inhibitor protein (or function) results in hereditary angioedema. • Deficiency of factor H is associated with development of membranoproliferative glomerulonephritis, hemolytic–uremic syndrome, and age-related macular degeneration. • Deficiency of mannose-binding lectin can result in susceptibility to bacterial infection in individuals with comorbid conditions (e.g., cystic fibrosis).

41

The Complement System The complement system comprises a collection of plasma proteins activated by microbes (Chapter 21). It helps mediate microbial destruction and inflammation.18 Complement activation can occur via three pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). In the CP, complement C1 detects immunoglobulin M (IgM), IgG1, or IgG3 bound to the surface of a microbe. C1 is composed of the C1q, C1r, and C1s subunits. These form multimeric complexes that recognize IgM or IgG bound to microbial surfaces. C1r and C1s are serine proteases. Activated C1s generates a C3 convertase composed of C4b and C2b (C4b2b) bound to the microbial surface. C3 convertase cleaves C3, generating C3b. C3b binds covalently to C4b2b, generating C5 convertase. C5 convertase then activates the late steps of complement activation leading to assembly of the membrane attack complex (MAC) and subsequent cytolysis. The AP is initiated by small amounts of C3b, which are spontaneously generated in plasma. C3b that remains unbound to a cell surface is rapidly hydrolyzed and inactivated. C3b bound to a microbe becomes a binding site for factor B. Bound factor B is cleaved by factor D, generating factor Bb that binds covalently to C3b, forming the AP C3 convertase, which activates the late steps of complement activation, as in the CP. The lectin pathway is activated by MBL or ficolins binding to microbial surfaces. MBL then binds to MBL-associated serine proteases (MASPs)-1, -2, and -3. MASP-2 cleaves C4 and C2 to activate the complement cascade, as in the CP (Fig. 3.1). Complement components also function as opsonins. Complement-coated microbes can be phagocytosed via complement receptors on phagocytes. Complement receptor type 1 (CR1) is a high-affinity receptor for the C3b and C4b fragments of complement and mediates the internalization of C3b- and C4b-coated particles. On erythrocytes, it mediates clearance of immune complexes from the circulation. Complement type 2 receptor (CR2, also known as CD21) is expressed on B cells and follicular dendritic cells (FDCs). It binds C3 proteolytic fragments, including C3d, C3dg, and iC3b. CR2 augments humoral immune responses by enhancing B-cell activation by antigen and by promoting trapping of antigen–antibody complexes in germinal centers.19 CR2 is also the receptor for Epstein-Barr virus (EBV), allowing EBV to enter B cells. Complement receptor 3 (CR3) is composed of a heterodimer of CD18 and CD11b and is expressed in polymorphonuclear neutrophils (PMNs) and monocytes or macrophages. CR3 binds to iC3b bound to the surface of microbes, leading to phagocytosis and destruction of the pathogen. Activation of complement via the AP can greatly enhance monocytegenerated TNF-α elicited by gram-positive bacteria, such as group B Streptococcus.20 There are multiple regulatory proteins within the complement pathways. C1-inhibitor (C1-INH) binds to, and inhibits, the enzymatic functions of C1r and C1s within the CP (21). Properdin stabilizes C3bBb complex, increasing the life span of the AP C3 convertase. Conversely, factor H inhibits the formation of, and degrades, C3bBb complex. Factor I inactivates C3b. CD55 (decay accelerating factor) and CD59 are cell surface, GPI-linked proteins that block complement-mediated cytolysis by inhibiting formation of C3bBb complex and binding of C9 to C5b678 complex, respectively. Paroxysmal nocturnal hemoglobinuria, an acquired defect in the PIGA gene that causes a deficiency of GPI-linked proteins, is the result of absent cell surface expression of CD55

42

Part one  Principles of Immune Response Classical pathway Antigen-antibody

Lectin pathway Microbial carbohydrates MBL and ficolins Mannose

Antibody

C4, C2 MASP1, 2, 3

Y C1q, r, s C4, C2

Factor B

C3b

C3

Factor D

C4b2b C3 convertase C4a, C2a CD55

Alternative pathway Microbial surfaces

C3bBb C3 convertase

C3 cleavage

C4b2b3b C3bBb3b C5 convertase C5 C5

Properdin

Factor I, H

C5b CD59

C5b-9

FIG 3.1  Complement Activation Pathways. The classical complement cascade is activated by antibody bound to microbial surfaces, which is a binding site for the C1 complex. The alternative pathway is activated by the binding of spontaneously generated C3b to microbial surfaces. Microbe-bound C3b binds factor B, which is converted to factor Bb, forming a C3 convertase. The lectin pathway is activated by the binding of mannose-binding lectin (MBL) to mannose residues on microbial surfaces. MBL binds MBL-associated serine proteases, which bind and cleave C4 and C2, forming a C3 convertase.

and CD59 that leads to hemolytic anemia caused by complementmediated lysis of red blood cells (RBCs). Complement Deficiency Diseases Deficiencies of early components of the complement pathway are associated with invasive bacterial infections caused by encapsulated organisms (Chapter 21). Lack of early components of the complement pathway are also associated with rheumatic disorders, including a lupus-like syndrome that may be caused by impaired immune complex clearance, impaired clearance of apoptotic cells, and loss of complement-dependent B cell tolerance (Chapter 50). Deficiency of factor I is also associated with increased incidence of invasive infection with encapsulated bacteria, as well as glomerulonephritis and autoimmune disease. Deficiency of C1-INH protein and function, either hereditary or acquired, leads to hereditary angioedema (HAE) or acquired angioedema (AAE) (Chapter 42). C1-INH inhibits C1, factors XIa and XIIa, and kallikrein. Dysregulation of these cascades leads to generation of vasoactive products that result in angioedema.21,22 Deficiencies of late components of complement, including C5 through C9, as well as factors B, D, and properdin create susceptibility to meningococcal infections.23 Deficiency of factor H function is associated with membranoproliferative glomerulonephritis (Chapter 68), hemolytic–uremic syndrome, and age-related macular degeneration (AMD) (Chapter 74).24 Deficiency of MBL is associated with increased susceptibility to bacterial infections in infancy and in individuals with other comorbid conditions, such as cystic fibrosis.25

CELLULAR INNATE IMMUNITY Polymorphonuclear Leukocytes PMNs are the most abundant leukocyte (Chapter 22). They have a short life span of ~6 hours in circulation, and in the healthy

KEY CONCEPTS Cellular Innate Immunity • Polymorphonuclear leukocytes (neutrophils) are the most abundant cells of the innate immune system, are short-lived, and are the earliest responders to infection. • Monocytes and macrophages are the predominant immune cells several days after an infection. • Activated neutrophils, monocytes, and macrophages kill phagocytosed bacteria through production of reactive oxygen intermediates and antimicrobial proteins and peptides (APPs). • Dendritic cells (DCs) are efficient in uptake and presentation of foreign antigen and provide a critical link between innate and adaptive immunity. • Natural killer (NK) cells can kill infected or malignant cells without prior activation. • Mast cells are present are found at the interface between host and environment and are first responders to microbes and recruit other inflammatory cells.

adult, ~109 PMNs are produced per hour. PMNs are readily identified by light microscopy by segmented nuclei divided into 3–5 lobules. Their cytoplasm contains four types of granules: azurophilic (or primary), specific (or secondary), gelatinase, and secretory. PMN granules contain a wide variety of APPs with a broad spectrum of antimicrobial activities (see Table 3.2). Azurophilic granules contain enzymes, such as proteinase 3, cathepsin G, and elastase as well as α-defensins and BPI. Specific granules contain lactoferrin and the proforms of cathelicidin peptides. Gelatinase granules are rich in gelatinase and are a marker of terminal neutrophil differentiation. Secretory granules contain a variety of receptors that are inserted into the cell membrane upon activation. Exocytosis of these receptors convert PMNs into cells responsive to inflammatory stimuli. PMNs are

CHAPTER 3  Innate Immunity the earliest responders to infection. Those not recruited to sites of infection undergo apoptosis and are cleared by the reticuloendothelial system. Individuals with severely low numbers of neutrophils ( IgG3 > IgG1 > IgG2 > IgG4. Binding of these Ab exposes sites in the Fc region for attachment of the first subcomponent of complement, C1q.13,14 C1 is a large calcium-dependent complex composed of C1q and two molecules each of the proenzymes, C1r and C1s. C1q is a 410-kDa protein with six globular heads connected by a collagenlike tail. For IgM, which is pentameric, binding to antigen creates a conformational change that exposes the C1q binding site in the Cµ3 domain. For IgG, at least two closely bound molecules are required to provide multiple attachment points for C1q binding to the Cγ2 domain. Similarly, CCRP or SAP molecules bound to ligand provide multiple C1q binding sites, resulting in CP activation. IgM, IgG, and CRP bind C1q through its globular head groups. Membrane lipids exposed on apoptotic cells or mitochondrial membranes, polyanions, nucleic acids, retroviruses, and endotoxins can also activate the CP.

FIG 21.3  Sequence of Protein Interactions in the Assembly of the Membrane Attack Complex (MAC). C5b, generated by a convertase cleaving C5, combines with C6 and C7 to form a hydrophobic complex capable of a membrane interaction. Binding of C8 allows the complex to insert further into the membrane and forms a site for C9 polymerization. C9 polymers (10–15) form a transmembrane pore to mediate cell lysis.

Once C1q binds to an activator through several globular heads, C1r is cleaved by an autocatalytic process. Activated C1r then cleaves and activates C1s, which, in turn, cleaves circulating C4. C4 and C3 are highly homologous proteins that share an unusual posttranslational modification known as an internal thioester bond (see Fig. 21.2).10,15 Cleavage of C4 releases the C4a fragment and exposes the reactive thioester bond in the larger C4b fragment. This allows C4b to attach covalently to nearby target structures, through either amide or ester bonds, to form amino or carboxyl groups on proteins and polysaccharides on cell surfaces, including Abs and a wide array of antigens. The exposed thioester bond is highly but transiently reactive as it is susceptible to rapid hydrolysis. For example, only about 5% of the C4b typically becomes attached to the target. Bound C4b provides an anchor site for C2 attachment, which is then also cleaved by C1s, releasing the smaller fragment C2b. The complex of C4b2a is termed the CP C3 convertase because it has the capacity to cleave C3, releasing C3a. The C2a component of the complex contains the active enzymatic site. C3 cleavage is similar to C4 cleavage in that the larger fragment, C3b, contains a thioester site (see Fig. 21.2) that mediates covalent attachment to nearby surface structures, including the antigen, the Ab, and the attached C4b. C3 is found at a three- to fourfold higher concentration in serum compared with C4, and its cleavage is amplified by the AP. Thus efficient complement activation will result in clusters of multiple bound C3b molecules that can be recognized by cellular receptors. C3b that attaches to C4b within the C3 convertase produces the trimolecular complex C4b2a3b, which is a C5 convertase. Cleavage of C5 produces C5a, which has potent inflammatory activity, and C5b, which initiates the formation of the MAC or, as it is also known, the terminal complement complex (TCC) (Fig. 21.3).

Lectin Pathway The LP is similar to the CP, except that it uses pattern recognition molecules, MBL, and ficolins-1, 2, and 3, instead of Ab to target activation.7,16 MBL is structurally similar to C1q, with a collagenlike region and globular heads. The globular heads of MBL are C-type lectin domains specific for repeating carbohydrate structures found on microorganisms. Like C1q, MBL and ficolins are in complex with serine proteases, MBL-associated serum

CHAPTER 21  The Human Complement System: Basic Concepts and Clinical Relevance proteases (MASPs), which are structurally and functionally similar to C1r and C1s. MASP-1 and -2 are active proteases, but only MASP-2 cleaves both C4 and C2 to generate C4b2a, the same C3 convertase as the CP. MASP-1 can supplement activation by cleaving C2 but not C4. Two nonproteolytic splice products of the MASP2 and MASP1/3 genes, sMAP and MAP-1, compete with MASP-1 and MASP-2 for binding to MBL to regulate the LP. Subsequent steps in the LP are identical to those in the CP. Although still controversial, MASP-1 and MASP-3 likely contribute to the AP by cleaving profactor D to active factor D.7,16,17

Alternative Pathway The AP uses proteins that are structurally and functionally homologous to those of the CP, but this pathway has unique features that play three important roles in the complement cascade. The surveillance role of the AP is mediated by a continuous low level of spontaneous activation that results from the hydrolysis of the C3 thioester bond.8 Hydrolyzed C3, C3(H2O), assumes a conformation similar to that of C3b and can bind factor B (homologous to C2), which is cleaved by factor D (homologous to C1s) to form a fluid-phase C3 convertase. This convertase cleaves C3 to generate C3b, which can covalently bind to nearby structures and provide the basis for a bound C3 convertase (C3bBb). Because C3b is both a part of this enzyme and a product of the reaction, a positive feedback loop that rapidly deposits more C3b is formed. This low-grade activation process is tightly regulated on host cells and tissues by plasma and membrane-bound complement regulatory proteins. It is the lack of such regulation that usually restricts AP activation to microbial targets. The plasma protein factor H (FH) is particularly important in controlling AP activation, both in the fluid phase and on “nonactivating” surfaces. The latter recruits FH through its binding sites for polyanions, including sialic acid and glycosaminoglycans. “Activating” surfaces, such as microbial polysaccharides, lipopolysaccharides, and foreign glycoproteins, provide C3b attachment sites that are protected from regulatory proteins. Similar to the CP, the AP C5 convertase (C3bBb3b) is formed when a second C3b attaches to the C3 convertase. The AP C3 and C5 convertases are stabilized by P (factor P or P), for which this pathway was originally named. More recently, an additional role for properdin (P) in initiating AP activation was rediscovered.8,18,19 P is a pattern-recognition molecule with specificity for microbes and damaged cells. Once bound, P can recruit fluid-phase C3b or C3b (H2O), independent of covalent binding, and thereby provide a platform for the assembly of the AP convertase. Thus P binding can direct AP activation, similar to MBL in the LP. P binding to certain Neisseria species potently activates the AP, and this may account for the susceptibility of P-deficient individuals to infection with N. meningitidis. The third important role of the AP is the amplification of C3b deposition and C5 convertase generation that is initiated by the CP or the LP.8 This function of the AP is critical in complement-mediated pathology, as it increases the generation of C5a and the MAC, the most inflammatory components of the system. It is this amplification role of the AP that makes it an attractive therapeutic target.

MEMBRANE ATTACK COMPLEX All three complement pathways merge with the cleavage of C5 into C5a and C5b. Although C5 is structurally homologous to C3 and C4, it lacks an internal thioester bond that allows

303

covalent attachment to surfaces. C3a and C5a are also structurally homologous and, as described below, are the most potent proinflammatory mediators of the complement system. C5b initiates the formation of the MAC (see Fig. 21.3), a complex of C5b, C6, C7, C8, and multiple (5–10) C9 molecules.20 This complex, as indicated by its name, penetrates membrane bilayers to form pores that disrupt the osmotic barrier, leading to swelling and lysis of susceptible cells. Lysis of Ab-sensitized erythrocytes by the MAC is the basis of the total hemolytic complement (THC) assay or CH50. C5b initiates the formation of the MAC without further proteolytic steps. C5b binds to C6, and this complex binds to C7. The C5b67 complex is lipophilic and associates with cell membranes, if available, or with serum lipoproteins. Once bound to a membrane, C5b67 recruits C8, and the complex penetrates more deeply into the membrane. However, efficient lysis requires C9, a pore-forming molecule with homology to perforin, a protein used by cytotoxic T cells and natural killer (NK) cells for killing virus-infected targets. The complex of C5b678 forms a nidus for C9 binding and polymerization. Although complement-dependent lysis of bacteria can be observed in vitro, many pathogens have evolved mechanisms to circumvent this activity of complement.21 Opsonization by C3b is the most potent mechanism for destruction (adherence followed by ingestion) of bacteria by the complement system. The sublytic MAC is proinflammatory because of its membrane perturbing capabilities for host cells and contributes to the deleterious effects of complement activation in inflammatory diseases.22

REGULATION OF COMPLEMENT ACTIVATION The complement cascade is rapidly activated and highly amplified by the generation of C3 and C5 convertases. There are three main levels of control that limit the potential harm that uncontrolled complement activation might cause: (1) the initiation step in the CP and the LP; (2) the C3 and C5 convertases of all three pathways; and (3) the assembly of the MAC. Both soluble and membrane-bound regulatory proteins serve these functions, which help terminate complement activation and direct it to appropriate targets.23

C1 Esterase Inhibitor C1 esterase inhibitor (C1-INH) is a plasma serine proteinase inhibitor (serpin). C1-INH covalently binds to activated C1r and C1s, irreversibly inhibiting their activity and thereby limiting CP activation. C1-INH inactivation of C1r and C1s also removes them from the C1 complex, exposing sites on the collagen-like region of C1q. Likewise, C1-INH inhibits MASP-1 and MASP-2, kallikrein, factor XIa, factor XIIa, and plasmin of the LP and the contact, coagulation, and fibrinolytic systems. Inherited deficiency of C1-INH is the basis of hereditary angioedema, a disease characterized by recurrent attacks of subcutaneous or submucosal edema (Chapter 42).24

Regulators of the C3 and C5 Convertases The C3 and C5 convertases are central to the generation of the inflammatory and opsonic products of complement activation and are highly regulated by fluid-phase and membrane-bound regulatory proteins. The membrane deposited C4b and C3b may be bound by the regulator to prevent an association with C2 or FB. The convertases themselves are complexes of two or three components, and one mechanism of regulation is the dissociation of these complexes. This type of regulation is termed decay

304

Part two  Host Defense Mechanisms and Inflammation

FIG 21.4  FI dependent cleavage of C3 showing the structures of the products and the required cofactors. The cofactor protein binds first and then the serine protease Factor I cleaves the C3b.

acceleration. A second mechanism of regulation is the enzymatic inactivation of the C4b and C3b components of the convertases (Fig. 21.4). This is accomplished by the plasma enzyme factor I (FI), which, however, only acts on C4b or C3b in complex with one of several regulatory proteins. The binding of regulatory proteins to C4b or C3b to enable FI cleavage is termed cofactor activity. Factor I FI (C3b inactivator, C3bINA) cleaves C4b and C3b into products that are recognized by specific cellular receptors (as discussed below). The sequential cleavages of C3b by FI to iC3b and C3dg are depicted in Fig. 21.4. C4b is cleaved in an analogous manner to C4d. (The iC4b intermediate is found only transiently.) The regulatory proteins that facilitate this cleavage by cofactor activity and those that inactivate C3 and C5 convertases by decayaccelerating activity are members of a family of structurally related proteins encoded within the regulators of complement activation (RCA) genetic locus.25 This family is characterized by a repeating structure that consists of subunits, termed complement control protein repeats (CCP), of about 60 amino acids with a conserved pattern of two disulfide bonds per repeat and are usually encoded by a single exon. Soluble Regulatory Proteins, C4b-Binding Protein, and FH C4b-binding protein (C4bp) and FH are fluid-phase regulatory proteins with both decay-accelerating and cofactor activities. C4bp is multimeric, being composed of seven identical subunits, each containing eight CCPs. FH is a single-chain protein composed entirely of 20 CCPs. C4bp is specific for C4b and the C4bcontaining convertases of the CP (C4b2b, C4b2b3b), whereas FH regulates C3b and C3b-containing convertases (C3bBb, C3bBb3b, C4b2b3b). FH is essential for regulation of C3 “tickover,” and FH deficiency results in an acquired deficiency of C3. Additional binding sites on FH that recognize polyanions, such as sialic acid and glycosaminoglycans, provide targeted regulation of AP activation on surfaces.23,26-28 Membrane Regulatory Proteins The RCA family includes the membrane regulatory proteins decay-accelerating factor (CD55, DAF), membrane cofactor protein (CD46, MCP), and complement receptors CR1 (CD35) and CR2 (CD21).25,29 CD55 (DAF) and CD46 (MCP), as their

names imply, have decay-accelerating and/or cofactor activity, respectively, that inhibits complement activation on cell membranes.25 Each has an extracellular domain composed exclusively of four CCPs. CD55, a glycophosphatidylinositol (GPI)–anchored protein, and CD46, a transmembrane protein, are widely distributed on cells in contact with blood, with the notable exception of erythrocytes that lack CD46. Soluble CD55 is also found in most biological fluids. Both protect cells from complementmediated lysis. CD35 (CR1) has decay-accelerating and cofactor activity and is a receptor for bound C3b. The function of CD35 as a complement receptor is discussed later in the chapter. Complement C2 receptor inhibitor trispanning (CRIT) is a non-RCA membrane regulator of the CP. CRIT was originally identified on Schistosoma and Trypanosoma parasites and later found to be widely expressed on human tissues and blood cells, except for neutrophils and erythrocytes.30 CRIT competes with C2 for binding to C4b, blocking the formation of the CP C3 convertase. Properdin In contrast to the regulatory proteins discussed above, the plasma protein P (factor P) stabilizes C3 and C5 convertases of the AP, increasing their activity.8,18,19 This enhancer of AP activation is found as noncovalently linked dimers, trimers, tetramers, and larger species composed of identical 56-kDa chains. The majority of this plasma protein consists of a series of six thrombospondin type 1 modules. P binds to C3b and to Bb, preventing the spontaneous or induced decay of the AP C3 and C5 convertases. Its multimeric structure promotes interaction with clustered C3b. As discussed above, bound P can also recruit C3b to provide a site of assembly for the AP C3 convertase.

Regulators of the Membrane Attack Complex The MAC is also regulated by both fluid-phase and membrane regulatory proteins.1,20,23,29 Soluble MAC Inhibitors: Vitronectin and Clusterin Soluble hydrophobic proteins block the incorporation of the MAC into membranes. Two well-characterized proteins with this activity are vitronectin (S protein) and clusterin (SP-40,40, apolipoprotein J).23,29 Vitronectin is in plasma and the extracellular matrix and binds to C5b-7. C8 and C9 can still bind to the complex, but membrane insertion and C9 polymerization are prevented. Soluble complexes of vitronectin and C5b-9 are in plasma during complement activation, and an enzyme-linked immunosorbent assay (ELISA) specific for this complex has been used to monitor activation of the MAC. Clusterin forms a complex with C5b-9, preventing membrane insertion. It is found in plasma, in the male reproductive tract, and on endothelial cells of normal arteries. It is also associated with amyloid deposits, including β amyloid in Alzheimer disease. Membrane MAC Inhibitor CD59 The primary membrane-bound inhibitor of the MAC is CD59.23,29 CD59 is a GPI-anchored protein expressed by most cells. CD59 binds to C8 and C9, preventing the incorporation and polymerization of C9.

COMPLEMENT RECEPTORS Many of the biological effects of complement activation are mediated by cellular receptors for fragments of complement

CHAPTER 21  The Human Complement System: Basic Concepts and Clinical Relevance

305

SIPRα. However, none has been definitively established as a receptor in the classic sense.1,5,16,31

Complement Receptor 1 (CR1, CD35) ss

CR3 CD11b CD18

CR1 CD35

CR2 CD21

CRIg

HS C=O O

SS

S S

iC3b

S S

SS

C3b

HS C=O O

C3dg HS C=O O

FIG 21.5  Receptors for Bound C3b and Its Cleavage Products. Receptors shown are CD35 and CD21 composed of CCP (SCR) subunits; CD11b/CD18 (CR3), a β2 integrin; and CRIg with one or two immunoglobulin domains. The specificities of the receptors are CD35 for C4b and C3b, (C4b > C3b), CRIg for iC3b > C3b; CD11b/CD18 for iC3b; CD21 for C3dg and C3d. CD11c/CD18 (CR4) is similar to CD11b/CD18 and is not shown. Receptors are not drawn to scale. Their molecular weights are listed in Table 21.1.

proteins. These include receptors for the small soluble complement fragments, C5a and C3a, and receptors for bound complement fragments, C1q and C4b and C3b and their cleaved fragments. Receptors are specific for C3b and for its further breakdown products generated by the enzymatic processing by FI in conjunction with the cofactor proteins mentioned above. The breakdown of C3b and intermediate products are shown in Fig. 21.4 and the receptors for these components in Fig. 21.5.

C1q Receptors C1q is one of a family of proteins termed soluble defense collagens, which includes the “collectins” (MBL, surfactant proteins A and D, conglutinin), and the ficolins. Each of these proteins is composed of a collagen-like linear stem region terminated by multiple globular recognition domains or head groups. The collectins recognize carbohydrates with their C-type lectin head groups, and the ficolins recognize acetyl groups on carbohydrates and other molecules with fibrinogen-like recognition domains. In contrast, the globular head groups of C1q do not recognize carbohydrates but, rather, bind to amino acid motifs on IgG, IgM, and pentraxins. In general, the soluble defense collagens broadly recognize pathogen-associated carbohydrate patterns and damaged or apoptotic cells. Reported direct effects of this group on leukocytes include the enhancement of phagocytosis, triggering of the respiratory burst, and regulation of cytokine responses. Several cell surface proteins have been proposed to facilitate these activities, including CD93 (C1qRp), CD35 (CR1), α2β1 integrin, calreticulin in complex with CD91, gC1q binding protein, and

There are five identified receptors for bound fragments of C3 and/or C4. CD35 is a large protein composed of a linear string of CCPs, a transmembrane region, and a short intracytoplasmic domain.32 Different allelic forms of CD35 are found, the most common being composed of 30 CCPs with a molecular weight of 190 kDa. These CCPs are organized into groups of seven, creating structures termed long homologous repeats (LHRs), each of which contains a single binding site. The predominant allele of CD35 contains two binding sites for C3b, three for C4b, and one for C1q. CR1 is expressed on human erythrocytes, monocytes and macrophages, neutrophils, B lymphocytes, a small percentage of T lymphocytes, eosinophils, FDCs, and glomerular podocytes. CD35 on primate erythrocytes provides a mechanism for clearing soluble immune complexes from the circulation. Although the number of receptors on each erythrocyte is low, the large number of erythrocytes provides the major pool of CR1 in the circulation. Soluble immune complexes that fix complement attach quickly to erythrocytes in the circulation, bypassing monocytes and neutrophils. These erythrocyte-bound complexes are taken to the liver, where they are transferred to Kupffer cells expressing Fc and complement receptors and destroyed. The erythrocytes exit into the circulation to pick up more immune complexes. This clearance pathway is impaired in patients with SLE because of decreased complement in the circulation, decreased CD35 on erythrocytes, and saturated Fc receptors in the liver and spleen. CD35 on monocytes and neutrophils promotes binding of microbes carrying C3b and C4b on their surface (immune adherence reaction), facilitating their phagocytosis through Fc receptors. CD35 can directly mediate the uptake of microbes when phagocytic cells have been activated by chemokines or integrin interactions with matrix proteins. CD35 is a member of the RCA family and has decay-accelerating and cofactor activity in addition to its function as a receptor. It differs from the membrane regulatory proteins DAF (CD55) and MCP (CD46) in its ability to also bind to C3b and C4b extrinsically (on targets other than the cell expressing it) and in its cofactor activity for iC3b processing. CD35 is the most effective cofactor for FI cleavage of C3b and iC3b to the smallest covalently bound fragment C3dg. C3dg is the major ligand for CR2 on B lymphocytes (described below). The cofactor activity of CD35 on B lymphocytes can process bound C3b to C3dg, facilitating binding to CR2 and lowering the threshold for B-cell activation.29,33,34

Complement Receptor 2 (CR2, CD21) CD21 is also an RCA family protein composed of 15–16 CCPs. CD21 has a limited range of expression that includes B lymphocytes, FDCs, and some epithelial cells. CD21 is specific for the smallest covalently bound C3 fragments, C3dg and C3d, and has weaker binding to iC3b. CD21 is also the Epstein-Barr virus (EBV) receptor on B cells and nasopharyngeal epithelial cells and binds to CD23, a low-affinity IgE receptor.33,34 CD21 on B lymphocytes serves a costimulatory role. It is expressed on mature B cells as a complex with CD19 and CD81 (TAPA-1). Coligation of CD21 and the B-cell antigen receptor induces the phosphorylation of CD19, activating several signaling pathways and strongly amplifying B-cell responses to antigen. This role of CD21 is believed to contribute to the strong adjuvant effect produced by attaching C3d to antigen.33,34

306

Part two  Host Defense Mechanisms and Inflammation

Complement Receptors 3 and 4 CR3 and CR4 are the β2 integrins commonly known as CD11b/ CD18 (Mac-1) and CD11c/CD18.34,35 β2 integrins are large heterodimers found on neutrophils and monocytes with multiple roles in adhesion to endothelium and matrix molecules as well as direct recognition of microbial pathogens. The binding activities of β2 integrins are regulated by cellular activation often through chemokine receptors. Both CD11b/CD18 and CD11c/CD18 are expressed primarily on neutrophils, monocytes, and NK cells and bind to iC3b and, to a lesser extent, C3b. CD11b/CD18 has been studied more extensively than CD11c/CD18. CD11b/ CD18 expression, clustering, and conformation are all rapidly upregulated by chemokine activation of neutrophils, leading to increased responses to ligand. CR3 plays an essential role in neutrophil attachment to and migration through activated endothelium to sites in inflammation and in the regulation of neutrophil apoptosis. Deficiency of the β2 chain (CD18) results in leukocyte adhesion deficiency, characterized by recurrent pyogenic infections, and defects in inflammatory and phagocytic responses. Complement receptors CD11b/CD18 and CD11c/CD18 provide an essential function for the removal of microbial pathogens following complement activation, since C3b processing to iC3b often occurs rapidly after deposition.

Complement Receptor of the Immunoglobulin Superfamily (CRIg) CRIg is a receptor for iC3b and C3b present on Kupffer cells in the liver as well as other tissue macrophages but is absent from splenic macrophages, peripheral blood cells, bone marrow–derived macrophages, and monocyte/macrophage cell lines.35,36 Two alternative-spliced forms of human CRIg were identified with one and two Ig domains. The mouse receptor has a single Ig domain. CRIg removes C3b or iC3b-opsonized particles from the circulation by the liver.

C5a and C3a Receptors During complement activation, the homologous proteins C3 and C5 are each cleaved near the amino-terminus of the α chains to release a soluble peptide fragment of approximately 8 kDa. These fragments are designated C3a and C5a. C5a may also be generated locally by direct cleavage of C5 by thrombin or leukocyte proteases.37 C3a and C5a are termed anaphylatoxins because of their ability to increase vascular permeability, contract smooth muscle, and trigger the release of vasoactive amines from mast cells and basophils.38-41 C5a is 10- to 100-fold more active than C3a. These peptides are also chemotactic: C5a is specific for neutrophils, monocytes, and macrophages, whereas C3a is specific for mast cells and eosinophils. Other biological activities of complement anaphylatoxins are summarized in Table 21.2. Structurally, anaphylatoxins are compact structures consisting of multiple helices cross-linked by disulfide bonds with more flexible carboxy-terminal regions. The C-terminal peptide of C3a interacts with the C3aR and can reproduce C3a agonist activity. In contrast, C5a interacts with the C5aR at multiple sites. Plasma carboxypeptidases cleave the C-terminal arginine from C3a and C5a producing the des-Arg forms. This inactivates C3a; however, C5adesarg retains much of its biological activity. The C5aR (CD88 and C5L2) and the C3aR are rhodopsin-type receptors with seven transmembrane-spanning domains coupled to G-protein signaling pathways. C5aR is expressed at high levels on neutrophils and is also found by macrophages, mast cells,

TABLE 21.2  Cellular Targets and Effects of

Complement Anaphylatoxins Targets Bearing Receptors C3a, C5a

Mast cells, basophils

C3a C5a

Eosinophils Endothelium

C5a

Neutrophils, monocytes/ macrophages, eosinophils, basophils, astrocytes Neutrophils, monocytes/ macrophages

C5a

C5a

Resident macrophages

C5a

Hepatocytes

C3a, C5a

Lymphocytes (antigenpresenting cells)

Effects Degranulation, release of vasoactive amines: contraction of smooth muscle, increased vascular permeability Chemotaxis, degranulation Increased adhesion of leukocytes; augmented chemokinesis and cytokine synthesis Chemotaxis

Priming: activation of receptors, assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; activation: degranulation, respiratory burst Regulation of FcγR expression (↑activating, ↓inhibitory) Acute phase protein synthesis Regulation of T-cell responses to antigen

basophils, smooth muscle cells, and endothelial cells. If C5a is generated locally, for example, in an extravascular site of infection, it helps induce an acute local inflammatory response, including vasodilation, edema, neutrophil chemotaxis, and activation of neutrophils and macrophages for enhanced phagocytosis and killing. The inflammatory activities of C5a can also contribute to complement-mediated pathology in some conditions, such as sepsis, acute respiratory distress syndrome, and ischemia/ reperfusion (I/R) injury, making the C5a–C5aR interaction an attractive therapeutic target. The C5L2 receptor binds to both C5a and C5adesarg. C5L2 was initially believed to be a default or decoy receptor for C5a because it is uncoupled from G proteins. Genetic deletion of C5L2 (Gpr77−/−) in mice resulted in enhanced neutrophil infiltration and cytokine production in the pulmonary Arthus reaction, supporting an antiinflammatory role for C5L2 in immune complex disease, where genetic deletion of C5aR is fully protective.40 However, studies in a cecal ligation and puncture (CLP) model of sepsis found increased survival in mice lacking either C5aR or C5L2.41 The results suggest an active proinflammatory role for C5L2 that requires C5a and results in the release of the inflammatory signal, high-mobility group box-1 protein (HMGB1) from phagocytic cells. Thus both C5aR and C5L2 may contribute synergistically to harmful inflammatory events during sepsis.

COMPLEMENT IN HOST DEFENSE AND IMMUNITY Complement in Host Defense Complement activation provides a coordinated response to infection that results in the opsonization of microbial pathogens and the attraction and activation of phagocytic cells to kill them.

CHAPTER 21  The Human Complement System: Basic Concepts and Clinical Relevance Complement-dependent opsonization is of greatest importance in infections with encapsulated extracellular bacteria, and individuals with deficiencies in Ab production, neutrophil function, or C3 share increased susceptibility to these organisms, including Streptococcus pneumoniae and Haemophilus influenzae. MBL deficiency is also associated with recurrent pyogenic infections in young children. In general, activation of complement by natural Ab or MBL results in C3b and iC3b deposition on these pathogens, overcoming the antiphagocytic effects of the capsule. Phagocytic cells ingest and kill the organisms using CD35, CD11b/CD18, and CD11c/CD18 receptors in conjunction with other innate and Fc receptors. C5aR signaling activates these receptors, leading to increased phagocytosis. Gram-negative bacteria are susceptible to complement-dependent lysis. This is evident in the increased incidence of disseminated neisserial infection in individuals deficient in C3, any of the MAC components, or P, as discussed below.

Complement in Inflammation An essential function of complement in host defense is the coordination of the local inflammatory response. C5a is the most potent complement product in this activity.39 Sublytic deposition of the MAC on endothelial cells and platelets and C3a interaction with the C3aR also contribute to the proinflammatory effects of complement activation. As discussed below, these potent inflammatory fragments of complement, when generated in high amounts or targeted inappropriately, result in many of the disease-related deleterious effects of complement. Local production of C5a at a site of infection occurs either through local complement activation or through direct cleavage of C5 by tissue macrophages or thrombin.37,38 This C5a is released and sets up a chemotactic gradient for neutrophils and macrophages. In addition, C5a activates endothelial cells to express P-selectin and synthesize chemokines, including interleukin-8 (IL-8). Interaction of C5a with mast cells releases vasoactive amines, increasing endothelial permeability. Neutrophils and macrophages are “primed” by interaction of C5a with its receptor. Priming includes enhancement of chemotaxis, activation of complement receptors for phagocytosis increased expression of activating FcγR, and assembly of the nicotinamide adenine dinucleotide phosphate (NADPH)–oxidase that is required for effective killing of microbes after phagocytosis. C5a also prevents neutrophil apoptosis, prolonging survival and contributing to local accumulation. Together, these actions result in the attraction and activation of potent antimicrobial cells and resolution of infection (Fig. 21.6).

307

Complement or protease activation

Limited local release of C5a

Neutrophil

Macrophage

Endothelial cell

Priming

↑ of activating ↓ of inhibitory FcγR

Expression of P-selectin

Activation of C receptors Assembly of oxidase

Chemokine (IL-8) and cytokine (IL-6) synthesis

Effective control of infection transmigration, phagocytosis, killing

FIG 21.6  C5a in Local Host Defense. Note that C5a can also be released by a direct protease event, such as by thrombin. IL-8, Interleukin 8.

defense against viruses is suggested by the multiple strategies used by viruses to evade complement.21,42 Several viruses produce complement regulatory proteins, including vaccinia virus complement control protein and herpes virus glycoprotein C, which facilitate breakdown of C3b and C4b. Some viruses, such as human immunodeficiency virus (HIV), incorporate complement regulatory proteins into the viral envelope, a strategy that is also used by other pathogens, such as Schistosoma.21,42,43 There are also many examples of complement receptors and membrane regulatory proteins being exploited as receptors for pathogens to invade cells. Examples of these include strategies of direct pathogen binding to receptors as well as deposition of C3 fragments followed by invasion through host C3 receptors.21

Role of Complement in Adaptive Immunity

Pathogen Evasion of Complement

Over the past 10 years there has been renewed interest in the role of the innate immune system in adaptive immune responses.1,8 The importance of complement in humoral immunity has been recognized since the observation that complement depletion of mice before immunization decreased Ab responses to thymusdependent antigens. Further studies have shown that complement receptors CR1 (CD35) and CR2 (CD21) are also required.33,34 In humans, these receptors are found together on B cells and FDCs. CD35 is also expressed on a number of other cell types (described above), including erythrocytes and phagocytic cells.

Further evidence of the host defense function of complement is the association of complement evasion strategies with virulence. Pathogenic gram-negative bacteria, such as Salmonella, have lipopolysaccharides with long O-polysaccharide side chains that promote rapid shedding of the MAC and prevent its insertion into the cell membrane. Neisseria species have several FH-binding components that help restrict AP activation and protect against lysis. Group A and B streptococci and S. pneumoniae have cell surface components (M protein, Bac or beta, PspC, Hic) that bind to FH and/or C4bp, restricting complement activation. Other organisms, including type 3 group B streptococci, elaborate sialic acid–containing capsules or cell walls to limit AP activation. Although complement deficiencies are not generally associated with viral infections, the importance of complement in host

Effects of Complement on the Humoral Immune Response Results obtained by experimental manipulation of C3, C4, and their receptors in mouse models indicate roles for these complement components at multiple levels in the humoral immune response.33,34 One caveat regarding these studies is that in the mouse CD35 and CD21 are alternative splice products of the same gene, and genetically deficient animals lack both receptors.34,44 In humans, CD35 and CD21 are encoded by separate genes. The first role of CD35/CD21 is in B-cell development, indicated by a pronounced defect in B-1-cell development in CD35/CD21–deficient mice. B-1 cells are generally found outside lymphoid follicles, have a restricted repertoire, and are essential in the production of natural Ab to pathogens, such as S.

308

Part two  Host Defense Mechanisms and Inflammation

KEY CONCEPTS Abuse by and Evasion Strategies of Complement by Pathogens: Some Examples 1. Bacteria Block C1, C3b deposition Streptococcus pneumoniae Block MAC access to plasma membrane Salmonella Limit access of C3b, iC3b to C receptors by capsule Streptococcus pneumoniae Haemophilus influenzae Block AP activation by sialylation Streptococcus agalactiae (GBS) type III, Neisseria Bind FH, C4BP to inhibit complement activation Streptococcus pneumoniae (Hic) Streptococcus pyogenes (GAS)(M protein) Neisseria Use CD55 (DAF), CD46 (MCP) for attachment to cells Streptococcus pyogenes (GAS)(M protein) Neisseria Escherichia coli Use complement receptors for entry Mycobacterium tuberculosis (CR3) Bacillus anthracis spores (CR3) 2. Viruses Express complement regulatory proteins homologous to those synthesized by the host Herpes simplex virus (glycoprotein C) Poxviruses (SPICE/VICE) Express unique complement regulatory proteins Flaviviruses (Dengue, West Nile) Use CD55 (DAF), CD46 (MCP) for attachment to cells Measles virus, adenovirus, herpes virus 6 (CD46) Picornaviruses, hantavirus (CD55) Use complement receptors for entry Epstein-Barr virus (CD21) Human immunodeficiency virus (CD35, CR3) 3. Parasites Express complement regulatory proteins Schistosoma (CRIT) Acquire complement regulatory proteins from host Schistosoma (CD55) Use complement receptors for entry Leishmania (CR1, CR3)

pneumoniae, and to self antigens exposed on damaged cells, such as phosphatidylcholine and DNA. Although the mechanism of this defect in CD35/CD21–deficient mice is not fully understood, these mice have an altered repertoire of natural Ab and B-1 cells.33,34,44 Decreased natural Ab may contribute to susceptibility to infection and autoimmune disease in hereditary complement deficiency (discussed below). A second role for complement in the Ab response is the well-described function of CD21 as a coreceptor for the mature B-cell response to antigen.33,34,44,45 As described above, CD21 is associated with the signaling complex of CD19 and CD81 (TAPA-1) in the B-cell membrane. Coligation of CD21 with the B-cell antigen receptor occurs naturally when the antigen activates complement and covalently binds C3dg. This coligation of the B-cell receptor with CD21 greatly decreases the threshold for B-cell activation and blocks Fas-initiated apoptosis of B cells. B cells activated by complement-opsonized antigen have increased ability to present antigen as well as survival and proliferation during encounters with T-dependent antigens.

The expression of CD35 and CD21 on FDCs is also important in the Ab response. FDCs trap antigen in the germinal centers and provide selection of somatically mutated high-affinity B-cell clones. Antigen trapped on FDCs also provides a source of long-term stimulation for maintenance of memory B cells. FDCs use complement receptors (CD35 and CD21) and FcγR to trap and retain antigen for these functions. Expression of CD21 on both FDC and B cells is required for effective affinity maturation of the Ab response and for the development and maintenance of memory B cells. Complement and T-Cell Activation Studies in primary pulmonary infection with influenza indicate that C3-deficient mice have a defect in influenza-specific CD4 and CD8 T-cell priming.45 CR1/2 deficiency had no effect. The mechanism is unknown but may be more efficient uptake and presentation of C3-opsonized virus by APC through CR3 and CR4 or stimulation of T-cell responses through the C3aR. Costimulation of human T cells in vitro through CD3 and CD46 leads to the development of T cells with a regulatory phenotype characterized by synthesis of IL-10 in the absence of other Th2 cytokines (IL-2, IL-4) (Chapter 9).46 The induction of regulatory T cells (Tregs) was seen in response to both anti-CD46 cross-linking and natural ligands (C3b dimers, streptococcal M protein). CD55-deficient mice showed enhanced T-cell responses to immunization and increased T cell–dependent autoimmune disease. These effects were complement dependent and apparently involve the loss of CD55 regulation of local complement synthesis by APCs during cognate interactions with T cells. One postulated mechanism is that CD55 inhibits the generation of C3a and C5a by APCs, preventing their interactions with C3aR and C5aR on T cells.47 Complement anaphylatoxins, C3a and C5a, have many important effects in inflammatory diseases that include attraction and activation of inflammatory cells, as well as regulation of APC and T-cell responses. Examples of these will be discussed in the sections below.

Role of Complement in Clearance of Apoptotic Cells Damaged tissue and dead and dying cells activate complement through several pathways. This can increase local inflammation and injury, as in I/R injury and hemolytic–uremic syndrome (HUS) (discussed below). Complement activation by apoptotic cells contributes to their opsonization and clearance and may prevent the development of autoimmunity. The deleterious consequences of complement activation following tissue damage are mainly attributable to AP-dependent generation of C5a and the MAC, whereas the beneficial effects are dependent on early CP components and innate recognition molecules.48,49 Necrosis, as occurs following ischemic tissue injury, exposes phospholipids and mitochondrial proteins that activate complement directly or indirectly. The pathways are different depending on the tissue involved.48 For example, renal reperfusion injury appears to be initiated by the AP, possibly secondary to the loss of regulatory proteins on tubular epithelial cells. Intestinal (I/R) injury is initiated by natural IgM Ab and requires both the CP for initiation and the AP for injury. MBL and CRP-initiated complement activation have been proposed to contribute to myocardial reperfusion injury after coronary artery ligation. Apoptotic cells are recognized by multiple receptors and opsonins.49,50 The association between early CP deficiencies and

CHAPTER 21  The Human Complement System: Basic Concepts and Clinical Relevance

309

COMPLEMENT DEFICIENCIES CR3

IgM iC3b C3b

Genetics and Incidence

CR1

CRP FcγR

C1q MBL

C1qR

SAP FcγR

TGF-β, IL-10

FIG 21.7  Pathways of Opsonization of Apoptotic Cells by Complement. Innate recognition of apoptotic cells by natural immunoglobulin M (IgM), cross-reactive protein (CRP), serum amyloid P (SAP), C1q, and mannose-binding lectin (MBL) is shown. Each reaction activates complement leading to opsonization by C3b and iC3b. In addition, C1q and MBL bind to collectin receptors, and CRP and SAP bind to FcγR on macrophages. Cytokine responses to apoptotic cells opsonized by complement include the antiinflammatory cytokines, transforming growth factor-β (TGF-β), and interleukin-10 (IL-10).

SLE (see below and Chapter 51) has been attributed to a failure of complement-dependent opsonization, resulting in accumulation of apoptotic cells and released autoantigens. Support for this hypothesis is provided by studies of mice deficient in C1q, IgM, or SAP, all of which develop autoantibodies against phospholipid and nuclear antigens characteristic of SLE, and by the therapeutic effect of CRP in mouse models of SLE.5 The role of complement in apoptotic cell recognition and uptake by macrophages is depicted in Fig. 21.7. MBL, C1q, and surfactant protein-D (SP-D) bind to apoptotic cells and facilitate clearance through direct binding to cellular receptors as well as complement activation.50 Natural IgM Ab, CRP, and SAP bind to phospholipids exposed on late apoptotic cells. All three proteins can also activate the CP generating C1q, C4b, C3b, and iC3b ligands for complement receptors. CRP and SAP also directly opsonize apoptotic cells for uptake through Fcγ receptors.51 Phagocytosis of apoptotic cells generally induces antiinflammatory cytokines transforming growth factor-β (TGF-β) and IL-10.52,53 Targeted Activation of Complement for Opsonization Interestingly, CRP and SAP also bind complement regulatory proteins, FH and C4bp, which helps limit complement activation to the deposition of opsonic components with little or no lysis or generation of C5a.53,54 This type of complement activation was also observed on acrosome-activated spermatozoa. In this case, the CP was activated by CRP from follicular fluid, resulting in bound C3b and iC3b, which are proposed to bind complement receptors on the egg and facilitate fertilization. Riley-Vargas et al. have proposed the acronym TRACS (targeted and restricted activation of the complement system) for this type of limited complement activation that occurs as part of normal processes, such as the acrosome reaction, and the recognition and removal of ischemic tissue and apoptotic cells.55

Complete genetic deficiencies of complement proteins are rare, with an estimated combined prevalence of 0.03% for any inherited complete deficiency (excluding MBL deficiency) in the general population.2-4,56-58 For most components, inheritance is autosomal and expression is codominant, so complete deficiency is homozygous recessive and heterozygotes express half levels. There are two C4 genes (C4A and C4B), so a range of partial deficiencies can be observed. All cases of C1-INH deficiency have been heterozygous, and P deficiency is X-linked. MBL is found in multiple allelic forms with different levels of expression ranging from 5 nanograms per milliliter (ng/mL) to more than 5 micrograms per milliliter (µg/mL) in plasma. Deficiencies specific to the LP are not detected by the screening assays described below but can be determined by specific assays.7 A 10% incidence of MBL deficiency and a single case of MASP-2 deficiency have been described. The most common clinical presentations of patients with complement deficiencies are recurrent infections with encapsulated bacteria, recurrent neisserial infections, and systemic autoimmune disease (Table 21.3). Populations with these disease manifestations have a much higher incidence of complement deficiency. For example, in Caucasian patients with SLE, the incidence of C2 deficiency is nearly 1%, 100-fold higher than in the general population. Screening of patients with autoimmune disease for complement deficiencies is useful, as these individuals are at higher risk for certain disease manifestations and may be at greater risk for infectious complications. Complement deficiency is found in as many as 20% of patients with recurrent disseminated neisserial infections. Evaluation of complement function is highly recommended in patients with recurrent or disseminated neisserial infections so that appropriate immunization and antibiotic prophylaxis can be initiated. Complement deficiencies are most readily detected by hemolytic screening assays (the CH50 and AH50), which determine the dilution of patient’s serum needed to lyse 50% of erythrocytes sensitive to the CP (CH50) or the AP (AH50).59 Deficiency of any C1 subcomponent, or any of the other CP components (C2–C8), will result in little or no lysis in the CH50 (CH50 values 1000 pg/ml Anemia and/or thrombocytopenia Hepatosplenomegaly Bone marrow cellularity >80% Spindle-shaped mast cells Myelofibrosis

Lymphocytic variant

PDGFRA -associated HES

Chronic eosinophilic leukemia

F/P positive by RT - PCR or FISH

Demonstrable cytogenetic abnormalities and/or blasts on peripheral smear

Familial

Clonal lymphocyte population by flow cytometry or PCR analysis of T-cell receptor usage

Undefined

Family history of documented persistent eosinophilia of unknown cause

Benign Asymptomatic with no evidence of organ involvement

Overlap*

EGID eosinophilic pneumonia, eosinophilia myalgia syndrome, and other organrestricted eosinophilic disorders

Associated**

CSS Systemic mastocytosis, inflammatory bowel disease, sarcoidosis, HIV, and other disorders

Complex

Episodic

Symptomatic with organ dysfunction but does not meet criteria for myeloproliferative or lymphocytic variant

Cyclical angioedema and eosinophilia

FIG 24.4  Classification of Hypereosinophilic Syndromes Based on a Workshop Summary Report. Specific syndromes discussed at the workshop are indicated in bold. *Incomplete criteria, apparent restriction to specific tissues/organs. †Peripheral eosinophilia, >1500/mm3 in association with a defined diagnosis. ‡ Presence of the FLPL1/PDGFRA (F/P) mutation. § Clonality analysis based on the digestion of genomic DNA with methylation-sensitive restriction enzymes followed by polymerase chain reaction (PCR) amplification of the CAG repeat at the human androgen receptor gene (HUMARA) locus at the X chromosome. CSS, Churg-Strauss syndrome (now called eosinophilic granulomatosis with polyangiitis); EGID, eosinophil gastrointestinal diseases; FISH, fluorescence in situ hybridization. (From Klion AD, Bochner BS, Gleich GJ, et al. Approaches to the treatment of hypereosinophilic syndromes: a workshop summary report. J Allergy Clin Immunol 2006; 117:1294, with permission from the American Academy of Allergy, Asthma and Immunology.)

Some patients with HES, termed myeloproliferative variants of HES, exhibit features common to myeloproliferative disorders, including elevated vitamin B12 and lactate dehydrogenase (LDH) levels, splenomegaly, cytogenetic abnormalities, myelofibrosis, anemia, myeloid dysplasia, and often elevated serum level of mast cell tryptase. In many patients with myeloproliferative HES, the molecular defect has been identified as a chromosome 4 deletion that yields a fusion gene encoding a FIP1LI/PDGFRA (PDGF-α receptor) (F/P) protein that constitutively expresses receptor kinase activity. This fusion gene can be diagnostically evaluated by reverse transcription–polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH) (Chapter 96). Importantly, the majority of patients with this fusion mutation, which constitutes a form of chronic eosinophilic leukemia (CEL), respond to therapy with imatinib, which is considered

the first line of therapy for FIP1LI/PDGFRA-positive HES.26 For patients with any evidence of cardiac involvement, including elevated troponin levels, it is recommended that glucocorticoids be administered along with initiation of imatinib therapy. Other patients with eosinophilia without F/P mutations have also responded to imatinib, indicating that other receptor tyrosine kinase mutations can underlie some of these CEL/myeloproliferative forms of HES.27 The presence of more than four myeloproliferative features commonly seen in mutation-positive disease, predicted response in those without known mutations. Some of these features included dysplastic eosinophils, vitamin B12 level >1000 picograms per milliliter (pg/mL), tryptase level ≥12 nanograms per milliliter (ng/mL), anemia/thrombocytopenia, hypercellular marrow, and spindled mast cells, reticulin fibrosis, and dysplastic megakaryocytes on bone marrow biopsy.28 In addition, clonal

358

Part two  Host Defense Mechanisms and Inflammation

abnormalities in the eosinophil lineage have been reported in a few patients (see Fig. 24.4). Another variant form of HES is a lymphoproliferative form resulting from clonal expansions of lymphocytes, often CD4+CD3− Th2-like lymphocytes, which elaborate IL-5.29 These aberrant T cells can be sought by flow cytometry or T-cell receptor (TCR) analysis. These patients, who may have elevated IgE levels, usually do not develop eosinophilic endomyocardial disease but are at risk for developing T-cell lymphomas.29 In addition to these recognized variants, there are a substantial number of patients with HES for whom the etiologies of the eosinophilia remain unknown.26 Some such patients develop no signs or symptoms of disease and can be monitored without therapy.30 For those who require therapy, including those with lymphoproliferative variants,29 glucocorticosteroids are the mainstay of treatment.26 With glucocorticoid therapy, partial or complete remission of eosinophilia within 1 month has been reported to occur in 85% of patients.26 Second-line agents include hydroxyurea and IFN-α.26 The neutralizing anti-IL-5 monoclonal antibody (mAb), mepolizumab, has beneficial and steroid-sparing effects in those with FIP1L1-PDGFRA negative hypereosinophilic syndromes,31 but it is approved by the US Food and Drug Administration (FDA) for treating severe eosinophilic phenotype asthma but not yet approved for hypereosinophilic syndromes. Anti-CD52 mAb (alemtuzumab) and allogeneic hematopoietic cell transplantation have been used for particularly severe and refractory HES. In contrast to older reports, with earlier diagnosis and therapy and with more varied and targeted therapeutic options, morbidity and, particularly, mortality in HES syndromes have been reduced.

Eosinophilia With Tumors or Leukemias The F/P-positive and related chromosomal fusion gene mediated myeloproliferative variants of HES are forms of CEL.27 Eosinophilia is a characteristic of the M4Eo subtype of acute myeloid leukemia, having the common M4 characteristic of chromosomal 16 abnormalities. Other forms of eosinophilic leukemia, often with specific cytogenetic and molecular genetic abnormalities, have been recognized.27 Eosinophilia may accompany chronic myelogenous leukemia (often with basophilia) but is uncommon with acute lymphoblastic leukemia. Eosinophilia may be observed in some patients with lymphoma, including Hodgkin disease, especially the nodular sclerosing form, T-cell lymphoblastic lymphoma, and adult T-cell leukemia/lymphoma. A small proportion of patients with carcinomas, especially of mucin-producing epithelial cell origin, have associated blood and tissue eosinophilia. Eosinophilia may accompany angioimmunoblastic lymphadenopathy, mycosis fungoides, Sézary syndrome, and lymphomatoid papulosis. Eosinophilia occurs in about 20% of patients with systemic mastocytosis and may be the presenting finding in the absence of cutaneous manifestations.

ORGAN SYSTEM INVOLVEMENT AND EOSINOPHILIA Eosinophilic syndromes limited to specific organs, such as eosinophilic pneumonias or eosinophilic GI disorders (EGIDs; Chapter 46), characteristically do not extend beyond their own target organ, and hence lack the multiplicity of organ involvement often found with non–organ-specific hypereosinophilic syndromes. They also do not have the predilection to develop

secondary eosinophil-mediated cardiac damage, for reasons that are not known.

Pulmonary Eosinophilias Blood eosinophilia can infrequently accompany pleural fluid eosinophilia, a nonspecific response seen with various disorders, including trauma and repeated thoracenteses. In addition, several pulmonary parenchymal disorders can be associated with eosinophilia (see Table 24.2).32 Helminth parasites are responsible for four forms of eosinophilic lung disease.24,32 The first form, Loeffler syndrome, is marked by blood eosinophilia, eosinophilic patchy pulmonary infiltrates that appear and resolve over weeks, and, at times, bronchospasm, and is typically caused by those helminth parasites (Ascaris lumbricoides/Ascaris suum), and less commonly hookworm and Strongyloides that migrate through the lungs early in their developmental lifecycle.24 Stool examinations are not helpful, as the pulmonary response is elicited by infecting larval forms months before productive egg-laying from later adult stages begins in the intestines. Diagnosis is made on epidemiological grounds.24 The second form of helminth-induced lung disease is the syndrome of tropical pulmonary eosinophilia, which develops in a minority of patients infected with lymphatic-dwelling filarial species.23 This syndrome is characterized by marked blood eosinophilia, a paroxysmal nonproductive cough, wheezing, occasional weight loss, lymphadenopathy, and low-grade fevers. On chest X-rays, increased bronchovesicular markings, diffuse interstitial lesions 1–3 mm in diameter or mottled opacities, usually more prominent in lower lung fields, are common. Patients have markedly increased numbers of blood and alveolar eosinophils, and elevations in both total serum IgE and antifilarial antibodies. A third form of helminth-induced lung disease is caused by helminths that invade the pulmonary parenchyma, notably lung flukes that cause paragonimiasis. The fourth form of lung disease is caused by larger than usual numbers of helminth organisms that are carried hematogenously into the lungs. Examples include schistosomiasis, trichinellosis, and larva migrans. Bronchopulmonary aspergillosis constitutes another type of eosinophil-associated pulmonary disease. Two forms of idiopathic eosinophilic pneumonia are recognized.32 In chronic eosinophilic pneumonia, patients may exhibit peripheral pulmonary infiltrates that can extend across lobar fissures. Chronic eosinophilic pneumonia is of unknown etiology and is responsive to corticosteroids but is prone to relapse. An acute form of eosinophilic pneumonia, which manifests as fever, pulmonary infiltrates, and respiratory insufficiency, is diagnosable by finding eosinophils in bronchoalveolar lavage (BAL) fluids or on lung biopsy. Acute eosinophilic pneumonia, which often follows new exposures to cigarette or other smoke or dusts, responds to corticosteroid treatment and does not relapse. The major vasculitis associated with eosinophilia is eosinophilic granulomatosis with polyangiitis (EGPA, formerly called ChurgStrauss syndrome) (Chapter 58).33 Late-onset asthma, eosinophilia, and at times transient pulmonary infiltrates antedate the development of systemic vasculitis in about half the cases. Pulmonary involvement is seen in almost all patients, and pulmonary infiltrates occur in three-quarters of them. Nasal and sinus involvement is common. Corticosteroids, anti-IgE mAb, or anticysteinyl leukotriene agent therapies for asthma may mask

CHAPTER 24  Eosinophils and Eosinophilia the evolution of EGPA. Neurological, cutaneous, cardiac, and GI organ involvement is common.33 Cardiac involvement includes pericarditis and small vessel cardiac vasculitis, and, much less commonly, endomyocardial thrombosis and fibrosis. Diverse drugs are capable of eliciting pulmonary eosinophilia. More commonly implicated medications include nonsteroidal antiinflammatory drugs (NSAIDs) and antimicrobial medications. Likewise, toxic agents, including those from occupational exposure, can be responsible for pulmonary eosinophilia. Each of these reactions has a defined etiological stimulus and hence differs from idiopathic and other eosinophilic diseases, but the clinical presentation of drug- and toxin-elicited pulmonary eosinophilias can resemble other forms of pulmonary eosinophilia, including acute or chronic eosinophilic pneumonia.

Skin and Subcutaneous Diseases A number of cutaneous diseases can be associated with heightened blood eosinophils,34 including atopic dermatitis, blistering disorders including bullous pemphigoid, drug reactions, and two diseases associated with pregnancy: (i) herpes gestationis and (ii) the syndrome of pruritic urticarial papules and plaques of pregnancy. Eosinophilic pustular folliculitis is seen mostly in patients with HIV infections and in those treated for hematological malignancies or after bone marrow transplantation. In patients with cutaneous involvement and eosinophilia, angiolymphoid hyperplasia with eosinophilia and Kimura disease, eosinophilic cellulitis (Wells syndrome), eosinophilic fasciitis, and eosinophilic pustular folliculitis can be differentiated on the basis of histopathology of biopsied lesions. Another syndrome, episodic angioedema with eosinophilia, is characterized by recurring episodes of angioedema, urticaria, fever, and marked blood eosinophilia. This syndrome responds to glucocorticosteroid therapy.

Gastrointestinal Diseases EGIDs (Chapter 46), including eosinophilic esophagitis, eosinophilic gastroenteritis, and eosinophilic colitis, represent a heterogeneous collection of disorders in which there may be eosinophilic infiltration of the mucosa, the muscle layer or the serosa, the last of which can lead to eosinophilic ascites. Peripheral blood eosinophilia may occur in EGIDs, although with eosinophilic esophagitis, peripheral blood eosinophil counts are often normal. Eosinophils are present in the lesions of collagenous colitis and ulcerative colitis, but blood eosinophilia is usually absent. GI eosinophilia elicited by intestinal helminths and eosinophilic enterocolitis as a result of hypersensitivity reactions to medications must be excluded in patients with these diseases who have tissue eosinophilia.

Rheumatological Disorders Of the various forms of vasculitis, only two are commonly associated with eosinophilia. The principal eosinophil-related vasculitis is EGPA, formerly called Churg-Strauss syndrome (as discussed above; and in Chapter 58). Cutaneous necrotizing eosinophilic vasculitis with hypocomplementemia and eosinophilia is a distinct vasculitis of small dermal vessels that are extensively infiltrated with eosinophils. This form of vasculitis may occur in patients with connective tissue diseases. In addition, eosinophilia may uncommonly accompany rheumatoid arthritis itself but more commonly results from adverse reactions to treatment medications (including NSAIDs, gold, and tetracyclines) or concomitant vasculitis.

359

Immunological Disorders CLINICAL PEARLS Eosinophilia and Drug Reactions Drug Reactions

Examples

Interstitial nephritis

Semisynthetic penicillins, cephalosporins Nitrofurantoin, sulfas, nonsteroidal antiinflammatory drugs (NSAIDs) Dantrolene Semisynthetic penicillins, tetracyclines Allopurinol, phenytoin Aspirin L-Tryptophan contaminant syndrome Ampicillin, penicillins, cephalosporins Granulocyte macrophage–colonystimulating factor (GM-CSF), interleukin (IL)-2 Minocycline, allopurinol, anticonvulsants

Pulmonary infiltrates Pleuropulmonary Hepatitis Hypersensitivity vasculitis Asthma, nasal polyps Eosinophilia–myalgia Asymptomatic Cytokine-mediated

DRESS (drug reaction with eosinophilia and systemic symptoms)

Adapted from Weller PF. Eosinophilia and eosinophil-related disorders. In: Adkinson NF, Jr., Yunginger JW, Busse WW, et al., eds. Allergy: Principles and Practice, 6th ed. Philadelphia, Penn.: Mosby; 2003:1105.

Adverse reactions to medications are a common cause of eosinophilia. Although often considered as hypersensitivity reactions, in most instances of drug-associated eosinophilia, the mechanism leading to eosinophilia is not understood. Eosinophilia may develop without other manifestations of adverse drug reactions, such as rashes or drug fevers. In addition, drug-induced eosinophilia may be associated with distinct clinicopathological patterns in which eosinophilia accompanies drug-induced diseases that are characteristically limited to specific organs with or without associated blood eosinophilia. When organ dysfunction develops, cessation of drug administration is necessary. Drug-induced interstitial nephritis may be accompanied by blood eosinophilia, and eosinophils may be detectable in urine. Unlike granulocyte–colony-stimulating factor (G-CSF) therapy, therapy with GM-CSF can lead to prominent blood and tissue eosinophilia. Administration of IL-2 or of IL-2-stimulated lymphocytes can be followed by the development of eosinophilia, most likely as a result of stimulated production of IL-5. Reactions to medications, often anticonvulsants, minocycline, and allopurinol, can elicit DRESS (drug reaction with eosinophilia and systemic symptoms).34 In addition to cutaneous eruptions, fever, lymphadenopathy, hepatitis, nephritis, atypical lymphocytosis, GI tract involvement, and eosinophilia are common but variable elements of this drug-induced syndrome, which can be fatal. The triggering medication must be stopped, and corticosteroids are often administered. Some primary immunodeficiency syndromes are associated with eosinophilia.35 Hyper-IgE syndrome is characterized by recurrent staphylococcal abscesses of the skin, lungs, and other sites; pruritic dermatitis; hyperimmunoglobulinemia E; and eosinophilia of blood, sputum, and tissues. Eosinophilia is characteristic of Omenn syndrome, combined immunodeficiency with hypereosinophilia (Chapter 35). Infiltration of eosinophils accompanies rejection of lung, kidney, and liver allografts. Tissue and blood eosinophilia occur early in the rejection process, and eosinophil counts and

360

Part two  Host Defense Mechanisms and Inflammation

eosinophil granule protein levels (in urine, BAL fluids, and involved allograft tissues) have correlated with prognosis, severity, and response to rejection therapy.

Endocrine Diseases The loss of endogenous adrenoglucocorticosteroid production in Addison disease, adrenal hemorrhage, or hypopituitarism can cause increased blood eosinophilia, although usually not more than mild to moderate.

Other Causes of Eosinophilia The syndrome of atheromatous cholesterol embolization is at times associated with hypocomplementemia, eosinophilia, and eosinophiluria. Rarely, cases of hereditary eosinophilia among family members have been recognized. Irritation of serosal surfaces can be associated with eosinophilia, and related diseases can include Dressler syndrome; eosinophilic pleural effusions; peritoneal and, at times, blood eosinophilia developing during chronic peritoneal dialysis; and perhaps the eosinophilia that follows abdominal irradiation.

THERAPEUTIC PRINCIPLES Therapy of Specific Eosinophilic Diseases Eosinophil-Associated Diseases With Identifiable Etiologies Parasitic infections Treat causative parasite Drug-reaction related Terminate eliciting medication eosinophilias Adrenal insufficiency Corticosteroid replacement therapy Allergic/atopic diseases Varied, may include topical or inhaled corticosteroids Distinct Eosinophilic Syndromes Involving Specific Organs Eosinophilic pulmonary diseases: Acute eosinophilic pneumonia Corticosteroids Chronic eosinophilic pneumonia Corticosteroids, interferon-α Eosinophilic granulomatosis with Corticosteroids, interferon-α polyangiitis Hypereosinophilic Syndromes F/P-positive myeloproliferative Imatinib Lymphoproliferative and other Corticosteroids, interferon-α, hydroxyurea, anti–interleukin (IL)-5 monoclonal antibody (mAb), other

EVALUATION OF EOSINOPHILIA Because a diversity of disorders can be accompanied by eosinophilia, evaluation of the patient with eosinophilia requires a consideration of features based on the patient’s history, physical examination, and other laboratory, radiographic, or diagnostic tests.26,36,37 An initial approach can focus on identifying eosinophilic diseases that have a defined treatable etiology. These include infections with helminth parasites, and for these, the approach should be guided by information obtained from patient history about potential exposures; results of the history and physical examinations with regard to signs and symptoms of any clinically apparent associated illness; results of standard biochemical and radiographic tests for evidence of organ involvement; and results of specific parasitological tests, including potentially stool, urine,

blood, sputum, or tissue examinations, as well as results of serological tests.24 The duration and magnitude of the eosinophilia may suggest some entities, especially if it is prolonged or markedly elevated (see Table 24.1). Other causes of eosinophilia that are amenable to treatment include eosinophilia secondary to medications, for which cessation of the offending drug may be indicated if the eosinophilia is accompanied by organ damage. Likewise, if eosinophilia is secondary to glucocorticosteroid deficiency, diagnostic testing can corroborate this deficiency and lead to the administration of replacement corticosteroids and consequent resolution of the eosinophilia. Because allergic diseases usually are associated with at least some degree of eosinophilia, clinical and laboratory evidence of such disease should be sought. If the eosinophilia is not attributable to allergic diseases, parasitic infections, medications, or steroid deficiency, further evaluation will be guided by whether the patient has evidence of organ disease and, if so, which organs are involved (see Table 24.2). This is germane, for instance, in defining whether the patient has a distinct eosinophilic pulmonary, GI, or cutaneous syndrome. Bone marrow examinations in most patients with eosinophilia are not usually informative, revealing only evidence of enhanced eosinophilopoiesis; but bone marrow should be examined if there is suspicion of a hematological malignancy or myeloproliferative disorder. For patients with sustained eosinophilia who meet the criteria for HES, diagnostic testing should aim to identify which variant form of HES the patient may have, in which case, bone marrow examination is often needed (see Fig. 24.4).

ON THE HORIZON Identify the causes of those hypereosinophilic syndromes for which etiologies currently remain unknown. Delineation of the signaling mechanisms that govern the agonist-specific, differential secretion of cytokines that are preformed and stored within eosinophil granules and secretory vesicles. Continue to evaluate the therapeutic efficacies of anticytokine therapeutics, including anti–interleukin (IL)-5 neutralizing antibodies, in the treatment of the varied forms of eosinophilic diseases. Identify biomarkers that are predictive of eosinophil-mediated tissue damage and that can be used for clinical diagnostic and therapeutic monitoring tools.

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Blanchard C, Rothenberg ME. Biology of the eosinophil. Adv Immunol 2009;101:81–121. 2. Rosenberg HF, Dyer KD, Foster PS. Eosinophils: changing perspectives in health and disease. Nat Rev Immunol 2013;13:9–22. 3. Spencer LA, Weller PF. Eosinophils and Th2 immunity: contemporary insights. Immunol Cell Biol 2010;88:250–6. 4. Shamri R, Xenakis JJ, Spencer LA. Eosinophils in innate immunity: an evolving story. Cell Tissue Res 2011;343:57–83. 5. Nussbaum JC, Van Dyken SJ, von Moltke J, et al. Type 2 innate lymphoid cells control eosinophil homeostasis. Nature 2013;502:245–8. 6. Bochner BS. Siglec-8 on human eosinophils and mast cells, and Siglec-F on murine eosinophils, are functionally related inhibitory receptors. Clin Exp Allergy 2009;39:317–24. 7. Bozza PT, Magalhaes KG, Weller PF. Leukocyte lipid bodies—biogenesis and functions in inflammation. Biochim Biophys Acta 2009;1791:540–51.

CHAPTER 24  Eosinophils and Eosinophilia 8. Melo RCN, Perez SAC, Spencer LA, et al. Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils. Traffic 2005;6:866–79. 9. Hogan SP, Rosenberg HF, Moqbel R, et al. Eosinophils: biological properties and role in health and disease. Clin Exp Allergy 2008;38:709–50. 10. Spencer LA, Szela CT, Perez SA, et al. Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially. J Leukoc Biol 2009;85:117–23. 11. Melo RC, Weller PF. Piecemeal degranulation in human eosinophils: a distinct secretion mechanism underlying inflammatory responses. Histol Histopathol 2010;25:1341–54. 12. Spencer LA, Melo RCN, Perez SAC, et al. Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion. Proc Natl Acad Sci USA 2006;103:3333–8. 13. Neves JS, Perez SA, Spencer LA, et al. Eosinophil granules function extracellularly as receptor-mediated secretory organelles. Proc Natl Acad Sci USA 2008;105:18478–83. 14. Neves JS, Weller PF. Functional extracellular eosinophil granules: novel implications in eosinophil immunobiology. Curr Opin Immunol 2009;21:694–9. 15. Ueki S, Melo RC, Ghiran I, et al. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans. Blood 2013;121:2074–83. 16. Klion AD, Nutman TB. The role of eosinophils in host defense against helminth parasites. J Allergy Clin Immunol 2004;113:30–7. 17. Swartz JM, Dyer KD, Cheever AW, et al. Schistosoma mansoni infection in eosinophil lineage-ablated mice. Blood 2006;108:2420–7. 18. Padigel UM, Hess JA, Lee JJ, et al. Eosinophils act as antigen presenting cells to induce immunity to Strongyloides stercoralis in mice. J Infect Dis 2007;196:1844–51. 19. Fabre V, Beiting DP, Bliss SK, et al. Eosinophil deficiency compromises parasite survival in chronic nematode infection. J Immunol 2009;182:1577–83. 20. Akuthota P, Wang HB, Spencer LA, et al. Immunoregulatory roles of eosinophils: a new look at a familiar cell. Clin Exp Allergy 2008;38:1254–63. 21. Wang HB, Ghiran I, Matthaei K, et al. Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells. J Immunol 2007;179:7585–92. 22. Chu VT, Frohlich A, Steinhauser G, et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat Immunol 2011;12:151–9.

361

23. Wu D, Molofsky AB, Liang HE, et al. Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science 2011;332:243–7. 24. Wilson ME, Weller PF. Eosinophilia. In: Guerrant RL, Walker DH, Weller PF, editors. Tropical infectious diseases: principles, pathogens and practice. 3rd ed. Philadelphia, Penn.: Elsevier; 2011. p. 939–49. 25. Klion AD, Bochner BS, Gleich GJ, et al. Approaches to the treatment of hypereosinophilic syndromes: a workshop summary report. J Allergy Clin Immunol 2006;117:1292–302. 26. Ogbogu PU, Bochner BS, Butterfield JH, et al. Hypereosinophilic syndrome: a multicenter, retrospective analysis of clinical characteristics and response to therapy. J Allergy Clin Immunol 2009;124:1319–25. 27. Valent P, Gleich GJ, Reiter A, et al. Pathogenesis and classification of eosinophil disorders: a review of recent developments in the field. Expert Rev Hematol 2012;5:157–76. 28. Khoury P, Desmond R, Pabon A, et al. Clinical features predict responsiveness to imatinib in platelet derived growth factor receptor alpha-negative hypereosinophilic syndrome. Allergy 2016;71:803–10. 29. Lefevre G, Copin MC, Staumont-Salle D, et al. The lymphoid variant of hypereosinophilic syndrome: study of 21 patients with CD3-CD4+ aberrant T-cell phenotype. Medicine (Baltimore) 2014;93:255–66. 30. Chen YY, Khoury P, Ware JM, et al. Marked and persistent eosinophilia in the absence of clinical manifestations. J Allergy Clin Immunol 2014;133:1195–202. 31. Rothenberg ME, Klion AD, Roufosse FE, et al. Treatment of patients with the hypereosinophilic syndrome with mepolizumab. N Engl J Med 2008;358:1215–28. 32. Akuthota P, Weller PF. Eosinophilic pneumonias. Clin Microbiol Rev 2012;25:649–60. 33. Comarmond C, Pagnoux C, Khellaf M, et al. Eosinophilic granulomatosis with polyangiitis (Churg-Strauss): clinical characteristics and long-term follow-up of the 383 patients enrolled in the French Vasculitis Study Group cohort. Arthritis Rheum 2013;65:270–81. 34. Long H, Zhang G, Wang L, et al. Eosinophilic skin diseases: a comprehensive review. Clin Rev Allergy Immunol 2016;50:189–213. 35. Ben m’rad M, Leclerc-Mercier S, Blanche P, et al. Drug-induced hypersensitivity syndrome: clinical and biologic disease patterns in 24 patients. Medicine (Baltimore) 2009;88:131–40. 36. Williams KW, Milner JD, Freeman AF. Eosinophilia associated with disorders of immune deficiency or immune dysregulation. Immunol Allergy Clin North Am 2015;35:523–44. 37. Klion A. Hypereosinophilic syndrome: current approach to diagnosis and treatment. Annu Rev Med 2009;60:293–306.

CHAPTER 24  Eosinophils and Eosinophilia

361.e1

MULTIPLE-CHOICE QUESTIONS 1. Which of the following may NOT contribute to or elicit eosinophilia? A. CD3−, CD4+ aberrant lymphocyte subsets B. FIP1L1-PDGFRα chromosomal gene rearrangement C. Giardia lamblia infection D. Strongyloides stercoralis infection 2. A 45-year-old male who has had eosinophilia of between 2500 and 5000/ mm3 documented for the past 6 months has been referred to you. Your evaluation could include: A. Flow cytometry for lymphocyte phenotyping B. Serum immunoglobulin E (IgE) C. Serum troponin D. Strongyloides enzyme-linked immunosorbent assay (ELISA) serology E. All of the above

3. Which three cytokines promote eosinophil development in bone marrow and sustain eosinophil viability? A. Transforming growth factor (TGF)-β, interleukin (IL)-2, IL-4 B. IL-7, IL-2, IL-13 C. Granulocyte macrophage–colony-stimulating factor (GMCSF), IL-3, IL-5 D. Interferon (INF)-γ, IL-8, IL-10

25  Host Defenses to Viruses Barry T. Rouse, Scott N. Mueller

Viruses as obligate intracellular parasites require their host to replicate them and to facilitate their spread to others. In humans, most clinically relevant infections were derived from other animals, and this process continues. Recent examples include human immunodeficiency virus (HIV), Ebola virus, severe acute respiratory syndrome (SARS) virus, and Zika virus. Viral infections are rarely lethal, even if they are highly cytolytic to individual cells. Mortality commonly occurs when viruses jump species, when the virus undergoes a major antigenic change (i.e., influenza viruses), or when host immunity is compromised. HIV (Chapter 39) represents one of the most dramatic human examples of an exotic virus that kills its host. However, HIV kills slowly, providing ample time to spread to new hosts and an effective strategy for persistence in the species. Death or dire consequences following virus infection in mammals with inadequate immunity are well illustrated by observations that fetuses or neonates, especially if deprived of passive immunity, succumb to many agents well tolerated by healthy adults. The science of viral immunology seeks to understand mechanisms of virus–host interactions with a view to applying this knowledge to the design of effective vaccines and immunomodulators that control virus infections. These objectives are facilitated by an increasing wealth of immunological techniques, an expanding array of genetically manipulated animal models, and an abundance of high throughput technologies, which generate data that can be subjected to complex computational analysis. Such analyses can yield signatures indicative of optimal immunogenicity and vaccine efficacy or failure and can explain the variable outcome of infections in individual hosts. In most situations, defense against viruses involves multiple immune components, and the impact of a single mechanism varies greatly according to the method by which individual viruses enter, replicate, and spread within the host. In this chapter, we highlight the principal means by which the host achieves immunity after infection by viruses. Table 25.1 presents an overview.

VIRAL ENTRY AND INFECTION Access to target tissues presents numerous obstacles for entry and infection by most human viruses. Most effective of these are the mechanical barriers provided by skin and the mucosal surfaces, as well as the chemically hostile environment of the gut (Fig. 25.1). A number of common human viral pathogens enter through the gastrointestinal tract, including rotavirus, enteric adenoviruses, and hepatitis A virus (HAV). These are usually spread via person-to-person contact or contaminated food and water. Respiratory infections caused by influenza viruses,

rhinoviruses, coronaviruses, measles virus, varicella-zoster virus (VZV), and respiratory syncytial virus (RSV) are often spread by aerosol transmission, as well as person-to-person contact. Many of the herpes viruses target the skin or the mucosae, such as herpes simplex virus (HSV) and VZV. HSV, in particular, can infect the oral and genital mucosae, the eye, and skin through small cuts and abrasions. Other herpes viruses, such as EpsteinBarr virus (EBV) and cytomegalovirus (CMV), target mucosae. CMV can also spread vertically from mother to baby or rarely via blood transfusions. Human papillomavirus (HPV) targets skin and mucosae and causes warts and may transform cells, inducing cancers, such as cervical cancer. Some viruses, such as West Nile virus, Dengue virus, Semliki forest virus, and Zika virus, can enter through skin via insect vectors. HIV and hepatitis B virus (HBV) are commonly spread via sexual contact. HIV, HBV, and hepatitis C virus (HCV) can also infect humans by direct entry into the bloodstream via transfusions or contaminated needles. Most human viruses replicate only in certain target tissues, this being mainly the consequence of viral receptor distribution. Many viruses use two receptors, such as the use of the CD4 coreceptor and the chemokine receptor CCR5 on T cells by HIV. After attachment to a cellular receptor, viruses may fuse with the cell membrane or be endocytosed and then gain entry into the cytoplasm or nucleus by fusing with the vesicular membrane (enveloped viruses, such as HSV and HIV), or translocate across the cell membrane or induce lysis of the endocytic vesicle once in the cytoplasm (nonenveloped viruses, such as Norwalk virus and poliovirus).1 Viruses then utilize host cell machinery and specialized virally encoded proteins to replicate rapidly within the cell. Once they have multiplied within the cell, many viruses induce cytolysis to facilitate release of new infectious virions (e.g., poxviruses, poliovirus, and herpes viruses). Other viruses are released from infected cells by budding through the cell membrane in the absence of cell death (e.g., HIV and influenza virus). Having entered the body, however, viruses encounter numerous innate defenses and activate the components of adaptive immunity. The latter usually assures that clinical disease, if not infection, will not become evident. Successful exploitation of these defenses through the use of vaccines (Chapter 90) remains a central challenge for many human viruses, particularly those that cause chronic infections, such as HIV and HCV.2

INNATE IMMUNITY TO VIRUSES Viral infection induces an extensive array of defense mechanisms in the host. Innate defenses come into play to block or inhibit

365

366

Part THREE  Host Defenses to Infectious Agents

TABLE 25.1  Viral Infections and Immunity

TABLE 25.2  Sensors of Viral Infection

Viral Event

Obstacles

Time Course

Toll-Like Receptors (TLRs)

Transmission

Mechanical and chemical barriers Innate immunity

0

Infection and replication Infection stopped or spreads Infection controlled Sterile immunity Viral persistence if infection not controlled

Viral antigens transported to lymphoid tissues Specific antibodies and cell-mediated immunity Immune memory Immune disruption or evasion

0→ Within 24 hours 4–10 days 14 days to years Weeks to years

TLR3 TLR7 and TLR8 TLR9 TLR2 TLR4

RIG-I-Like Helicases (RLHs) RIG-I MDA-5

Respiratory tract • Influenza virus • RSV • Rhinoviruses • Coronaviruses • Adenoviruses parainfluenza virus • VZV • Measles virus Skin entry and infection • HSV • Human papillomavirus • West Nile virus

Influenza virus, VSV, HCV, JEV, MV, RSV, Sendai virus, EBV Poly(I:C), MV, Sendai virus, VSV, MCMV, Picornaviruses

NOD-Like Receptors (NLRs) NLRP3

Ocular infection • HSV • Adenoviruses

dsRNA, MCMV, VSV, LCMV, HSV, EBV ssRNA, Influenza virus, HIV, VSV dsDNA, HSV, MCMV MV hemagglutinin protein, HSV, HCMV MMTV envelope protein, RSV

Gastrointestinal tract • Rotavirus • Adenoviruses • Hepatitis A virus • Caliciviruses

Genitourinary tract • HSV • HIV • HBV • CMV • Human papillomavirus

FIG 25.1  Common Routes of Entry and Infection for Human Viral Pathogens. CMV, cytomegalovirus; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HSV, herpes simplex virus; RSV, respiratory syncytial virus; VZV, varicella-zoster virus.

initial infection, to protect cells from infection, or to eliminate virus-infected cells. Innate mechanisms occur well before the effectors of adaptive immunity become active, but they are critical for the initiation of adaptive immunity via the elicitation of inflammation that promotes immune cell activation. The innate immune defenses are initiated via pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs)3 (Chapter 3). These include transmembrane receptors of the Toll-like receptor (TLR) family, two families of intracellular receptors including the NOD-like receptors (NLRs) and the RIG-I–like helicases (RLHs), as well as the sensor molecule absent in melanoma-2 (AIM2). Additionally, the molecules cyclic guanosine monophosphate–adenosine monophosphate (GMP-AMP) synthase (cGAS), DDX41, IFI16, and Z-DNA–binding protein 1 (ZBP1) can sense cytosolic DNA (Table 25.2). These cellular sensors promote the expression of interleukin-1 (IL-1) and IL-18, type I (α/β) interferon (IFN-I), and a variety of IFNstimulated genes and inflammatory cytokines, and chemokines. TLRs are cell surface or endosomal membrane–bound proteins expressed by numerous cells, including dendritic cells (DCs), macrophages, lymphocytes, and parenchymal cells. Expression of TLRs is largely inducible in most cell types, although some (TLR7/8/9) are constitutively expressed at high levels by specialized plasmacytoid DCs for rapid IFN production. Different TLR molecules recognize specific viral products, such as single- and

NOD2 Other sensors AIM2 ZBP1 (DAI) IFI16 cGAS

Influenza virus, Sendai virus, Adenovirus, Vaccinia virus Influenza virus, VSV, RSV Vaccinia virus, MCMV Cytosolic dsDNA, HSV Cytosolic dsDNA, HSV Cytosolic dsDNA, HSV

AIM2, absent in melanoma-2; IFI16, Gamma-interferon-inducible protein Ifi-16; cGAS, cyclic GMP-AMP Synthase; ZBP1, Z-DNA-binding protein 1; DAI, DNA-dependent activator of IFN; dsRNA, double-strand RNA; EBV, Epstein-Barr virus; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HSV, herpes simplex virus 1/2; JEV, Japanese encephalitis virus; LCMV, lymphocytic choriomeningitis virus; MCMV, murine cytomegalovirus; MDA-5, melanoma differentiation-associated gene; MMTV, mouse mammary tumor virus; MV, measles virus; NLR, NOD-like receptor; RLH, RIG-I-like helicase; RSV, respiratory syncytial virus; ssRNA, single-strand RNA; TLR, Toll-like receptor; VSV, vesicular stomatitis virus.

double-stranded RNA (TLR 3 and TLR7/8, respectively) or double-stranded DNA (TLR9). The RLHs retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene (MDA-5) mediate cytoplasmic recognition of viral nucleic acids. These activate mitochondrial antiviral signaling (MAVS) proteins to stimulate IFN-I production and activate inflammasomes, which are molecular complexes that facilitate the activation of caspases and induce the production of proinflammatory IL-1β and IL-18.4 NLRs are a second class of cytosolic sensors of PAMPs that activate inflammasomes via the adapter protein ASC. These include the NLRP (or NALP), NOD, and IPAF/NAIP receptors. Three major inflammasomes have been shown to be involved in antiviral immunity: the NLRP3 inflammasome, the RIG-I inflammasome, and the AIM2 inflammasome.3 The innate defense system consists of multiple cellular components and many specialized proteins. The longest known and best-studied antiviral proteins are the α/β IFNs, which act by binding to the type I IFN receptor and result in the transcription of more than 100 IFN-stimulated genes. One consequence of this “antiviral state” is the inhibition of cell protein synthesis and the prevention of viral replication.5 Multiple leukocyte subsets are involved in innate defense, including macrophages, DCs, neutrophils, natural killer (NK) cells, natural killer T cells (NKT cells), and γδT cells. Furthermore, tissue cells, including fibroblasts, epithelial cells, and endothelial cells, express PRRs and respond to viral infection via the production of innate cytokines, including IFN-I and IL-1. IFN-I is a critical link between the innate and adaptive immune system, via activation of DCs and T cells, as well as protecting T cells from NK cell-mediated attack.6 IFN-Is can also activate NK cells and induce other cytokines that promote

CHAPTER 25  Host Defenses to Viruses KEY CONCEPTS Major Antiviral Innate Defense Mechanisms Acting to block infection: Natural antibodies Complement components Some cytokines and chemokines Acting to protect cells from infection: Interferon-α/β Interferon-γ (IL-γ) IL-1, IL-18 Acting to destroy or inhibit virus-infected cells: Natural killer (NK) cells Natural killer T cells (NKT cells) Macrophages Neutrophils γδ T cells Nitric oxide Involved in regulating antiviral inflammatory response: ILs-1, 6, 10, 12, 18, 23, 33 Transforming growth factor (TGF)-β Chemokines (CCL2, 3, 4, 5)

NK responses, such as IFN-γ and IL-12. NK cells produce proinflammatory cytokines; they can kill infected cells and interact with DCs, and are an important component of innate defense against viruses. NK cells can protect against some herpes viruses, which downregulate major histocompatibility complex (MHC) expression in the cells they infect. NK cells are also important in resistance to mouse and human CMV and possibly to HIV, influenza virus, and Ebola virus.7 NK cells have also recently been shown to possess traits of adaptive immunity and, like T and B cells, can form populations of memory cells.8 NK cells are regulated by an array of activating and inhibitory receptors, whose expression and function are just beginning to be understood. Uninfected cells are usually protected from NK cell cytolysis as they deliver negative signals, such as high expression of MHC molecules. In contrast, virus-infected cells are killed either because they deliver positive signals or because they lack adequate MHCnegative signals. NK cells may also control excessive immune responses to viruses by killing CD4+ T cells and indirectly regulating cytotoxic T lymphocyte (CTL) responses. NKT cells may provide some antigen-specific innate immune protection against certain viruses, such as influenza virus.9 Several classes of innate host proteins function in antiviral defense. These include natural antibodies, which may play a role in defense against some viral infections, as well as pentraxins and complement proteins.10 Some viruses may be directly inactivated by complement activation or be destroyed by phagocytic cells that bind and ingest complement-bound virions. Several proinflammatory cytokines and chemokines induced by virus infection also play key roles in defense. Foremost among these is IL-1 and other members of the IL-1 family, including IL-18 and IL-33.11 These cytokines influence both innate and adaptive immune cells and play critical roles in antiviral defense. Other antiviral cytokines are produced early following infection, such as TNF-α, IFN-γ, IL-12, IL-6, and chemokines, such as MIP-1α. In particular, IL-12 is a potent inducer of IFN-γ from NK cells. Inflammatory chemokines may also play an important role in innate antiviral defense by orchestrating macrophage, neutrophil, DC, and NK cell responses at the site of infection. Not only are these components of innate immunity involved in mediating initial protection against viruses; several components (e.g., the

367

PRRs; the cytokines IFN-I, IL-I, IL-33, and IL-12; and phagocytes, including macrophages, monocytes, and DCs) serve to shape the nature and effectiveness of the subsequent adaptive response to viral pathogens. For instance, DCs require innate signals, such as IFN-I and IL-12, for maturation and optimal T-cell activation. Furthermore, CD8+ T cells responding to viruses need IFN-I and IL-33 signals for expansion and memory formation. Thus both the magnitude and the type of innate response induced by virus infection have a marked influence on the generation of adaptive immune responses.

ADAPTIVE IMMUNITY TO VIRUSES Innate immunity generally only slows, rather than stops, viral infection, allowing time for the adaptive immune response to begin. The two major divisions of adaptive immunity, antibodymediated and T-cell–mediated, are mainly directed at different targets. Antibodies usually function by binding to free viral particles and, in so doing, block infection of the host cell (Chapter 15). In contrast, T cells act principally by recognizing and destroying virus-infected cells or by orchestrating an inflammatory response that includes several antiviral components (Chapters 16, 17). As all viruses replicate within cells and many can spread directly between cells without reentering the extracellular environment, resolution of infection is reliant more on T-cell function than on antibody function. However, broadly neutralizing antiviral antibodies have the potential to be effective therapies against many different human infections, including HIV, influenza viruses, and Ebola virus. Recent advances have allowed researchers to isolate and identify human monoclonal antibodies (mAbs) against these and other pathogens,12 offering promise of new therapies as well as significant insight for vaccine design. Antiviral antibodies are also very important as an immunoprotective barrier against reinfection. It is the presence of antibodies at portals of entry— most often mucosal surfaces—that is of particular relevance to influenza, HSV, and HIV infections.13 Yet, how to generate vaccines that induce optimal antibody responses, including broadly neutralizing antibodies, remains an important unsolved problem. Initiation of adaptive immunity is closely dependent on early innate mechanisms that activate antigen-presenting cells (APCs), principally subsets of DCs. APCs and lymphocytes are drawn into lymphoid tissues by chemokine and cytokine signals and are retained there for a few days to facilitate effective intercellular interactions. The architecture of the secondary lymphoid tissues supports the coordinated interactions among the cells of the adaptive immune system14 through a network of supportive stromal cells and local chemokine gradients15(Chapter 2). The induction events occur in lymph nodes draining an infection site or in the spleen if virus enters the bloodstream. The passage of viral antigens to lymph nodes usually occurs in DCs. Some viruses are able to compromise the function of APCs, such as HSV and measles virus, which can inhibit DC maturation. B-cell activation occurs following antigen encounter in the B-cell follicles, and possibly the T-cell zones, in the spleen or lymph nodes. Some activated B cells become short-lived plasma cells, whereas others move to the edges of the B-cell follicles and interact with antigen-specific helper CD4+ T cells via presentation of antigenic peptides on B-cell MHC class II molecules. These Bcl6-dependent CD4 T follicular helper (Tfh) cells are specialized for providing help for B-cell responses and are needed to promote and regulate B-cell responses.16 Activated B cells initiate germinal center (GC) reactions with the help of CD4 Tfh cells, ensuring

Part THREE  Host Defenses to Infectious Agents

somatic hypermutation and affinity maturation for the selection of high-affinity, antibody-producing, long-lived plasma cells, as well as memory B cells.17 At the molecular level, upregulation of the transcription factors Blimp-1, XBP-1, and IRF-4 dictates plasma cell formation, whereas Pax-5 expression delineates B cells destined for GC reactions and the memory B-cell lineage. Antibody binding to epitopes expressed by native proteins at the surface of free virions usually blocks viral attachment or penetration of target cells. Sometimes the consequence is viral lysis (with complement proteins also involved), opsonization, or sensitization for destruction by Fc receptor–bearing cells that mediate antibody-dependent cellular cytotoxicity (ADCC). Occasionally, however, Fc receptor binding of antibody-bound virus may facilitate infection and result in more severe tissue damage. This occurs in Dengue fever and may happen in some instances in HIV infection. The antibody involved in the protection of mucosal surfaces in humans is predominantly secretory immunoglobulin A (IgA), but serum-derived IgG may also be protective, particularly in such sites as the vaginal mucosa.13 Both antibody isotypes act mainly to block infection of epithelial cells, although in some instances, the antibody may transport antigen from within the body across epithelial cells to the outside. Mucosal antibody persists for a much shorter period compared with serum antibody, which explains, in part, why immunity to mucosal pathogens is usually of much shorter duration compared with immunity to systemic viral infections.

Primary infection

Time A Naive

CD62Lhi CCR7hi CD69lo

B

KEY CONCEPTS Antiviral T- and B-Cell Immunity Effector Systems

Recognized Molecules

Antibody

Surface proteins or virions

Antibody + complement

Surface proteins expressed on infected cells

Mucosal antibody (IgA) CD4+ T cells

Surface proteins or virions

CD8+ T cells

Viral peptides (10–20 mers) presented on MHC class II surface, internal or nonstructural proteins presented by APCs Viral peptides (8–10 mers) presented on MHC class I surface, internal, or nonstructural proteins presented on infected cells or by cross-presentation

Control Mechanisms Neutralization of virus, opsonization, or destruction of infected cells by ADCC Infected cell destruction by ADCC or complement-mediated lysis Viral neutralization, opsonization, and transcytosis Antiviral cytokine and chemokine production; help for CD8+ T-cell and B-cell responses; killing infected cells; regulatory functions to reduce immunopathology Killing infected cells or purging virus without cell death; antiviral cytokine and chemokine production

ADCC, antibody-dependent cellular cytotoxicity; APC, antigenpresenting cell; IgA, immunoglobulin A; MHC, major histocompatibility complex.

Recall infection

Expansion Contraction Memory

Antigen - specific T cells

368

Effector

CD62Llo CCR7lo

90–95% death

TRM

TEM

TCM

CD62Llo CCR7lo CD69lo

CD62Lhi CCR7hi CD69lo

CD62Llo CCR7lo CD69hi

FIG 25.2  Expansion/Contraction/Memory Phases of Adaptive Immunity and Memory Cell Subsets. (A) Dynamics of primary and secondary (recall) T-cell responses to viral infection. Both primary and recall T-cell responses undergo expansion and contraction phases, followed by stable immune memory. Recall responses induce a larger effector pool and reduced contraction further boosting the memory pool. (B) Effector and memory T-cell differentiation. Antigen stimulation expands effector cells, most of which die during the contraction phase. Effector memory T (TEM) cells that are formed gradually convert to central memory T (TCM) cells over time, with corresponding changes in surface marker expression. Some effector T cells develop into resident memory T (TRM) cells that persist in the tissues and do not reenter the circulation.

Like B-cell responses, T-cell responses to viral infections also begin within lymphoid tissues. Specific CD8+ CTL precursors recognize antigen in the context of MHC class I–peptide antigen complexes on DCs. The CD8+ T cells become activated, proliferate, and differentiate into effectors. Expansion of these naïve antigenspecific precursors is considerable, often exceeding 10 000-fold, and results in an effector population that can account for 40% or more of a host’s total CD8+ T-cell population (Fig. 25.2). Various factors, including antigen and APCs, costimulatory molecules (e.g., CD28 and 4–1BB), and inflammatory cytokines (e.g., IFN-I and IL-12) are required to program the development of functional effector lymphocytes. In some infections, CD4+ T-cell help is also important to prime robust CTL responses via signals, including CD40 that are delivered to DCs.18 Activated CTL effectors then exit lymphoid organs and access almost all body locations via the bloodstream. However, effectors do not stay activated for long once the virus is cleared, and approximately 95% die by a process termed activation-induced cell death.

CHAPTER 25  Host Defenses to Viruses Following this contraction phase, the remaining cells differentiate into memory cells, which remain as a more or less stable population in the host for many years. They represent an expanded pool of CTL precursors that can be activated upon secondary encounter with antigen and provide enhanced protection upon reinfection with the same virus (see next section). Although much of our knowledge of T-cell responses to viruses has been obtained from murine studies, it is increasingly clear that the fundamental principles are the same or similar in humans.19 T-cell immunity against a particular virus involves both CD4+ and CD8+ T-cell subsets that recognize peptides derived from viral antigens bound to surface MHC proteins (class II and class I, respectively) (Chapters 5, 6). Complexes of viral peptides bound to MHC class II proteins are generated by APCs from scavenged and processed virus-infected cells or viral particles. Antigen–MHC class I complexes are expressed on the surface of infected cells, and antigen can also be transferred to APCs from infected cells by a process known as cross-presentation. Recent experiments in mice have also demonstrated a role for transfer of antigen between DCs as they migrate from infected tissues to the lymphoid tissues. Multiple subsets of DCs exist and specialize somewhat in antigen presentation on MHC-I or MHC-II.20 During the process of activation, T cells can receive signals from multiple DC types in a temporally controlled sequence that coordinates CD4+ and CD8+ T-cell interactions.18 Use of MHC class I and class II tetramers to directly visualize antigen-specific CD8+ and CD4+ T-cell responses, respectively, has demonstrated the significant size of T-cell responses to viruses, such that the majority of the activated T cells seen at the peak of the response are virus-specific. CTLs function by recognizing virus-infected cells and killing them; this often involves perforins and cytotoxic granules containing granzymes. Effector CTLs can also induce death in target cells following engagement of the Fas ligand on the CTL with Fas on target cells. Both pathways lead to apoptosis of the target cell, involving the degradation of nucleic acids, including those of the virus. Alternatively, CD8+ T cells also mediate defense through the release of various cytokines after antigen recognition. Some of the cytokines and chemokines most highly produced by CTLs include IFN-γ, TNF-α, lymphotoxin-α, and RANTES (CCL5) (Chapters 9, 10). These cytokines can have multiple antiviral effects on infected cells and on the cells around them, including purging of virus from infected cells without killing the cells. This is particularly important for such viruses as HSV, which infects nonrejuvenating cells, such as nerve cells. CD4+ T cells are involved in antiviral defense as well as being modulators of inflammatory reactions to viruses. Multiple functional subsets of CD4+ T cells are recognized based largely on the types of cytokines produced when they recognize antigen. CD4+ T cells are more broadly reactive than CD8+ T cells; they recognize larger peptides processed from viral proteins and are restricted by MHC class II. These CD4+ T cells participate in antiviral immunity in several ways. They can act as helper cells for the development of high-affinity antibody responses and for more functional CD8+ T-cell responses.16,21 Additionally, CD4+ T cells act as effectors and orchestrate inflammatory reactions, which either serve a protective function or, in some cases, become prolonged causing chronic tissue damage (Chapter 16). The latter can happen in HCV-mediated hepatitis and HSV-mediated stromal keratitis. Occasionally, CD4+ T cells can mediate direct cytotoxicity, but they are less effective than CD8+ T cells. The principal subsets of CD4+ T cells involved in inflammatory reactions are T helper-1 (Th1) cells (producing mainly IFN-γ,

369

tumor necrosis factor [TNF]-α, IL-2) and Th17-producing cells (IL-17a and IL-22). A third effector subset, Th2 cells producing (IL-4, IL-5, and IL-13), also participates in inflammatory reactions, although in the case of viruses, these are usually more tissue damaging than protective. This situation can occur in responses to RSV infection. Regulatory T cells (Tregs) are a further subset of CD4+ T cells of particular importance, since these cells largely act to regulate the function of effector subsets and, in so doing, influence the severity and duration of inflammatory reactions22 (Chapter 18). Tregs produce antiinflammatory cytokines, such as IL-10 and TGF-β, and can be distinguished from other CD4+ subsets by their expression of a unique transcription factor FoxP3. The balance of CD4+ T-cell subset representation in response to a virus infection is critical. In situations where responses become overtly tissue damaging and chronic, the balance favors effector subsets. In such situations, changing the balance to favor Tregs can result in diminished lesions.

IMMUNOLOGICAL MEMORY Immunological memory is a cardinal feature of adaptive immunity. The goal of vaccinology is to induce long-lived immunological memory to protect against reinfection (Chapter 90). Following infection with certain viruses, memory can be exceptionally long-lived, potentially for the life of the host (e.g., yellow fever and smallpox viruses).19,23 Memory is defined by the persistence of specific lymphocytes and antibody-producing plasma cells rather than that of antigen to induce continuous lymphocyte activation. Humoral memory to viruses involves long-lived plasma cells in bone marrow, which provide a continuous low-level source of serum antibody. This maintenance of humoral immunity also involves a population of homeostatically maintained memory B cells, which may be required to maintain stable numbers of long-lived plasma cells over time. The pool of memory T cells is regulated by low-level homeostatic division controlled by the cytokines IL-7 and IL-15. For memory CD8+ T cells, IL-7 is primarily important for survival, whereas IL-15 is crucial for low-level proliferation to maintain the size of the memory T-cell pool.

KEY CONCEPTS Principles of Antiviral Immunity Many human viral infections are successfully controlled by the immune system. Certain emerging viruses may overwhelm the immune system and cause severe morbidity and mortality. Other viruses have developed mechanisms to overwhelm or evade the immune system and persist. Individuals with defects in innate or adaptive immunity demonstrate more severe viral infections. T-cell immunity is more important for control than are antibodies in many viral infections. Antibodies are important to minimize reinfection, particularly at mucosal sites. Immune memory is often sufficient to prevent secondary disease, although not in all viral infections. Tissue-specific immune memory may be important to rapidly protect against reinfection at peripheral sites (e.g., skin and mucosae).

Immunological memory is defined by a pool of antigen-specific cells whose increased frequency enables rapid control of viral reinfection (see Fig. 25.2). IL-7Rα-expressing effector T cells are the precursors of this memory pool. This population of cells, which constitutes about 5–10% of the effector pool, preferentially

370

Part THREE  Host Defenses to Infectious Agents

survives the contraction phase and gradually differentiates into a stable memory population.24 Upon reinfection, these memory cells can be rapidly activated and, by virtue of their increased frequency, mediate more rapid clearance of the viral pathogen. Moreover, repeated stimulation of memory cells via multiple infections with the same virus, or prime-boost vaccine regimes, further increases the size of the antigen-specific memory T-cell pool.25 Restimulation also affects the activation status and tissue distribution of memory T cells, which may enhance protection from viral infection in mucosal and other tissues. Experiments in humans and mice have demonstrated that memory T cells are heterogeneous. Memory T cells were divided into effector memory (TEM) and central memory (TCM) subsets, defined by expression of two surface molecules involved in T-cell migration: CD62L and CCR7.24 The CD62LloCCR7lo TEM subset is found primarily in nonlymphoid tissues and the spleen, whereas the CD62LhiCCR7hi TCM subset is largely present in lymph nodes and the spleen. The current model predicts that effector T cells form the TEM subset and that these cells gradually convert to a TCM phenotype over time (Fig. 25.2B). Although the conditions that control the rate of this conversion are unknown, it is likely that the amounts of antigen and inflammatory signals received during the effector phase greatly influence this. It has also been shown that CD4+ T-cell help is required for the generation of long-lived memory CD8+ T cells, via interactions with DCs.21 Studies suggest that TCM are capable of mounting stronger proliferative responses following reinfection. Tissue-specific homing of TEM cells permits them to enter sites of potential viral infection, such as skin and mucosae. However, we now know that many memory T cells found at sites of previous viral infections take up long-term residence in tissues.26 This includes skin, intestines, lungs, the liver, and the brain. These resident memory T cells (TRM cells) are sequestered from the circulation and provide rapid protection against viruses, such as HSV, in skin, where they localize with a unique dendritic morphology and undergo slow surveillance of the tissue (Fig. 25.3). Notably, activation of

TRM cells can trigger enhanced early inflammation to drive local immunity. This is in contrast to TEM cells, which continue to migrate through nonlymphoid tissues, rather than being sequestered in peripheral tissues, and also differs from the CD8+ and CD4+ TCM, which migrate largely through lymphoid organs (spleen and lymph nodes). These differences may define the physiological raison d'être for these memory T-cell subsets, highlighting that measurement of memory T cells in human peripheral blood is a poor representation of the total-body memory T cell pool. TRM cells can be detected in tissues by using markers, such as CD69 and CD103, although these are imperfect identifiers, including in human tissues. TRM cells in different anatomical locations share a common genetic signature and require common transcription factors for their formation. Yet, these cells also adopt unique gene expression that is imprinted by the tissue environment, and presumably imparts specialized functions on TRM cells in each location. However, memory in certain peripheral tissues, such as lungs, appears to wane over time, suggesting that memory T cells may not persist in sufficient numbers in this site. This rationalizes a need for vaccines that induce optimal numbers of memory T cells in tissues as well as blood.

IMMUNE EVASION AND IMMUNITY TO CHRONIC VIRAL INFECTIONS Many, if not all, viruses employ immune blunting or delay tactics to circumvent aspects of the immune system, allowing them time to replicate further or escape detection (Table 25.3).27 One such mechanism may involve killing or infecting APCs. Viruses may also delay or prevent apoptosis induced by CTLs within infected cells. Other viral evasion measures aimed at the CD8+ T cell–mediated antiviral defense system inhibit antigen processing, thereby minimizing effector CTL induction. To escape CTL killing, many viruses also downregulate the MHC molecules on the surface of infected cells. In addition, viruses may produce various mimics or modulators/inhibitors of cytokines, chemokines, or

Epidermis

TABLE 25.3  Mechanisms and Examples of

Viral Immune Evasion

CD8+TRM

CD4+TEM

Basement membrane

Mechanism

Example

Interference with viral antigen processing and presentation Evasion of NK cell function

HSV (ICP47), EBV (EBNA-1), HIV (Nef, Tat), HPV (E5), CMV (UL6) HIV (Nef), EBV (EBNA-1), CMV (UL40, UL18) Adenovirus (RID complex and E1B), HIV (Nef), EBV (BHRF-1) HIV EBV (IL-10 homologue), CMV(US28 chemokine receptor homologue), vaccinia virus (IL-18-binding protein), HIV (Tat chemokine activity) HSV, pox viruses HSV, vaccinia virus Influenza virus, HIV Measles virus, VZV and HSV (neurons) HIV, HCV, HBV

Inhibition of cell apoptosis

CD8+TRM

CD4+TEM

Dermis

FIG 25.3  Unique Subsets of Memory CD8+ and CD4+ T Cells Reside Within Peripheral Tissues, at Sites of Previous Viral Infection, and Provide Rapid Protection Against Reinfection. Resident memory CD8+ T cells (TRM) remain localized in the epidermis in skin after herpes simplex virus (HSV) infection. Resident memory CD4+ T (TEM) cells continue to migrate through the dermal layers of skin, with access to blood and lymphoid tissues.

Destruction of T cells Interference with antiviral cytokines and chemokines

Inhibition of complement action Inhibition of DC maturation Frequent antigenic variation Infection of immune privileged site Immune exhaustion

CMV, cytomegalovirus; DC, dendritic cell; EBV, Epstein–Barr virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HSV, herpes simplex virus; IL-18, interleukin-18; NK, natural killer; RID, receptor internalization and degradation; VZV, varicella-zoster virus.

CHAPTER 25  Host Defenses to Viruses other components of the immune system or their receptors. Viruses also resort to antigenic hypervariability to escape antibody or T-cell recognition. This can occur during transmission from host to host (e.g., influenza virus), or within hosts during chronic infection through the generation of viral escape mutants. The latter is particularly important for HIV and HCV infections. The success of many viral pathogens rests in their ability to subvert the host immune response. The most successful human viruses can escape the immune system and persist for the life of the host.28 Two well-studied examples of this are CMV and EBV. T-cell responses to these viruses are prominent and readily detectable in humans, and yet the immune system is unable to clear either pathogen completely. However, these viruses generally remain undetectable in immunocompetent individuals. Other viral infections, such as those caused by the herpes viruses HSV and VZV, are marked by periods of latency when no virus can be detected. Yet, periods of viral reactivation, often triggered by stress, can lead to episodes of disease. These are controlled by the immune response, which plays a central role in controlling herpes virus latency.29 Many of the most medically important human viruses are associated with persistent viremia. These include those causing chronic infections, such as HIV, HCV, HBV, and human T-lymphotropic virus (HTLV), among others. Such chronic viral infections are marked by high levels of persisting antigen and can result in skewed T-cell immunodominance hierarchies, altered tissue localization of immune cells, and severely impaired T-cell function.30 This altered T-cell function is hierarchical and results in functional T-cell defects ranging from reduced cytokine production and altered proliferative capacity (exhaustion) to death (deletion) of the responding T cells (Fig. 25.4). Sustained viral antigen levels and inflammation are responsible for this immune dysfunction. This is in stark contrast to normal memory T-cell development, which occurs in the absence of persisting antigen (see previous section). Studies have demonstrated that signaling through multiple inhibitory receptors expressed on the cell surface contributes to exhaustion during chronic infections.30 This includes the receptor programmed death (PD)-1, expression of which may be essential for preventing excessive immunopathology by effector T cells and yet appears to contribute directly to failed immunity to HIV infection and other chronic human viral infections. Although the molecular mechanisms of exhaustion remain unclear, differential involvement of transcription factors and altered gene expression define Cytokines/ Proliferative Antigen killing potential load

PD-I expression

Functional T cells Partial exhaustion Full exhaustion Deletion (death)

FIG 25.4  Hierarchical Model of T-Cell Exhaustion During Persistent Viral Infection. T-cell function (cytokine production, killing, and proliferative potential) is negatively influenced by increasing levels of antigen. Low levels of persistent antigen may lead to partial loss of function and intermediate levels of programmed death (PD)-1 expression. High, sustained levels of antigen over time can lead to full loss of function, high levels of PD-1, and eventually cell death (deletion).

371

exhausted T cells. These studies implicated multiple inhibitory receptors as a potential therapeutic targets, and although combinations of these checkpoint inhibitor blockade therapies are proving highly beneficial to the treatment of certain cancers,31 similarly efficacious responses have yet to be demonstrated during chronic virus infection.

OUTCOMES OF VIRUS INFECTION: IMMUNITY OR IMMUNOPATHOLOGY Typically, individual humans respond to a virus infection in different ways. When the common cold or even pandemic influenza infection occurs, only a small percentage of exposed persons may develop overt clinical disease. In the prevaccine days, poliomyelitis was a much-feared consequence of poliovirus infection, but only a very small percentage of infected persons developed the paralyzing complications. Similarly, only an unfortunate few develop life-threatening meningoencephalitis following infection with the insect-transmitted West Nile virus. It is particularly characteristic of chronic viral infections that clinical expression is highly variable. With HCV, for example, in 70–80% of patients, some form of chronic liver disease develops, and the virus is not cleared. However, in up to 30%, the infection is controlled, the virus is cleared, and immunity to reinfection develops. The latter group of individuals make a type of immune response that includes protective antibodies along with an appropriate pattern of T-cell responsiveness.32 We do not fully understand the reasons for the varying outcomes of virus infections in different persons, and almost certainly multiple factors are involved. Many of these factors impact the response pattern made by the innate immune system, which, in turn, affects the magnitude and type of adaptive immune response that occurs. Some of the circumstances that do influence the outcome of infection include genetic susceptibility of the host, the age of the host when infected, the dose and route of infection, the variable induction in the host of antiinflammatory cells and proteins, and the presence of concurrent infections and past exposure to cross-reactive antigens.32

IMMUNOPATHOLOGY AND AUTOIMMUNITY Immune responses against virus-infected cells often result in tissue damage, especially if cell killing is involved or if there is extensive recruitment and activation of inflammatory cell types, such as macrophages and sometimes neutrophils. If the response is brief and is quickly repaired, it is usually deemed an immunoprotective event. A prolonged tissue-damaging effect resulting from an immune reaction against viruses is considered immunopathology. Such situations most commonly involve persistent viruses, which are themselves often mildly cytodestructive in the absence of an immune reaction. Chronic tissue damage initiated by viruses can also result in development of an autoreactive and an occasionally oncogenic response. For example, some autoimmune diseases may be initiated or exacerbated by viral infections, but no named virus has been regularly incriminated as a cause of human autoimmune disease.33 Circumstantial evidence exists for a virus link in multiple sclerosis (MS), insulindependent diabetes, and possibly systemic lupus erythematosus (SLE). In MS, many viruses have been isolated from patients, although no specific one has been tied to the disease etiology. The current hypothesis is that viral infections set up an

372

Part THREE  Host Defenses to Infectious Agents

TABLE 25.4  Lesions Resulting

From Immunopathology Primarily involving CD8+ T cells acting as cytotoxic T lymphocytes or sources of proinflammatory cytokines

Primarily involving CD4+ T cells that produce Th1 cytokines

Involvement of CD4+ T cells that produce Th2 cytokines Involvement of antibody

Murine lymphocytic choriomeningitis virus; Hepatitis B virus (HBV)–induced chronic hepatitis Coxsackie B virus–induced diabetes Coxsackie B virus–induced myocarditis Demyelination caused by some strains of mouse coronavirus and Theiler virus Demyelination caused by some strains of mouse coronavirus and Theiler’s virus; Herpes simplex virus (HSV)–induced stromal keratitis Respiratory syncytial virus (RSV)– induced pulmonary lesions Glomerulonephritis in chronic hepatitis B Dengue hemorrhagic fever

inflammatory environment that may exacerbate or tip the balance toward disease in genetically susceptible individuals. Immunopathological reactions involving viruses have several mechanisms, but T cells are usually involved as orchestrators of inflammatory events (Table 25.4). The clearest example of immunopathology involving a virus is lymphocytic choriomeningitis virus (LCMV) in the mouse. This model has dominated ideas and has set several paradigms in viral immunology in general. The first virus-induced immunopathological lesions recognized were glomerulonephritis and arteritis, noted in mice persistently infected with LCMV. The lesions were assumed to represent inflammatory reactions to tissue-entrapped immune complexes that activate complement. Similar immune complex– mediated lesions occur in other infections, including lung lesions found in severe influenza, respiratory syncytial virus infection, viral hepatitis, and arthritis. However, only rarely have viral antigens been shown to contribute to the antigen component of the complex. An example where the inclusion of viral antigen in immune complexes has been demonstrated is chronic HBV infection of humans. Autoimmune diseases, such as SLE, also result from immune complex–mediated tissue damage. However, evidence linking viruses to the etiology or pathogenesis of SLE is scarce, since the immune complexes in SLE do not appear to include viral antigens at any stage. Thanks largely to the LCMV model, it is clear that CD8+ T-cell recognition of viral antigens can result in tissue damage. In LCMV infection, damage occurs in the leptomeninges of immunocompetent mice infected intracerebrally. Hepatitis can also occur in mice infected intravenously. Neither lesion becomes evident if the CD8+ T-cell response is suppressed. CD8+ T cell–mediated immunopathology can be a causative mechanism of chronic hepatitis associated with HCV and HBV infection, although the tissue damage also involves inflammatory CD4+ T cells. Additional viral immunopathology models where lesions result primarily from CD8+ T-cell involvement include myocarditis and insulin-dependent diabetes associated with coxsackie B virus infection. In both instances, CD8+ T cells mainly orchestrate events, but tissue damage may result from the bystander effects of cytokines and other molecules, such as lipid mediators,

FIG 25.5  Example of Herpetic Stromal Keratitis (Hsk) in the Human Eye After Herpes Simplex Virus-1 (HSV-1) Infection. Inflammation of the eye and eyelid can be observed, as well as neovascularization and substantial necrosis, ulceration, and opacity of the cornea.

metalloproteinases, and components of the oxygen burst. Although coxsackie virus can be a cause of diabetes in the mouse, attempts to relate viral infection directly to the etiology of human diabetes have so far failed.

CLINICAL RELEVANCE Hypothesized Role of Viruses in Autoimmunity Molecular mimicry: similar epitopes shared by virus and host Bystander activation: chronic release of cytokines and host antigens activates local autoreactive lymphocytes Viral persistence: chronic viral antigen presentation on host cells leads to prolonged immunopathology

Immunopathological reactions against viruses can also involve subsets of CD4+ T cells, which can be either Th1 or Th17 or both. One well-studied example involves persistent infection with Theiler virus in mice.34 This infection causes a demyelinating syndrome that resembles the autoimmune disease experimental allergic encephalomyelitis. In both situations, CD4+ T cells that produce Th1 cytokines appear to serve as pathological mediators. Furthermore, in both models an increase in the involvement of myelin-derived autoantigens occurs as the disease progresses. Once again, such observations indicate the possible role of a virus in an autoimmune disease. With the Theiler virus model, the virus persists in the nervous system and chronically stimulates CD4+ T cells to secrete an array of cytokines. The demyelinating events appear to result from cytokine action on oligodendrocytes. Myelin components, such as myelin basic protein, proteolipid protein, and myelin oligodendroglial glycoprotein, may be released and can participate as additional antigen in immunoinflammatory events. This scenario is referred to as epitope spreading. Another model of virus-induced immunopathology that mainly involves the Th1 subset of CD4+ T cells is stromal keratitis caused by HSV infection (Fig. 25.5).35 The pathogenesis of this immunopathological lesion is unusual in that it occurs and progresses when viral antigens can no longer be demonstrated. The chronic immunoinflammatory lesions are mainly orchestrated by CD4+ T cells, but multiple early events induce the subsequent

CHAPTER 25  Host Defenses to Viruses pathology. Viral replication, the production of certain cytokines and chemokines (IL-1, IL-6, IL-12, and CXCL8), recruitment of inflammatory cells (e.g., neutrophils), and neovascularization of the avascular cornea all precede immunopathology. Recently, it has become evident that Th17 T cells participate in stromal keratitis lesions. The role of Th17 T cells as orchestrators of inflammatory reactions has been a major research focus, especially in lesions of autoimmune diseases.36 When Th17 T cells are the principal mediators of tissue damage, abundant neutrophils are recruited to inflammatory sites, with such cells being mainly responsible for tissue damage. A further mechanism of viral-induced immunopathology and autoimmunity is molecular mimicry.33 Molecular mimicry represents shared antigenic epitopes, either B- or T-cell antigen, between the host and virus. This concept originated with streptococci and their association with rheumatic fever. With human autoimmune disease, there is little direct support for viral molecular mimicry; however, some animal models have been used to prove the theoretical case, where a viral antigen is expressed as a self-protein in the islet cells of the pancreas. In this model, subsequent infection with the virus induces diabetes. However, this is not true mimicry and may be more closely related to viral antigen persistence in a model such as Theiler disease. As discussed previously, the outcome of a T-cell response to a virus may be critically dependent on the balance of the T-celltype response thus, tissue damage is likely to be more severe and prolonged if CD8+ or Th1 and Th17 CD4+ T cells are predominant. Lesions become milder and may resolve when the balance favors Tregs. Accordingly, therapeutic approaches that can shift the balance of T cells are under trial.

KEY CONCEPTS Phases of Immunity Affected by Regulatory T Cells (Tregs) Interference with antigen presentation by dendritic cells Inhibition of T-cell proliferation Inhibition of molecules involved in tissue-specific migration of effector cells Inhibition of T-cell effector functions in lymphoid and nonlymphoid tissues

TRANSLATIONAL RESEARCH OPPORTUNITIES Reversing T-cell exhaustion in patients suffering from chronic infections or cancer will be a key clinical target in the near future. The discovery of multiple inhibitory receptors on exhausted T cells (e.g., PD-1, LAG-3, 2B4, TIM-3) has provided the opportunity to selectively improve T-cell function through blockade of these inhibitory receptors. This may be combined with blockade of immunosuppressive cytokines (e.g., IL-10) or enhancement of signals stimulatory to the response (e.g., IL-7 therapy), as well as with more traditional antiviral therapies and vaccination. The challenge that lies ahead will be in determining which combination of inhibitory and stimulatory signals will need to be manipulated in different diseases and in different groups of patients. The design of a new generation of vaccines to target diseases, such as HIV and influenza, may require tailor-made solutions for patients who respond poorly to vaccination or respond improperly, as with adverse effects, such as autoimmune reactions. High-throughput approaches now allow for the generation of a molecular signature of vaccination or infection.37 Such systems’

373

biology approaches are expected to result in novel screening for immune protection parameters after vaccination. In the near future, this should also assist in the formulation of new vaccines containing key immune activators, such as those that stimulate certain subsets of T cells or induce appropriate homing molecule expression on these cells to direct them to tissues where they are required to mediate protection (e.g., mucosal sites, or skin).

ON THE HORIZON Pressing Issues in Need of Solutions Design of new vaccines that induce broadly neutralizing antibodies Design of new vaccines that induce tissue-resident and circulating memory T-cell subsets Overcoming immune dysfunction during chronic viral infections for successful viral clearance Improving the efficacy of vaccines to viruses using systems biology approaches Therapies for reducing immunopathology during viral infections

In some individuals, viral infections cause mild, or sometimes debilitating, tissue damage. Factors that influence whether a viral infection results in immunopathology varies from individual to individual. These factors include age, the route of infection, preexisting immunity, host genetics, and the host’s viral burden or virome. Our knowledge of the influence of these factors on the outcome of viral infection is expected to improve rapidly in the coming decades. Recent advances have shed considerable light on the various proinflammatory and antiinflammatory mediators produced during viral infections. These represent key targets for novel therapies in the near future via the use of small-molecule inhibitors.

CONCLUSIONS Humans are infected by many pathogenic viruses. In most cases, these infections are controlled by the immune system with limited damage to the host. However, certain viruses, particularly in cases where the host’s immune system is impaired, can cause significant damage to the host’s tissues. As our understanding of the mechanisms underlying innate immune defenses, antigen presentation, T- and B-cell responses, and Tregs continues to improve, so too does the ability to design better vaccines and therapies to boost the immune control of viral infections. Although this remains a challenging goal, particularly for many human viruses, such as HIV, HCV, and HSV, these rapid advances continue to provide many avenues for further investigation.

ACKNOWLEDGMENTS Barry T. Rouse is supported by grants from the National Institutes of Health and Scott N. Mueller by the Australian Research Council and the Australian National Health and Medical Research Council. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Marsh M, Helenius A. Virus entry: open sesame. Cell 2006;124:729–40. 2. Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol 2010;10:787–96.

374

Part THREE  Host Defenses to Infectious Agents

3. Iwasaki A. A virological view of innate immune recognition. Annu Rev Microbiol 2012;66:177–96. 4. Kanneganti TD. Central roles of NLRs and inflammasomes in viral infection. Nat Rev Immunol 2010;10:688–98. 5. Garcia-Sastre A, Biron CA. Type 1 interferons and the virus-host relationship: a lesson in detente. Science 2006;312:879–82. 6. Crouse J, Kalinke U, Oxenius A. Regulation of antiviral T cell responses by type I interferons. Nat Rev Immunol 2015;15:231–42. 7. Jost S, Altfeld M. Control of human viral infections by natural killer cells. Annu Rev Immunol 2013;31:163–94. 8. O’Sullivan TE, Sun JC, Lanier LL. Natural killer cell memory. Immunity 2015;43:634–45. 9. Godfrey DI, Uldrich AP, McCluskey J, et al. The burgeoning family of unconventional T cells. Nat Immunol 2015;16:1114–23. 10. Bottazzi B, Doni A, Garlanda C, et al. An integrated view of humoral innate immunity: pentraxins as a paradigm. Annu Rev Immunol 2010;28: 157–83. 11. Garlanda C, Dinarello CA, Mantovani A. The interleukin-1 family: back to the future. Immunity 2013;39:1003–18. 12. Corti D, Lanzavecchia A. Broadly neutralizing antiviral antibodies. Annu Rev Immunol 2013;31:705–42. 13. Iwasaki A. Antiviral immune responses in the genital tract: clues for vaccines. Nat Rev Immunol 2010;10:699–711. 14. Qi H, Kastenmuller W, Germain RN. Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue. Annu Rev Cell Dev Biol 2014;30:141–67. 15. Mueller SN, Germain RN. Stromal cell contributions to the homeostasis and functionality of the immune system. Nat Rev Immunol 2009;9: 618–29. 16. Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol 2011;29:621–63. 17. Corcoran LM, Tarlinton DM. Regulation of germinal center responses, memory B cells and plasma cell formation-an update. Curr Opin Immunol 2016;39:59–67. 18. Bedoui S, Heath WR, Mueller SN. CD4+ T cell help amplifies innate signals for primary CD8+ T cell immunity. Immunol Rev 2016;272: 52–64. 19. Ahmed R, Akondy RS. Insights into human CD8+ T-cell memory using the yellow fever and smallpox vaccines. Immunol Cell Biol 2011;89: 340–5.

20. Merad M, Sathe P, Helft J, et al. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 2013;31:563–604. 21. Laidlaw BJ, Craft JE, Kaech SM. The multifaceted role of CD4+ T cells in CD8+ T cell memory. Nat Rev Immunol 2016;16:102–11. 22. Veiga-Parga T, Sehrawat S, Rouse BT. Role of regulatory T cells during virus infection. Immunol Rev 2013;255:182–96. 23. Amanna IJ, Slifka MK, Crotty S. Immunity and immunological memory following smallpox vaccination. Immunol Rev 2006;211:320–37. 24. Mueller SN, Gebhardt T, Carbone FR, et al. Memory T cell subsets, migration patterns, and tissue residence. Annu Rev Immunol 2013;31: 137–61. 25. Jameson SC, Masopust D. Diversity in T cell memory: an embarrassment of riches. Immunity 2009;31:859–71. 26. Mueller SN, Mackay LK. Tissue-resident memory T cells: local specialists in immune defence. Nat Rev Immunol 2016;16:79–89. 27. Finlay BB, McFadden G. Anti-immunology: evasion of the host immune system by bacterial and viral pathogens. Cell 2006;124:767–82. 28. Virgin HW, Wherry EJ, Ahmed R. Redefining chronic viral infection. Cell 2009;138:30–50. 29. Rouse BT, Kaistha SD. A tale of 2 alpha-herpesviruses: lessons for vaccinologists. Clin Infect Dis 2006;42:810–17. 30. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15:486–99. 31. Baumeister SH, Freeman GJ, Dranoff G, et al. Coinhibitory pathways in immunotherapy for cancer. Annu Rev Immunol 2016;34:539–73. 32. Rouse BT, Sehrawat S. Immunity and immunopathology to viruses: what decides the outcome? Nat Rev Immunol 2010;10:514–26. 33. Fujinami RS, von Herrath MG, Christen U, et al. Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease. Clin Microbiol Rev 2006;19:80–94. 34. Olson JK, Ercolini AM, Miller SD. A virus-induced molecular mimicry model of multiple sclerosis. Curr Top Microbiol Immunol 2005;296: 39–53. 35. Biswas PS, Rouse BT. Early events in HSV keratitis—setting the stage for a blinding disease. Microbes Infect 2005;7:799–810. 36. Korn T, Bettelli E, Oukka M, et al. IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485–517. 37. Pulendran B. Systems vaccinology: probing humanity’s diverse immune systems with vaccines. Proc Natl Acad Sci USA 2014;111:12300–6.

CHAPTER 25  Host Defenses to Viruses

374.e1

MULTIPLE-CHOICE QUESTIONS 1. Many of the most medically important human viruses are associated with persistent viremia, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). What are the effects of high levels of persisting antigen on the immune response? A. Increased killing capacity by cytotoxic T cells B. Altered tissue distribution and impaired functions of T cells C. Upregulation of T-cell proliferation D. Enhanced cytokine production by virus-specific T cells E. Reduced expression of coinhibitory molecules by the responding virus-specific T cells 2. Many viral infections can cause immunopathological reactions, such as herpetic stromal keratitis induced by herpes simplex virus. What is a major parameter in the pathogenesis of this disease? A. Inhibition of inflammatory cell recruitment into the infected tissues B. Recruitment of cytotoxic CD4 T cells that cause ocular pathology C. Recruitment and retention of pathogenic CD8 T cells in the late stage of the response D. Recruitment and retention of pathogenic Th1 CD4 T cells into the ocular tissues E. Reduction in Th17 CD4 T cells in the ocular tissues

3. Immunological memory is a cardinal feature of adaptive immunity to virus infection. Memory T cells can be divided into multiple subsets. Which of the following statements accurately describe the major subsets of memory T cells? A. Effector memory T cells migrate through lymphoid tissues, central memory T cells migrate through nonlymphoid tissues, and tissue-resident memory T cells circulate in blood. B. Effector memory T cells migrate through nonlymphoid tissues, central memory T cells persist in nonlymphoid tissues, and tissue-resident memory T cells persist predominantly in lymphoid tissues. C. Effector memory T cells persist in nonlymphoid tissues and do not enter blood, central memory T cells persist in lymphoid tissues, and tissue-resident memory T cells migrate predominantly through nonlymphoid tissues. D. Effector memory T cells migrate exclusively in blood, central memory T cells migrate through lymphoid tissues, and tissue-resident memory T cells are retained in nonlymphoid tissues. E. Effector memory T cells migrate through nonlymphoid tissues, central memory T cells migrate through lymphoid tissues, and tissue-resident memory T cells are retained in tissues.

26  Host Defenses to Intracellular Bacteria Stephen T. Reece, Stefan H.E. Kaufmann

The evolutionary relationship between humans and bacteria is so intimate that it is impossible to imagine the development of one without the other.1 Although this coexistence is generally mutually beneficial, clear boundaries do exist between the two and are intensely defended. We tend to think of the human host as the defender and bacteria as transgressors of these boundaries. Evolution of human immunity has been accompanied by evolution of ingenious bacterial mechanisms to not only survive its onslaught but also to manipulate it to enhance survival. This idea is instructively mirrored in the lifestyle of intracellular bacteria. These bacteria actively seek out an environment inside human cells where they can flourish; this is not an easy environment in which to survive. Human cells have developed an ability to differentiate bacterial from host components and direct host cells to clear the invader. The most successful intracellular pathogens have adapted to the intracellular environment of a particular cell target, proliferate only slowly, and can live for long periods completely undetected by the immune system, as we see in the case of the tuberculosis (TB) bacterium Mycobacterium tuberculosis. In other instances, for example, in listeriosis, intracellular infection is more explosive, with the rich intracellular environment harnessed to rapidly amplify bacterial growth. In some cases, intracellular bacteria live for a very long time in the human body, sometimes for a person’s entire lifetime. A wide spectrum of pathologies ensues from intracellular infection, making most intracellular bacteria highly clinically relevant. Moreover, new concepts on the influence of intracellular bacteria on host cell differentiation point to their ability to change infected cell phenotype to enhance survival. This chapter evaluates the current interpretation of this fascinating interplay between human and microbe, sheds light on how the human immune system functions, and how cellular phenotype can be molded in cells whose fates were previously believed to be strictly predetermined. Finally, such insights can inform new therapeutic and prophylactic approaches to keep intracellular bacterial infections under control.

BALANCE OF PROTECTION AND PATHOLOGY DEFINES THE CHRONIC NATURE OF INTRACELLULAR BACTERIAL INFECTION Some bacteria, such as Listeria monocytogenes, are fully eradicated once the host immune response has reached its peak activity. Most often, the intracellular habitat provides a protective niche that promotes persistent infection in the face of an ongoing immune response. Here, the bacteria can persist for long periods

CLINICAL PEARLS Distinguishing Clinical Characteristics of Infections With Intracellular Bacteria Nonsterilizing immunity Persistent bacteria, sometimes latent infection Formation of long-lasting tissue granulomas containing low numbers of viable bacteria Critical role of T cells in protection, role of antibodies less well established but likely to play an as-yet unappreciated role Critical role of immune response in pathology Lack of effective vaccines Host-directed therapies toward enhancing antimicrobial mechanisms while limiting host pathology

without causing clinical signs of illness, but bacterial growth can be reactivated to cause disease if the immune response becomes compromised. This occurs in M. tuberculosis infection, resulting in disease years or decades after primary infection. In fact, disease need not arise from infection at all. In many regions, for example, the majority of adults harbor M. tuberculosis without suffering from clinical disease. However, disease can develop directly after primary infection, during maturation of the immune response, or with regression once the immune response is sufficiently strong. Yet, sterile eradication of the pathogen is rarely achieved: bacteria persist latently, and illness may reemerge at a later time. For example, Rickettsia prowazekii may persist for decades after convalescence from typhus to cause Brill-Zinsser disease later. Several intracellular bacteria possess components that can profoundly influence the course of disease, for example, the lipopolysaccharides (LPSs) of brucellae and salmonellae. Chronic persistence inside host cells, however, depends on the target cell remaining intact and physiologically active. Accordingly, many intracellular bacteria are of low toxicity and do not have dramatic direct effects on their host. Instead, pathogenesis is largely determined by the immune response. Classic examples of this concept include granuloma liquefaction in acute TB, which severely affects lung function, and eye scarring as a consequence of chronic or recurring Chlamydia trachomatis infection that ultimately leads to trachoma. The survival of intracellular bacteria has major consequences for pathology. Although many intracellular bacteria show some organ tropism, dissemination to other organs frequently occurs, resulting in different disease forms. For example, TB is generally manifested in the lung in 80% of cases, yet many other organs can be affected. In contrast to other Salmonella enterica serovars,

375

376

Part THREE  Host Defenses to Infectious Agents

TABLE 26.1  Major Infectious Diseases Caused by Intracellular Bacteria Disease

Pathogen

Prevalence

Incubation Time

Route of Infection

Target Cell

Years (latency after primary infection and disease reactivation) Weeks (miliary TB) Years

Inhalation of bacteria-containing microdroplets

Macrophage

Smear infection through mucosa/ inhalation

Macrophage Schwann cell

7–10 days

Fecal–oral

Macrophage

Worldwide

Weeks to months

Macrophage

Worldwide

Days to months

Zoonosis; cows, goats, pigs; inhalation, gut, skin abrasion Fecal–oral

Worldwide Western hemisphere Worldwide

2–10 days 1 week

Inhalation Tick bite

1–3 weeks

Sexual intercourse

Macrophage Vascular endothelial cell Smooth muscle cell Epithelial cell

Africa

Conjunctivitis: 1–3 weeks Trachoma: years Bacillary angiomatosis Peliosis hepatitis Endocarditis Bacteremia with fever Neuroretinitis: 1–3 weeks

Eye

Epithelial cell

Flea, sandfly, or mosquito bite; animal scratch or bite

Erythrocyte Endothelial cell

Granulomatous Intracellular Bacteria Tuberculosis

Mycobacterium tuberculosis

Worldwide

Leprosy

Mycobacterium leprae

Typhoid fever Brucellosis

Salmonella enterica serovars Typhi and Paratyphi Brucella spp.

South America Africa India Southeast Asia Worldwide

Listeriosis

Listeria monocytogenes

Macrophage Hepatocyte

Nongranulomatous Intracellular Bacteria Legionnaires’ disease Rocky Mountain spotted fever Urogenital infection Conjunctivitis, trachoma Cat scratch disease

Legionella pneumophila Rickettsia rickettsiae Chlamydia trachomatis serovars D-K Chlamydia trachomatis serovars A-C Bartonella henselae B. quintana B. bacilliformis

Worldwide

the serovars Typhi and Paratyphi are not restricted to the gastrointestinal (GI) tract but are disseminated to internal organs, primarily the liver and spleen. In these cases, the type of clinical disease depends markedly on the infected tissue type.

KEY CONCEPTS Characteristic Features of Intracellular Bacterial Infections Persistence of bacteria inside mononuclear phagocytes (i.e., macrophages) Low to absent bacterial-mediated toxicity to the host Protection requires cytokine-mediated activation of infected phagocytes Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced by antigen-specific T cells are key cytokines for protection

INTRACELLULAR BACTERIAL INFECTIONS OF CLINICAL RELEVANCE (Table 26.1) Granulomatous Infections Tuberculosis The major entry of tubercle bacilli into the human body is via inhalation into the lung. These inhaled bacteria are then engulfed by alveolar macrophages, which transport the pathogens to the lung interstitia. Their exact fate after these events is enigmatic. Moreover, most infections in humans result in an asymptomatic

carrier state or the so-called latent TB infection (LTBI). Infection with M. tuberculosis starts with the so-called Ghon complex, characterized by a caseous lesion in the midlung as well as in the draining lymph nodes. These primary lesions can progress, but their development rarely causes disease directly.2 Moreover, bacteria from these sites can disseminate to other regions of the lung and systemically, causing disease of the kidneys, liver, and central nervous system (CNS). Containment of the primary lesions, which leads to LTBI, is a function of an effective, predominately cellular antitubercular immune response. Infection of immunocompromised patients, notably those with acquired immunodeficiency syndrome (AIDS), or newborns frequently results in systemic disease (miliary TB). TB represents a major health problem worldwide, including an increasing incidence in many industrialized countries. In 2015, the World Health Organization (WHO) estimated that 10.4 million active TB cases were diagnosed worldwide and close to 1.8 million people died of the disease.3 The much larger estimated number of 2 billion individuals infected with M. tuberculosis well illustrates the dissociation of infection from disease. The emergence of multidrug-resistant strains and extremely drug-resistant strains has complicated treatment with currently available antibiotic therapy, and even when treatment is successful, recurrence of disease can occur. The currently available live whole cell vaccine BCG (Bacille Calmette–Guérin), an attenuated strain derived from the etiological agent of bovine TB Mycobacterium bovis, shows only low and variable protection against pulmonary TB.

CHAPTER 26  Host Defenses to Intracellular Bacteria Leprosy Mycobacterium leprae is most likely transmitted by contact with patients who shed microorganisms in nasal secretions and lesion exudates. It primarily affects the nerves and the skin, frequently leading to stigmatizing deformities. In skin, bacilli target keratinocytes, histiocytes, and macrophages, whereas in peripheral nerves, Schwann cells are the major target for entry. Leprosy is a spectral disease. The tuberculoid pole is characterized by rigorous T-cell responses, which succeed in restricting microbial growth in well-defined lesions containing few bacilli. In contrast, at the lepromatous pole, bacterial growth is unrestricted and lesions contain abundant bacilli within macrophages lacking signs of activation. Several types of immunosuppression have been implicated in this latter type of disease. Infection of Schwann cells promotes nerve damage and anesthesia. This results in injuries and secondary infections that significantly exaggerate the disease. Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, some 216 000 new cases were reported in 2013. This suggests that active transmission of M. leprae is still occurring and that more effective interventions are required to prevent it.4 Atypical Mycobacterial Infections Mycobacterial species present in the environment are typically unable to persist within activated macrophages and thus rarely cause disease in individuals with competent immune status.5 As a consequence of human immunodeficiency virus (HIV) infection, however, nontuberculous mycobacteria (NTM), primarily Mycobacterium avium/Mycobacterium intracellulare, have gained clinical importance, and these infections are recognized as one of the most common complications of AIDS in industrialized nations. Mycobacterium scrofulaceum occasionally causes lymphadenitis in children, and Mycobacterium kansasii primarily causes infections in older men with preexisting lung disease. Mycobacterium ulcerans causes a severe subcutaneous infection characterized by chronic skin ulcerations, known as Buruli ulcer. This pathology is caused—at least in part—by elaboration of a mycolactone toxin by the bacillus that exhibits highly cytopathic effects.6 Buruli ulcer is most predominant in West African countries that accounted for most of the 2251 cases reported globally in 2014.7 Typhoid or Enteric Fever Salmonella enterica serovars Typhi and Paratyphi A, Paratyphi B, and Paratyphi C are leading causes of community-acquired bloodstream infections in low- and middle-income countries. The route of transmission is fecal–oral and largely occurs via contaminated water sources. Bacteria are disseminated within mononuclear phagocytes (MPs) from the GI tract to macrophagerich organs, particularly the liver, spleen, and lymph nodes. Accordingly, typhoid is characterized by systemic symptoms, such as prolonged fever and malaise, with sustained bacteremia, although diarrhea or constipation may also be present. In some cases, an asymptomatic carrier state can persist as a result of chronic infection of the gallbladder, which maintains the environmental reservoir of infection in endemic areas. Typhoid fever remains a major cause of morbidity and mortality, with approximately 21 million new cases and over 190 000 deaths per year worldwide.8

377

Gastroenteritis S. enterica serovars Typhimurium and Enteritidis, often referred to as nontyphoidal salmonella (NTS), are the major causes of salmonella gastroenteritis in humans, which occurs mainly as a result of the ingestion of contaminated food or water. The bacteria rapidly cross the intestinal epithelia and replicate in the lamina propria, inducing an influx of polymorphonuclear neutrophils (PMNs), which is generally sufficient to resolve the infection within 7 days. In rare cases, bacteria enter the bloodstream and cause systemic bacteremia, most notably in patients with AIDS, where death can occur as a result of septic shock.8 Listeriosis Listeriosis monocytogenes is increasingly recognized to cause foodborne gastroenteritis. Clinical listeriosis affects mainly pregnant women, older adults, fetuses, and neonates. Disease manifestations are most severe in patients with a compromised immune system, in whom the CNS becomes involved and fatal bacteremia can result. Additionally, as these bacteria are able to cross the placenta, listeriosis is a major cause of perinatal and neonatal disease, typically resulting in abortion. Listeria outbreaks are sporadic with low incidence but high fatality and affect high-income countries, such as the United States. Brucellosis Brucellosis is the most common global zoonosis of humans with approximately 500 000 cases per year.9 It is caused by Brucella abortus, B. melitensis, or B. suis, which primarily infect cows, goats, and pigs, respectively. The bacteria are transmitted to humans via inhalation, through abraded skin or the GI tract. Lesions are primarily found within macrophage-rich tissues, especially the spleen and bone marrow. Human brucellosis is characterized by systemic symptoms, particularly undulant fever. Although the disease often remains subclinical, it becomes chronic in some patients, and relapses and remissions may occur. Interest in brucellosis has increased in the last 5 years because of elevated levels of detection resulting from better surveillance. Lymphogranuloma Venereum Lymphogranuloma venereum (LGV), a sexually transmitted disease, is highly prevalent in Africa, Southeast Asia, and Latin America. LGV has recently emerged as infection of sexually active homosexual men in Europe and the United States. It is caused by the L1, L2, and L3 serotypes of Chlamydia trachomatis, which are disseminated from the urogenital tract to local lymph nodes and then to skin. Accordingly, LGV is characterized by lymph node swelling and skin lesions, which are accompanied by systemic complications.10 Melioidosis Burkholderia pseudomallei is a gram-negative bacillus and the causative agent of melioidosis, endemic in Southeast Asia and Northern Australia. The disease can be acquired through inhalation and ingestion or through cuts in the skin. Susceptible hosts can suffer abscess formation in multiple organs and, in some cases, disseminated infection, resulting in septic shock accompanied by pneumonia. There are an estimated 165 000 cases of melioidosis per year globally, resulting in approximately 89 000 deaths.11 Tularemia This rare zoonosis in humans caused by Francisella tularensis is mainly found in rabbits and has recently gained wider recognition

378

Part THREE  Host Defenses to Infectious Agents

because of its potential for dual use.12 Infection can be spread to humans via contaminated animals or tick bites. This gramnegative bacterium survives in macrophages and primarily causes acute pneumonia as well as skin sores, with subsequent involvement of lymph nodes.

Nongranulomatous Infections

Legionnaires’ Disease or Legionellosis Legionnaires’ disease is caused by Legionella pneumophila, an environmental bacterium that persists within amoeba living in water reservoirs (e.g., air-cooling systems), from where it is spread aerogenically. Infection is exacerbated by a compromised immune status. Characteristically, Legionnaires’ disease presents as atypical pneumonia associated with general symptoms and is complicated by extrapulmonary infection, renal failure, and lung abscesses. Cases of Legionnaires’ disease in the United States increased from 0.39 to 1.36 per 100 000 people from 2000 to 2011.13 Chlamydial Urethritis, Cervicitis, and Conjunctivitis C. trachomatis serovars D–K enter and persist in epithelial cells of the urogenital tract, causing cervicitis and urethritis. In women, infertility can develop as a result of chronic or recurrent infection. In neonates, congenital infection during birth may result in conjunctivitis and pneumonia. Urogenital infections by chlamydiae occur worldwide and are now considered the most common sexually transmitted bacterial disease, with an estimated 100 million new infections occurring annually.14 Trachoma Smear infections of the eye with C. trachomatis serovars A, B, and C cause inclusion conjunctivitis. As a consequence of multiple chronic infections and of the resulting immune response, scars develop that eventually injure the cornea, leading to trachoma. Approximately 84 million people are infected with C. trachomatis worldwide, 7.6 million of whom suffer from visual impairment.15 Chlamydia Pneumoniae C. pneumoniae (formerly known as C. trachomatis TWAR strain) is the cause of mild respiratory disease in young adults and may cause serious infections in older, more debilitated patients. Atypical pneumonia may also be caused by Chlamydia psittaci, although this zoonosis, transmitted by birds, is relatively rare. Typhus Rickettsia prowazekii, R. typhi, and R. tsutsugamushi cause diseases of varying severity.16 They are transmitted by arthropods and infect vascular endothelial cells at the site of an insect bite or scratch, causing skin reactions. Subsequently, pathogens are disseminated to the central organs, and more general symptoms develop. Globally, typhus is of minor importance. Rocky Mountain Spotted Fever, Ehrlichiosis Rocky Mountain spotted fever is caused by Rickettsia rickettsii. Infection of the vascular endothelium leads to systemic symptoms and skin manifestations that may be followed by shock and neurological complications.16 Worldwide, this disease, as well as Mediterranean spotted fever caused by Rickettsia conorii, is of minor importance; as is probably Ehrlichiosis, a newly emerging zoonosis transmitted by ticks and caused by various Ehrlichia spp., mainly E. chaffeensis.17 Disease manifestations include generalized symptoms, such as fever and muscle pain.

Bartonella Bartonella spp. represent gram-negative facultative intracellular pathogens transmitted by insect vectors, such as fleas, sandflies, and mosquitoes.18 The most clinically relevant species are B. henselae, B. quintana, and B. bacilliformis. B. henselae causes cat scratch disease (CSD) resulting in local lymphadenopathy in the lymph node draining the scratch site accompanied by fever, headache, and splenomegaly. Oculoglandular involvement (Parinaud syndrome), encephalopathy, neuroretinitis, or osteomyelitis can occur, albeit in rare cases. In immunosuppressed patients, bacillary angiomatosis and peliosis can occur, characterized by pseudotumoral proliferation of endothelial cells. Bacteria persist within erythrocytes with the intracellular location providing a protective niche.

GRANULOMA PATHOLOGY AS HALLMARK OF INTRACELLULAR BACTERIAL INFECTION KEY CONCEPTS Balance of Protection and Host Pathology in Granulomas Macrophage activation results in bacterial death (protective) Intracellular bacterial killing by “killer molecules” from T cells (protective) Lysis of infected macrophages by T cells results in release of bacteria and killing by more effective effector cells (protective) or bacterial dissemination (pathogenic) Development of central necrosis in granulomas results in death of tissue and bacteria (protective/pathogenic) Fibrotic encapsulation of granuloma results in containment of infection (protective) Overexuberant tissue fibrosis and necrosis (pathogenic) Liquefaction of central necrotic tissue in granulomas results in bacterial replication, cavity formation, and transmission of bacteria (pathogenic and contagious)

A characteristic feature of many infections caused by intracellular bacteria is the eventual need for tissue remodeling by the host at the site of infection. Granulomas are the result of an inability to rapidly clear host tissue of intracellular bacteria and represent a fascinating site of the host–pathogen interface (Fig. 26.1). The longevity of the granuloma depends directly on the continuous presence of the microbial pathogen, and the lesion generally disappears after its sterile eradication. Granulomas form the focus of the coordinated cross-talk between different types of T cells, B cells, and infected and uninfected mononuclear phagocytes (MPs) and dendritic cells (DCs). Even if the immune system fails to completely eliminate bacteria inside the granuloma, the latter performs a protective function by containing microbes within distinct foci and preventing their dissemination. At the same time, the granuloma can be detrimental to the host because it can interfere with physiological organ functions.19 More detailed study of cellular phenotype within granulomas is starting to establish how cellular differentiation is orchestrated and how the granuloma develops. Granulomatous lesions are generally initiated by nonspecific inflammatory signals mediated by bacterial products, chemokines, and proinflammatory cytokines that are produced by endothelial cells and MPs at the site of infection. Inflammatory phagocytes (of both monocytic and granulocytic origin) are attracted to the site of microbial replication, and an infiltrative, sometimes exudative, lesion develops. Following the accumulation and

CHAPTER 26  Host Defenses to Intracellular Bacteria

A

Solid granuloma

B

Necrotic granuloma

C

Caseous granuloma

379

FIG 26.1  Development of Granuloma Pathology and Implications for Tuberculosis (TB). This figure depicts three distinct yet continuous stages of granuloma pathology in the lung due to Mycobacterium tuberculosis infection. (A) Solid granuloma: Composed largely of T cells and infected and uninfected MPs. These granulomas are defined by a lack of central necrosis and likely are representative of an ability to control M. tuberculosis replication. (B) Caseous/necrotic granulomas: These structures contain a central region of demarcated necrotic cell death. Bacteria are often detected within the caseous necrotic region and in proximal cells, notably mononuclear phagocytes (MPs). Since calcified caseous granulomas containing few bacteria have been observed, development of central necrosis may be a consequence of antibacterial mechanisms resulting in sacrifice of host cells to contain infection. (C) Cavity formation: These structures result from inability of caseous granulomas to contain bacterial replication. The acellular necrotic region, containing a large number of extracellular bacteria, increases in size and can liquefy and empty into the lung airways, resulting in transmission of viable bacteria via cough. Therefore granuloma formation is central to humanto-human spread of TB. Dissemination of bacteria through the bloodstream results in disease manifestation in other organs, such as the meninges and the urinary bladder.

activation of increasing numbers of MPs and DCs, this lesion takes an increasingly structured granulomatous form. A significant number of B cells is also found, which seem to influence granuloma morphology. Once specific T cells have been attracted to the lesion, it transforms into a productive granuloma that provides the most appropriate tissue site for antibacterial protection. Here, activation of MPs by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) inhibits microbial growth. However, unbridled macrophage activation can have tissue-damaging effects, and mechanisms within the granuloma tightly regulate these effects. Eventually, the granuloma is encapsulated by a fibrotic wall, and its center becomes necrotic. Both tissue reactions are primarily protective, the former by promoting bacterial containment and the latter by reducing the nutrient and oxygen supply to the pathogen. The combined effects of chronic macrophage activation, persistence of intracellular bacteria, and hypoxia likely lead to enhanced cell death in the center of granulomas, resulting in the formation of a caseum. Caseation may favor the local replication of normally facultative intracellular bacteria in the cellular detritus, as well as microbial dissemination to distant tissue sites and to the environment to transmit infection. Hypoxia also has pronounced effects on enzyme functions that can dictate macrophage phenotype.

THE INTERDEPENDENCE OF INNATE AND ADAPTIVE IMMUNITY IN PROTECTION AGAINST INTRACELLULAR BACTERIA Innate Immune Mechanisms as First-Line Defense The interaction between host cell and pathogen that defines the intracellular lifestyle consists of a number of different layers. The first layer that differentiates intracellular bacteria from other bacteria, notably commensal bacteria that colonize the host but do not cause infection, is that of host cell entry. Extracellular bacteria are typically engulfed by professional phagocytes, which

380

Part THREE  Host Defenses to Infectious Agents

include tissue macrophages, DCs, and PMNs. This uptake is enhanced by host components of the complement system and antibodies, which bind to complement receptors (CRs) and Fc receptors, respectively, on professional phagocytes.20 M. tuberculosis actively targets macrophages, where it must counteract numerous antimicrobial mechanisms operative in these cells (see below). Intracellular bacteria also use elaborate mechanisms to enter nonprofessional phagocytes, by which they must subvert host endocytic processes that are normally engaged in traffic of cellular cargoes. In some cases, this provides a less hostile environment because of their inability to efficiently mobilize antibacterial effector mechanisms. Bartonella spp., unique among intracellular bacteria, can enter red blood cells, thus allowing transmission via blood-sucking insect vectors. This represents a particularly advantageous niche as red blood cells lack the machinery to drive the adaptive immune responses required for protection. Entry into nonphagocytic host cells requires bacteria to induce their own internalization. Bacteria that colonize the GI tract (i.e., L. monocytogenes or salmonellae) or mucosal membranes of the urogenital tract (i.e., C. trachomatis) must mediate tight adhesion to the host cell membrane and be capable of mediating the uptake process. Broadly, two processes are utilized by bacteria to induce uptake into a nonphagocytic cell. The “zipper” mechanism is mediated by binding of a bacterial cell surface protein binding a cognate receptor on the host cell membrane. L. monocytogenes entry into intestinal epithelial cells depends on engagement of InIA to E-cadherin to mediate uptake. Salmonellae and C. trachomatis use a “trigger” mechanism to induce internalization and inject multiple factors into the host cell cytoplasm to mediate uptake. These proteins are delivered by the needle-like structures that form part of bacterial type III secretion systems (T3SSs). These injected proteins then target host proteins involved in host cell signaling and actin remodeling to induce bacterial entry. The C. trachomatis–secreted proteins Tarp, CT166, and CT694 reversibly stimulate the Rho-family guanosine triphosphatase (GTPase) Rac1 to trigger internalization. Similarly, salmonellae inject T3SS factors to stimulate the Rhofamily GTPases Cdc42 and Rac1. The success of these mechanisms of induced uptake enables intracellular bacteria to persist inside diverse cell types. Rickettsia spp., C. trachomatis, M. leprae, and L. monocytogenes ultimately target vascular endothelial cells, epithelial cells, Schwann cells, and hepatocytes, respectively, as their preferred intracellular habitats. To prevent intracellular infection, the host depends on its ability to discriminate between host and bacterial molecules. As already mentioned, bacteria targeting the intracellular environment often do so via mucosal surfaces already populated by commensal organisms (the microbiome) that do not alert host defenses. The host must, therefore, discriminate between commensal and pathogenic bacteria via recognition of conserved molecular motifs of bacteria, named pathogen-associated molecular patterns (PAMPs). This occurs via host receptors broadly defined as pattern recognition receptors (PRRs, Table 26.2). The bestcharacterized group of PRRs is that of the so-called Toll-like receptors (TLRs). The TLR system constitutes an innate scanning mechanism of microbial pattern recognition to distinguish between a wide spectrum of bacteria and viruses. TLRs are present as homo- or heterodimers on the plasma membrane or within intracellular endosome/phagosome compartments.21 PAMPs of bacterial origin comprise di- and triacyl lipoproteins, LPSs, and flagellin, which are recognized by TLR-2/6, TLR-2/1, TLR-4/4, or TLR-5/5, respectively. The vast array of mycobacterial cell wall

TABLE 26.2  Major Pattern Recognition

Receptors Involved in Sensing of Intracellular Bacteria Pattern Recognition Receptor

Location

Ligand

Plasma membrane Plasma membrane Plasma membrane Plasma membrane Plasma membrane Endosome Endosome

Triacyl lipoprotein PGA, porins, LAM LPS Flagellin Diacyl lipoprotein ssRNA CpG DNA

SR-A

Plasma membrane

MARCO CD36 LOX-1 SREC

Plasma Plasma Plasma Plasma

LPS, LTA, CpG DNA, proteins LPS, proteins Diacyl lipoprotein Protein Protein

Toll-Like Receptors TLR1 TLR2 TLR4 TLR5 TLR6 TLR7 (human TLR8) TLR9

Scavenger Receptors membrane membrane membrane membrane

C-Type Lectins DC-SIGN

Plasma membrane

MINCLE

Plasma membrane

LPS, ManLAM, capsular polysaccharide Mycobacterial cord factor: TDM

NOD-Like Receptors NOD1

Cytoplasm

NOD2 NLRP1 NLRP3 NLRC4 Naip5

Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm

D-glutamyl-mesodiaminopimelic acid MDP MDP RNA, LPS, LTA, MDP Flagellin Flagellin

Cytoplasm Cytoplasm

dsDNA dsDNA

Cytoplasm

dsDNA

AIM2-Like Receptors AIM2 IFI16

STING/cGAS Pathway cGAS

Note: We omit PRRs (e.g., TLR3, which binds viral produced double-stranded RNA) not classically associated with intracellular bacteria. AIM2, absent in melanoma-2; CD36, cluster of differentiation 36; CpGDNA, cytosine-phosphatidyl-guanine DNA; DC-SIGN, dendritic cell–specific intercellular adhesion molecule-3-grabbing nonintegrin; dsDNA, double-stranded DNA; LAM, lipoarabinomannan; LOX-1, lipoxygenase-1; LPS, lipopolysaccharide; LTA, lipoteichoic acid; ManLAM, mannose lipoarabinomannan; MARCO, macrophage receptor with collagenous structure; MDP, muramyl dipeptide; MINCLE, macrophage-inducible C-type lectin; NLR, NOD-like receptor; NOD, nucleotide-binding domain; PGA, peptidoglycans; PRR, pattern recognition receptor; SR, scavenger receptor; ssRNA, single-stranded RNA; SREC, scavenger receptor expressed by endothelial cell-I; TDM, trehalose dimycolate; TLR, Toll-like receptor.

lipids, such as lipoarabinomannan (LAM), trehalose dimycolate (TDM), and phosphatidylinositol mannosides (PIMs) bind either TLR-2 or TLR-4. Lipoteichoic acid (LTA) of gram-positive bacteria is recognized by TLR-2. TLR-9 binds low-methylated bacterial DNA containing CpG motifs within endosomes. Scavenger receptors and C-type lectins are also PRRs and function at the cell membrane.22 Scavenger receptors were first defined by their ability to transport modified forms of lowdensity lipoproteins inside cells, indicating their ability to also

CHAPTER 26  Host Defenses to Intracellular Bacteria interact with host molecules. However, receptors, such as SR-A, MARCO, CD36, LOX-1, and SREC, can bind a wide array of bacterial molecules, such as lipids, CpG DNA, and proteins (see Table 26.2 for binding specificities). SR-A is important for clearance of extracellular bacteria from the spleen and liver. MARCO expressed on alveolar macrophages is implicated in clearance of pneumococcal bacteria preventing pneumonia. C-type lectins are similarly membrane-expressed and include DC-specific intercellular adhesion molecule–grabbing nonintegrin (DC-SIGN); mannose receptor; dectin-1; dectin-2, which chiefly recognize fungal components; and MINCLE, which recognizes trehalose dimycolate (TDM), the cord factor of M. tuberculosis. It has been suggested that scavenger receptors and C-type lectins are required to bind and internalize the bacillus, whereas it is primarily the TLRs that discriminate between the pathogens and initiate the necessary intracellular signaling events. It should, however, be noted that intracellular signaling events can also be triggered by other interactions, such as ligand binding to macrophage mannose receptor (MMR), dectin 1, or DC-SIGN. Far from a one-ligand, one-receptor binary mechanism of sensing and signaling, PRRs often collaborate to produce multiprotein complexes. CD14, MD2, and TLR4 collaborate for LPS sensing and signaling. Similarly, MARCO and TLR2 synergize to recognize TDM. To allow signaling, these complexes interact with adaptor proteins containing immunoreceptor tyrosine-based activation motif (ITAM)-like or Toll/interleukin-1 receptor (TIR) domain motifs. TLR signaling occurs via the adaptor proteins MyD88, TIRAP/Mal, and Trif. These molecules then orchestrate a downstream signaling cascade, which culminates in induced patterns of gene transcription that mediate innate and, ultimately, adaptive immune mechanisms that aim at combating the intracellular bacteria. The cellular cytoplasm is monitored for presence of molecules of bacterial origin by a further group of PRRs, the nucleotide oligomerization domain protein-like receptors (NLRs). These molecules are characterized by presence of a nucleotide-binding domain and leucine-rich repeat motifs. Molecules from this group recognizing bacterial components are nucleotide-binding oligomerization domain (NOD)–containing proteins NOD1 and NOD2, NOD-like receptor P1 (NLRP1), NLRP3, and Naip5. Other cytosolic PRRs include the absent in melanoma-2-like receptor (ALR) family, cyclic guanosine monophosphate–adenosine monophosphate (cGMP-AMP) synthase (cGAS), and stimulator of interferon (IFN) genes (STING), all of which can be activated by bacterial DNA (see Table 26.2). Engagement of NLRs and ALRs leads to activation of the multiprotein complex called the inflammasome, leading to cleavage of pro-IL-1β and pro-IL-18 to produce their active forms. In addition, activation of the NLRs, NOD1, and NOD2 results in inflammatory cytokine secretion. Certain PRRs are also receptive to certain endogenous “danger” signals produced by tissues undergoing stress, damage, or cell death. These signals are triggered by self-proteins, named danger-associated molecular patterns (DAMPs), include endogenous heat shock proteins, host nucleotides, and the chromatin component HMGB1. Therefore PRRs mediate signals not only emanating from intracellular bacteria but also from host cells damaged by the infection process. Understanding how PAMP and DAMP PRR signaling meshes to produce a coherent disease-specific output remains an exciting challenge for future research. As already alluded to, the culmination of PRR collaborative sensing and signaling is the induction of inflammatory genes

381

that induce innate immune mechanisms and, subsequently, the mobilization of the adaptive immune response. These include cytokines that act both locally and systemically and are important mediators of protection against intracellular bacteria via specific signaling through engagement of host cell surface receptors. Such engagement mobilizes both critical mechanisms of host protection and orchestration of adaptive immune responses.

Macrophage Training by Epigenetic Mechanisms Following infection with, for example, M. tuberculosis, macrophages express elevated effector mechanisms over long periods. More recent findings have revealed that this is caused, at least in part, by epigenetic changes that are induced in MPs during infection. Similar training can also occur after vaccination with live vaccines. Moreover, BCG vaccination was shown to induce changes in methylation patterns of the NOD2 gene in humans. These epigenetic changes led to increased production of proinflammatory cytokines by MPs, namely, TNF and IL-1β, a tendency that persisted for 3 months after vaccination. Furthermore, this enhanced innate immunity led to increased resistance not only against M. tuberculosis but also against other bacterial pathogens. Indeed, it has been speculated that epigenetic alterations in MPs following vaccination are responsible for the nonspecific reduction of mortality in BCG-vaccinated infants in resource-poor regions of the world.23

Cytokines as Mediators of Defense Against Intracellular Bacteria We have already mentioned that a range of cytokines are induced by the signaling mechanisms that result from engagement of PRRs. These serve by both enhancing intracellular mechanisms of bacterial killing and mobilizing adaptive immune responses, representing the next layer of host defense. Because these responses allow an amplification of the initial innate immune responses, they must be carefully regulated by the host to prevent extensive tissue pathology. In fact, we might view the development of a granuloma as the sequela of a balance between bacterial killing mechanisms and the need to restrict tissue pathology orchestrated by adaptive immunity. At the onset of infection, initial cytokine secretion occurs in the cell type that initially encounters intracellular bacteria and on initiation of signaling cascades by PRRs. These small molecules can act locally and systemically to directly instruct cells, to produce antibacterial molecules, to combat intracellular infection, and to both increase numbers of immune cells and direct the composition of the cellular infiltrate that will ultimately attempt to resolve intracellular bacterial infection. Cytokines are ultimately produced by multiple cell types, including adaptive T cells, B cells, unconventional T cells, MPs, DCs, PMNs, and even epithelial and endothelial cells. We will first consider the hierarchy by which these cytokines act in the control of intracellular bacterial infection and the antibacterial mechanisms they regulate. We will then return to the generation and regulation of the cells that produce them. IFN-γ, TNF-α, IL-12, and IL-18 By far, the cytokine with the clearest demonstrable potency against intracellular bacteria is IFN-γ. Extensive studies on the activation of antibacterial effector functions in macrophages have revealed a central role for IFN-γ. Accordingly, IFN-γ neutralization with antibodies, or deletion of the IFN-γ gene by homologous recombination, markedly exacerbates infectious diseases, such as

382

Part THREE  Host Defenses to Infectious Agents

KEY CONCEPTS T Cell–Mediated Mechanisms Underlying Protection IFN-γ- and TNF-α-mediated activation of phagocytes to kill bacteria by means of: Reactive oxygen intermediate (ROI) and reactive nitrogen intermediate (RNI) Delivery of lysosomal hydrolytic enzymes and antimicrobial peptides to the bacteria-containing phagosome Autophagy Formation and maintenance of granulomas T cell–mediated response controls but does not eradicate the pathogen

listeriosis, TB, or typhoid in experimental animals. For macrophages harboring intracellular bacteria, namely, M. tuberculosis, signaling with IFN-γ is a game-changer, summoning infected macrophages to escalate antimicrobial mechanisms. The action of TNF-α appears to augment IFN-γ and is also important in control of intracellular infection. This has been demonstrated in humans through use of blocking of TNF-α by antibodies as antiinflammatory therapy. Such treatments can activate TB in individuals with LTBI. Despite this, these potent protective effects of IFN-γ and TNF-α come at a price. The need to kill intracellular bacteria often leads to death of the host cell as collateral damage. In part, the host manages this by controlling how the cell dies. Excessive TNF-α leads to less regulated necrotic cell death, benefiting M. tuberculosis. For this reason elaborate host mechanisms have evolved to maintain TNF-α at optimal levels to control infection. The host enzyme leukotriene A4 hydrolase (LT4H) catalyzes synthesis of a highly proinflammatory lipid leukotriene B4. In the event of enzyme deficiency, an antiinflammatory lipid lipotoxin A4 accumulates that counteracts effects of TNF-α. Two common variant promoters control expression of LT4H in humans, and homozygotes are associated with either high or low inflammation. In contrast, the heterozygotes show a balanced response to TNF-α associated with resistance against TB. Such a finding strongly suggests that genetic mechanisms can maintain an optimal level of TNF-α responsiveness of cells harboring intracellular bacteria, namely, M. tuberculosis. A central antimicrobial mechanism stimulated by IFN-γ and TNF-α is production of reactive nitrogen intermediates (RNIs) via the induction of nitric oxide synthase (NOS)2 and reactive oxygen intermediates (ROIs) via activation of nicotinamide adenine dinucleotide phosphate (NADPH)–dependent oxidative burst. IFN-γ also promotes antimicrobial effects associated with vitamin D and induces autophagy, a mechanism that plays an important role in host defense. It is now clear that the production of IFN-γ depends on prior activation by IL-12 and/or IL-18. IL-12, in concert with TNF-α, induces a cytokine loop resulting in the production of IFN-γ, which sustains the production of IL-12 and IL-18. These observations have been extended to humans, in whom mutations that affect IFN-γ signaling cause susceptibility to M. tuberculosis and salmonellae, as well as to BCG and commonly nonpathogenic mycobacteria, and are termed mendelian susceptibility to mycobacterial disease (MSMD). The mutations are located in genes that include IL12B and IL12RB, which encode subunit β of the IL-12 cytokine and its receptor, respectively, and IFNGR1 and IFNGR2, which encode the IFN-γ receptor. Further unraveling of the molecular basis of human genetic

susceptibility to intracellular bacterial infection will continue to illuminate our understanding of how immunity is similarly orchestrated across multiple infectious diseases. Proinflammatory Cytokines and Phagocyte Attraction The recruitment of more phagocytes to the site of infection represents a vital process in the resolution of infection. Phagocyte recruitment is achieved via the secretion by MPs and endothelial cells of cytokines of the IL-1 family, TNF-α, IL-6, and chemokines. Signaling via IL-1 cognates is considered closely related to that of the TLRs because of the close homology of the cytoplasmic domains of TLRs and IL-1 family receptors. The most studied member is IL-1β, which, in synergy with chemokines and TNF-α, increases the expression of adhesion molecules on the vascular epithelium, thereby promoting extravasation of the inflammatory cell infiltrate into infected tissues. Chemokines are a family of structurally related proteins. The positions of the first two cysteine residues in the protein sequence have been used to divide chemokines into four subfamilies: CC (MIP-1β, MCP-1, MCP-2, MCP-3), CXC (MIP-2, IL-8), C (lymphotactin), and CX3C chemokines (fractalkine), where C represents cysteine and X represents any amino acid other than cysteine. These molecules are critical in controlling the migration of PMNs (IL-8) and monocytes (MCP-1, also known as CCL2) from the bloodstream to infected tissue.24 Recently, the role of chemokines in intracellular infections has been increasingly appreciated, for example, with mice lacking the receptor for CCL2 being deficient in their ability to clear listeria infection. It has been suggested that in the early stages of infection, M. tuberculosis exploits a delay in the mobilization of T-cell immunity to recruit MPs to the site of infection, which preferentially serve as habitat because of a lack of local IFN-γ from T cells. Moreover, M. tuberculosis is thought to infect the relatively sterile lower airways. The lack of commensal bacteria could mean that M. tuberculosis uses cell surface phenolic glycolipid (PGL) to signal epithelial cells to produce the chemokine CCL2 in the absence of signaling via other PAMPs.25 This mechanism then recruits MPs that are more permissive for bacterial growth than those recruited by a more “global” MyD88dependent signaling of TLRs, requiring coengagement of PAMPs on commensal bacteria that are more abundant in the upper airways. The initial macrophage infiltrates could play an important role in early granuloma development.

Cytokine-Induced Host-Protective Mechanisms

Effector Molecules Activation of a membrane-bound NADPH oxidase by stimulation with IFN-γ or immunoglobulin G (IgG) initiates an oxidative burst that generates the ROIs O2−, H2O2, OH−, 1O2, and •OH radical (Table 26.3).26 In human PMNs and blood monocytes that possess myeloperoxidase, ROI activity is further augmented by the formation of hypochlorous acid. Oxidation and/or chlorination of bacterial lipids and proteins result in their inactivation and subsequent bacterial killing. The importance of ROIs in antibacterial defense is underlined by recurrent infections in patients whose phagocytes fail to generate an oxidative burst. Nitric oxide synthase 2 (NOS2) is an inducible cytosolic enzyme in professional phagocytes that delivers NO to the phagolysosome harboring bacteria while consuming O2 and − − L-arginine. NO is further oxidized to NO2 and NO3 . Nitrification and/or oxidation then inactivates bacterial molecules needed for bacterial growth.27 The formation of •NO is catalyzed by NOS2, which is promoted by both immunological stimuli, such

CHAPTER 26  Host Defenses to Intracellular Bacteria

383

TABLE 26.3  Antibacterial Effector Mechanisms of Activated Macrophages and Corresponding

Microbial Evasion Strategies Macrophage Effector Mechanism

Microbial Evasion Strategy

Production of ROIs

Uptake via complement receptors; production of ROI detoxifying molecules (superoxide dismutase, catalase); bacterial ROI scavengers (phenolic glycolipids, sulfatides, lipoarabinomannans) Inhibition of phagosome maturation via blockage of H+ATP pump, indirect effect of ROI-detoxifying molecules Egression into cytoplasm; Resistant cell wall Inhibition of phagosome maturation Modification of cell wall lipid A to resist defensins Expression of microbial siderophores to increase iron uptake Upregulation of bacterial tryptophan synthesis

Production of RNIs Autophagy, intraphagolysosomal killing Phagosomal acidification, phagosome–lysosome fusion Defensins Reduced iron supply (transferrin receptor downregulation, lipocalins) Tryptophan degradation

ATP, adenosine triphosphate; ROI, reactive oxygen intermediate; RNI, reactive nitrogen intermediate.

as IFN-γ and TNF, and microbial products, such as LPS, lipoteichoic acid, and mycobacterial lipids. RNIs exert their bactericidal activity by destroying iron-/sulfur-containing reactive centers of bacterial enzymes and by synergizing with ROIs to form highly reactive peroxynitrite (ONOO−). Despite being highly effective in killing intracellular bacteria, NO production relies on a continuous supply of L-arginine, which becomes limited because of competition with another macrophage enzyme, Arginase-1 (Arg-1). Arg-1 metabolizes L-arginine to produce urea and ornithine and demonstrates antiinflammatory activity. The competitive function of Arg-1 likely regulates collateral tissue damage caused by overexuberant RNIs. The final downstream product of NOS2 activity is citrulline, which is recycled to L-arginine by the enzymes argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl). A mouse deficient in macrophage Asl activity is unable to control mycobacterial infection, highlighting the importance of this recycling pathway. The central role of NOS2 in protection against intracellular bacteria is well established in murine models of infection. Whether NOS2 plays a similarly central role in humans is still unclear. Defensins are small lysosomal polypeptides that are microbicidal at basic pH and are particularly abundant in phagocytes. These include granulysin, present in granules of human natural killer (NK) and cytolytic T (CTLs) cells,28 and cathelicidin, which is regulated by vitamin D in a TLR-dependent manner and is converted by cleavage to the antimicrobial peptide LL-37. Apoptosis and Autophagy Apoptosis is a highly regulated form of cell death that is critical for control of cell turnover, a vital process for tissue homeostasis.29 Macrophage apoptosis also constitutes a defense mechanism, allowing removal of phagocytes containing intracellular bacteria without the need to generate significant inflammation. Apoptosis, in contrast to cellular necrosis, results in cell death without permeabilization of the host cell membrane. The process can be triggered by TNF-α signaling and augmented by IFN-γ, resulting in activation of cellular caspases, mitochondrial membrane permeability, and cytochrome c release. These processes result in cellular disintegration and generation of apoptotic bodies that are engulfed and digested by neighboring phagocytic cells. Apoptosis is protective against L. monocytogenes and Salmonella spp. and is inhibited by M. tuberculosis, which promotes necrotic cell death of infected cells to its benefit via mitochondrial membrane damage and by caspase-independent mechanisms during conditions of high bacterial burden in macrophages.

Noninfected cells engulf bacterial antigens associated with vesicles produced by apoptotic cells. Apoptosis as a prerequisite for this pathway is induced by many intracellular bacteria, including salmonellae, mycobacteria, and listeriae. This cross-presentation pathway in infections with intracellular bacteria adds an essential function to the physiological role of apoptosis in the maintenance of tissue integrity and growth. Upon signaling via IFN-γ, autophagy, a process common to all cells for removal of dysfunctional or damaged cellular organelles, can be harnessed to dispose of intracellular L. monocytogenes and M. tuberculosis in a process termed xenophagy. Signaling via members of the immunity-related guanosine triphosphatase (GTPase) family (IRG family) and the guanylatebinding protein family, TLR2 and TLR4 engagements and the active form of vitamin D3 all act to augment xenophagy. Formation of double-membrane autophagosomes that mature analogously to the phagosomal pathway and fuse with lysosomes that degrade bacteria contained within. The importance of this process is highlighted by polymorphisms in one of the three IRG families of genes in humans, IRGM, being associated with susceptibility to TB. Recently, a host-encoded microRNA, miRNA-155, was shown to potentiate xenophagy during intracellular mycobacterial infection by targeting an endogenous inhibitor of autophagy, Ras homologue enriched in brain (Rheb) by suppressing Rheb expression. Nutrient Deprivation Deprivation of required nutrients to intracellular bacteria is also a strategy employed by the host, markedly so within infected macrophages. Tryptophan degradation is achieved by the enzyme indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan to kynurenine (see Table 26.3). This reaction is induced by IFN-γ in both MPs and IFN-γ-responsive nonprofessional phagocytes and inhibits the growth of C. psittaci and C. trachomatis inside human macrophages and epithelial cells. Similarly, augmentation of NOS2 by IFN-γ and TNF-α depletes intracellular L-arginine, also required for growth of intracellular bacteria.20

EVASION FROM, INTERFERENCE WITH, AND RESISTANCE TO MICROBIAL KILLING Strategies Against Toxic Effector Molecules Many intracellular bacteria have exploited successful strategies against macrophage effector mechanisms (see Table 26.3). One

384

Part THREE  Host Defenses to Infectious Agents

mechanism of evasion is determined by the receptor that is used for pathogen entry into the host cell. Internalization via CRs inhibits the production of IL-12, a cytokine critical in facilitating macrophage activation. Engulfment by this receptor also bypasses activation of the oxidative burst, thereby avoiding ROI production. Similarly, engaging mismatch repair (MMR) and DC-specific intercellular adhesion (DC-SIGN) molecules for uptake triggers secretion of the suppressive cytokines IL-10 and TGF-β. Several intracellular bacteria also produce ROI detoxifiers, including superoxide dismutase and catalase, which nullify oxygen (O2) and hydrogen peroxide (H2O2), respectively. Finally, a number of small bacterial products, such as the phenolic glycolipid and LAM of mycobacteria, scavenge ROIs. Many of the strategies used to counteract the effects of ROIs also overlap in their effects on RNIs. A modification of lipid A renders gram-negative bacteria, including salmonellae, resistant to the effects of host antimicrobial peptides.

are Rab3, -4, -5, -9, -7, -11, and -14. These proteins are associated with different maturation stages of the phagosome and chiefly orchestrate membrane fusion events to allow delivery of vesicular protein cargo to the phagosomal compartment. The mycobacteria-containing phagosome acquires Rab5a but not the late endosomal marker Rab7a, which ultimately mediates fusion of the bacterial-containing phagosome with lysosomes that contain proteolytic enzymes active at low pH. By enabling arrest of this maturation, M. tuberculosis maintains its compartment at an early endosomal stage. This compartment does not acidify, partly because of a paucity of vacuolar H+ adenosine triphosphatase (ATPase); at the same time, it exchanges molecules with the plasma membrane, such as the transferrin receptor, to access iron. Activation of macrophages with IFN-γ restores the normal maturation of the mycobacterial phagosome, resulting in a drop in mycobacterial viability. Francisellae and brucellae are engulfed by phagosomes that acquire the early endosomal markers EEA1 and Rab5a.31 The Francisella-containing vacuole acquires late endosomal markers, but the pathogen escapes into the cytosol by perforating the late endosomal membrane. After a transient phagosomal stage, brucellae enter compartments enclosed by endoplasmic reticulum (ER) membranes to escape delivery to phagolysosomes.

Intraphagosomal Survival Inhibition of phagolysosome fusion represents a major intracellular survival strategy for a number of intracellular bacteria, including M. tuberculosis, Francisella spp., Brucella spp., and L. monocytogenes (Fig. 26.2). After engulfment, these pathogens manipulate the endocytic fate of the phagosome that contains them. This is achieved in part by manipulation of Rab GTPases, proteins required for normal endocytic trafficking, positioned in the phagosome membrane.30 Rab GTPases associated with phagosome maturation of the pathogen-containing phagosome

Phenotypic Plasticity of the Infected Cell The ability of intracellular bacteria to influence the phenotypic fate of both the cell in which it dwells and the cells within lesions that form as a result of unresolved infection is becoming increasingly

Brucella Golgi

Early BCV EEA1 Rab5

ER

Intermediate BCV Lamp-1 ER

Early endosome markers EEA1 Rab5 Late endosome markers Lamp-1, 2 Rab5

Replicative BCVs

Francisella

Actin

Listeria

Rab5 Modified phagosome Mycobacterium

FIG 26.2  Inhibition of Phagolysosome Fusion Represents a Major Survival Strategy for a Number of Intracellular Bacteria, Including Mycobacterium Tuberculosis, Francisella spp., Brucella spp., and Listeria Monocytogenes. The mycobacteria-containing phagosome acquires Rab5 but not the late endosomal markers Lamp-1 and 2, enabling arrest of maturation of this compartment at an early endosomal stage. Francisella spp. and Brucella spp. are engulfed by phagosomes that acquire the early endosomal markers EEA1 and Rab5. The Francisella-containing vacuole acquires late endosomal markers but escapes into the cytosol by perforating the late endosomal membrane. A similar strategy is adopted by L. monocytogenes. After a transient phagosomal stage, brucellae enter compartments enclosed by endoplasmic reticulum (ER) membranes to escape delivery to phagolysosomes. BCV, Brucella-containing vacuole.

CHAPTER 26  Host Defenses to Intracellular Bacteria appreciated. This has already been highlighted by the tendency of M. tuberculosis to use its own membrane lipids to exploit host chemotactic pathways to recruit bacterial growth-permissive macrophages to the site of infection; this process subsequently allows a proliferative head-start before adaptive immunity kicks in and amplifies intracellular defences by the action of cytokines, such as IFN-γ and TNF-α. Moreover, bacterial killing must be tempered inside the granuloma to prevent destruction of host tissue. This is achieved by balancing macrophage phenotypes ranging from a phenotype highly bactericidal, termed “classically” activated, to a phenotype that is more suppressive of inflammation and is associated with wound healing and fibrosis, termed “alternatively” activated. Tipping the balance one way or the other is detrimental for the host in terms of disease. Myeloid-derived suppressor cells (MDSCs) represent a certain stage of development of myeloid cells (both of monocytic and granulocytic lineage).32 Although most of our knowledge stems from their suppressive role in cancer, recent evidence suggests that they play a role in control of chronic infections, such as TB.33 They can be distinguished from canonical MPs and granulocytes by means of distinct surface markers. The granulocytic MDSCs are CD11b+ LY6Ghi Gr1int, whereas monocytic MDSCs are CD11b+ LY6Gneg LY6C+ Gr1hi. More recent findings point to host cell reprogramming resulting from intracellular infection. During intracellular infection of Schwann cells, M. leprae is able to downregulate genes active for the Schwann cell phenotype and upregulate genes that orchestrate differentiation to a “stem cell–like” phenotype. This stem cell–like property allows the infected cell to differentiate further to multiple mesenchymal cell states, such as skeletal cells or smooth muscle cells.34 This ability to regress and then reprogram an infected cell phenotype could play a role in spreading infection throughout the host during leprosy. Recently, mesenchymal stem cells (MSCs) were identified as an intracellular niche of M. tuberculosis in mice, and the equivalent human MSC phenotype could be readily infected in vitro. Because they reside in hypoxic niches and most antimycobacterial therapies are inactive in these conditions, it is feasible that MSCs could maintain the bacteria during long-term infection and could represent a protective niche from drug therapy. Dormant M. tuberculosis has also been detected in hematopoietic stem cells (HSCs) in mice and humans. HSCs are pluripotent, giving rise to both lymphoid and myeloid cell lineages in the blood. Clarification of the pathophysiological context of carriage of M. tuberculosis by both HSCs and MSCs is an exciting prospect.

Escape Into Cytoplasm A successful strategy for survival inside activated macrophages is egression from the phagosome into the cytoplasm, which has been exploited by L. monocytogenes and the various pathogenic Rickettsia spp. (see Fig. 26.2).35,36 This has the advantage of both avoiding the cellular defense mechanisms within the phagosome and providing the bacteria with a nutrient-rich environment. L. monocytogenes possesses several virulence factors to facilitate its escape from the phagolysosome, a pore-forming hemolysin (listeriolysin [LLO]) that acts together with a metalloproteinase, a lecithinase, and two phospholipases to efficiently promote the rupture of the phagosomal membrane and to spread to other cells. M. tuberculosis and M. leprae can also egress from the phagosome into the cytoplasm of macrophages and DCs, a behavior that is mediated by a mycobacterial protein secretion system ESX-1.37 Bacterial virulence factors secreted by ESX-1 may also contribute

385

to increased cell death and are not expressed by the vaccine BCG, which hence does not escape from the phagosome.

T LYMPHOCYTES AS SPECIFIC MEDIATORS OF ACQUIRED RESISTANCE Activated macrophages act as the nonspecific executors, whereas T lymphocytes are the specific mediators of acquired resistance against intracellular bacteria. The dramatic increase in the incidence of TB and other intracellular bacterial infections in patients with AIDS illustrate the central role of T lymphocytes in protection. For instance, 15 million individuals are coinfected with HIV and M. tuberculosis, and HIV increases the risk of developing TB by several orders of magnitude resulting in more than 1 million TB cases annually. At the site of microbial growth, T lymphocytes not only initiate the most potent defense mechanisms available, they also focus this response to the site of encounter, thus minimizing collateral damage to the host. Although protective T-cell responses are multifactorial, they can be reduced to a few principal mechanisms (Fig. 26.3). As previously mentioned, T cells inevitably also produce pathology through cytotoxic antimicrobial defense mechanisms. Moreover, pathogenesis of intracellular bacterial infection is highly influenced by T cells. It is therefore important that the T-cell response be tightly controlled and downregulated, when necessary. Regulatory mechanisms, including regulatory T cells (Tregs), are in place to limit immunopathology.19 Protective immunity involves the so-called conventional T-cell sets, CD4 αβ T cells, and CD8 αβ T cells, as well as unconventional T cells, such as γδ T cells, CD1-restricted αβ T cells, and T cells that recognize antigen in the context of other nonclassic MHC class I molecules, such as mucosal-associated invariant T (MAIT; Chapter 20) cells (see Fig. 26.3). Although these T-cell sets perform different tasks, substantial redundancy exists. Furthermore, these T-cell populations act in a coordinated way in close interaction with other leukocytes. Depending on the etiological agent and the stage of disease, the relative contribution of the different T-cell subsets to acquired resistance may vary. The conventional αβ T cells make up more than 90% and γδ T cells less than 10% of all lymphocytes in the blood and peripheral organs of humans and mice. However, γδ T cells represent a significant proportion of the intraepithelial lymphocytes in mucosal tissues, suggesting a particular role at this important port of microbial entry.

CD4 T Cells The CD4 T-cell population can be further subdivided into distinct subsets, according to their pattern of cytokine production and expression of unique transcription factors that control patterns of gene expression (Chapter 16). At least four major subsets exist, T-helper cell-1 (Th1), Th2, Th17, and Tregs. The first two subsets were discovered over 20 years ago and have been identified in both mice and humans: Th1 cells, which overwhelmingly produce IFN-γ and IL-2, and Th2 cells, which produce IL-4, -5, and -13. The Th1 subset can also be defined on the basis of the T-bet transcription factor and the signal transducer STAT4, whereas Th2 classification is consistent with expression of the transcription factor GATA-3 and signal transducer STAT5. Th17 cells express the retinoid orphan receptor γt (ROR-γt) transcription factor and the signal transducer STAT3. They produce the cytokines IL-17, IL-22, and granulocyte macrophage–colonystimulating factor (GM-CSF). Cytokines of the IL-17 family are strong inducers of granulopoiesis; of proinflammatory mediators,

386

Part THREE  Host Defenses to Infectious Agents ls

cel

nti

nve

co Un

lT ona

IL-10 TGF-β

γδ

IL-1β IL-6 IL-23

Phospholigand P

T

Treg

P

IL-6 IL-17 IL-21 IL-22

IL-27

Lipid Metabolite CD8

TGF-β IL-6 (mouse) IL-21 (human)

Conventional T cells

Cytosol Golgi

MHC I

Th17

TGF-β RA

CD1, MR

1

Peptide

2

IL-12 IL-18 IFN-γ

3 CD4

MHC II

IL-4 IL-25 IL-33

Phagosome

MHC ICOS-L (B7.h)

ICOS

B7.1 B7.2 CD28

PD-L TCR

IFN-γ LT/TNF-α IL-2 GM-CSF

Th1

PD-1

IL-4

Th2

B7.1 B7.2

CTLA-4

T cell Activation

Inhibition

FIG 26.3  T-Cell Stimulation During Infection. (A) Recognition of bacterial antigen by T cells. Antigen originating from intracellular bacteria is presented to conventional CD4 and CD8 T cells. Unconventional T cells including γδ T cells, mucosa-associated invariant T (MAIT) cells, and CD1-restricted T cells are also activated. Human γδ T cells recognize small molecules containing pyrophosphate residues; MAIT cells recognize bacterial metabolites, such as vitamin B2 derivatives, in the context of major histocompatibility complex (MHC)–related (MR) gene products; CD1restricted T cells recognize glycolipids in the context of CD1 molecules. (B) CD4 T cells can be subdivided into different T helper (Th) cells according to their cytokine expression pattern. Th1 cells are critical for protection against intracellular bacteria. They typically produce interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α), lymphotoxin (LT), interleukin (IL)-2, and granulocyte macrophage– colony-stimulating factor (GM-CSF). Th2 cells stimulate humoral immune responses via secretion of IL-4 and IL-5. Other cytokines produced by Th2 cells include IL-13 and IL-25. Th17 cells produce IL-6, -17, -21, and -22, which probably contribute to early protection. Regulatory T cells (Tregs) produce transforming growth factor β(TGF-β) and IL-10, which suppress immune responses. ICOS, inducible costimulatory molecule; PD-1, programmed death 1; PD-L, program death ligand; RA, retinoic acid; TCR, T-cell receptor. See Chapters 9 and 12 for details. (Modified from Kaufmann SHE, Parida SK. Tuberculosis in Africa: learning from pathogenesis for biomarker identification. Cell Host Microbe 2008;4:219–28, with permission of Elsevier.)

IL-4 IL-5 IL-13 IL-25

CHAPTER 26  Host Defenses to Intracellular Bacteria such as IL-6; and of the chemokines CXCL1, CXCL8, and CXCL6, which attract neutrophilic and eosinophilic granulocytes and prolong their survival.38 Th17 cells, too, have limited importance for protection in murine models against primary infection with mycobacteria, salmonellae, and listeriae. However, Th17 cells can drive more rapid Th1 responses against pulmonary TB in mice after vaccination, resulting in enhanced protection. IL-17 is also required for optimally protective Th1 responses during murine F. tularensis infection.39 Despite the convenience of defining T-cell populations in terms of subsets, recent evidence suggests considerable plasticity in cytokine production by T cells. This was first suggested by demonstration that all subsets could produce IL-10, which regulates potency of T-cell responses to limit host collateral damage during immune responses. IL-10 expression might be an intrinsic control mechanism common to all T cells. However, reduction in T-cell potency also favors chronic intracellular bacterial infection. T-cell subsets may acquire the ability to produce additional cytokines by expression of additional transcription factors or by remodeling chromatin structure.40 Future research will redefine T-cell behavior during intracellular bacterial infection.

CD8 T Cells Infection of mice deficient in specific T-cell subsets has conclusively demonstrated a role for CD8 T cells during listeriosis and TB.41 Furthermore, CD8 effector T cells have been identified in granulomas of patients with tuberculoid leprosy with low numbers of bacteria. The cytolytic potential of these T cells can serve two roles in infection with intracellular bacteria, namely, target cell killing or lysis of cells that are unable to control the infection, thus releasing the bacteria for phagocytosis by more activated cells. In humans, CD8 T cell–mediated killing is cell contact dependent and based on production of perforin, granzymes, and granulysin.42 Finally, CD8 T cells are also a potent source of IFN-γ and TNF-α, thus contributing to direct activation of infected macrophages to enhance protective mechanisms (see Fig. 26.3). CD8 T cells recognize antigenic peptides in the context of MHC class I gene products, which are responsible for presentation of antigens residing in the cytosol. Initially, therefore, it was mysterious how CD8 T cells were stimulated by intracellular bacteria, which were thought to have a uniquely restricted phagosomal residence. However, with the knowledge that many intracellular bacteria egress into the cytoplasm, one major mechanism for MHC class I processing became obvious: proteins secreted by bacteria in the cytoplasm undergo antigen processing and presentation similarly to newly synthesized proteins of viral or host origin.35 Yet, alternative contact points for MHC class I molecules and bacterial peptides exist. Cross-presentation by noninfected antigen-presenting cells (APCs) of antigens engulfed within apoptotic blebs from infected cells represents a critical pathway to induce CD8 T cells by phagosomal bacteria (see below). One major advantage of CD8 T cells over CD4 T cells is their recognition of antigen bound by MHC class I gene products, which are expressed by almost all host cells. Thus CD8 T cells recognize professional and nonprofessional phagocytes equally well.

Unconventional T Cells The relevance of the γδ T cells to antibacterial immunity is incompletely understood. Several studies indicate that γδ T cells

387

are rapid producers of IL-17 at sites of bacterial implantation. Transient participation of γδ T cells in protection and a unique requirement for γδ T cells in granuloma formation have been described for murine listeriosis and TB. Murine γδ T cells appear to recognize peptides presented by nonpolymorphic MHC class I molecules, whereas human γδ T cells respond to nonpeptidic phosphorylated metabolites, notably from the isoprenoid pathway of bacterial and host origin.43 MAIT cells are primarily localized at mucosal sites, and current evidence suggests that they play a role in control of bacterial infections in mucosal tissues, such as lung (M. tuberculosis) and gut (gram-negative bacteria) tissues. Antigenic ligands include derivatives of vitamin B2 (riboflavin), produced by many intracellular bacteria, including salmonellae and mycobacteria.44-46 CD1 comprises a group of nonpolymorphic MHC-related molecules that can present glycolipid antigens to unconventional T cells. In humans, group 1 CD1-restricted T cells respond to a variety of microbial glycolipids, including LAM, PIMs, mycolic acids, sulfatides, sulfoglycolipids, and lipopeptides.47 Group I CD1 molecules are absent in mice. The group II CD1 molecule CD1d is present in both humans and mice and controls development of natural killer T cells (NKT cells) that express the NK cell marker NK1.1. Upon antigen activation, these T cells rapidly produce cytokines and are capable of producing both IL-4 and IFN-γ. Bacterial antigens recognized by NKT cells, PIMs from mycobacteria, and glycosphingolipids from Ehrlichia and Sphingomonas spp. have been identified. NKT cells also respond to host endogenous lysosomal lipids loaded onto CD1d.48 In summary, unconventional T cells often recognize nonpeptidic ligands of bacterial origin, emphasizing that they play a particular role in immunity against bacteria, including intracellular bacteria. Because of the highly skewed T cell receptor, these T cells are specific for a limited variety of bacterial ligands. Because of their less demanding antigen recognition and activation requirements, unconventional T cells may fill a gap between prompt innate resistance and the delayed conventional T-cell response.

T-Cell Memory and Regulation of Immune Responses Long-term protective immunity against infectious agents relies on immune memory, which forms the basis for the success of all vaccines. Memory T cells can be divided into central memory T (TCM) cells and effector memory T (TEM) cells, based on differential surface phenotypic and tissue migration patterns.49 TEM cells accumulate in peripheral tissues where they express effector functions, whereas TCM cells persist in lymph nodes, where they rapidly develop into TEM cells after secondary antigen encounter. The tissue-resident memory T (TRM) cells are localized to mucosal sites, where they provide efficient protection against invading pathogens.50 Although it is generally accepted that memory T cells can survive in the absence of persistent antigen, little is known about the induction and maintenance of long-lasting T-cell memory in chronic infections with intracellular bacteria.51

B Cells Although antibodies produced by B cells appear to play a minor biological role in infections with intracellular bacteria, many facultative intracellular bacteria spend some time outside their host cells, where they are accessible to antibodies.52 Thus antibodies contribute to protection against salmonellae. Besides antibody production, B cells are potent APCs for soluble antigens, including

388

Part THREE  Host Defenses to Infectious Agents

lipids presented by CD1c, and secrete many cytokines otherwise associated with T cells, DCs, and macrophages. B-cell signaling via MyD88 during S. typhimurium infection has been associated with B-cell production of IL-10, and mice with B-cell-specific deficiency in MyD88 were more resistant to infection demonstrating that, like T cells, B cells can perform regulatory functions.53

KEY CONCEPTS How Might a Vaccine Work? Activation of innate immunity for instruction of appropriate acquired immune responses (PRRs) Activation of the appropriate array of T-cell populations T-cell secretion of appropriate cytokine combination Efficient development of T-cell memory Efficient activation of antibacterial effector mechanisms Best case scenario: sterile pathogen eradication Second best case scenario: driving the infection “deeper” into latency, thus efficiently preventing reactivation of disease

Regulatory T Cells Intracellular bacteria can cause detrimental inflammation and tissue damage, for example, as a result of IFN-γ- and TNFα-mediated Th1 responses. Under normal circumstances, control mechanisms are in place to limit immunopathology. Such countermeasures are elicited as part of the ongoing immune response during infection. The main cytokines that limit inflammation and control IFN-γ production are IL-10 and TGF-β. Although macrophages and DCs produce these cytokines, their main producers are Tregs, which are the prime cells involved in immune regulation. Natural Tregs are responsive to IL-2 because of high constitutive CD25 expression and are characterized by expression of the transcription factor FOXP3.54 Expansion of Tregs appears to be both antigen dependent and antigen independent. In addition, Tregs selectively express TLRs and can be activated, for example, by LPS and possibly other TLR ligands. This makes their immediate activation during bacterial infection a probable scenario. Although Tregs limit CD8 T-cell responses in experimental listeriosis, their general role in infections with intracellular bacteria has not been fully elucidated. Suppression of T-cell responses and anergy have been clinically documented for TB and leprosy. Although Treg functions can limit detrimental T-cell responses and immunopathology, they can also prevent elimination of bacteria, and hence are likely a key factor in promoting the persistent chronic state of infection with intracellular bacteria.

CONCLUDING REMARKS ON THE HORIZON Design of biomarkers to discriminate latent infection from active tuberculosis (TB) and to predict risk of progression to active TB Development and clinical testing of new drugs for multidrug-resistant/ extensively drug-resistant (MDR/XDR) TB and dormant M. tuberculosis Developing host-directed therapy (HDT) in adjunct to conventional drugs, notably for highly drug-resistant pathogens Development and clinical testing of new vaccines that protect against pulmonary TB in adults Reduction of the unequal burden of TB in developing and industrialized countries by public health measures, education, and socioeconomic advances

Our deepening understanding of the molecular events governing intracellular bacterial infections is allowing development of novel therapeutic and preventive approaches. The need for such interventions is becoming all the more pronounced in the face of increasing levels of antibiotic resistance of bacteria, such as M. tuberculosis, which render canonical drugs that specifically target bacterial molecular processes ineffective. A new approach, termed host-directed therapy (HDT) aims to develop new drugs or repurpose previously approved ones that are directed at host molecular processes.55 Such approaches include monoclonal antibodies (mAbs) to neutralize cytokines, such as TNF-α or IL-6, to abrogate tissue destructive inflammation, repurposed use of licensed drugs, such as ibuprofen and verapamil, to modulate inflammation and enhance antibiotic effectiveness, respectively, and use of immunostimulatory natural molecules, such as vitamin D3 to enhance bacterial killing by xenophagy. These approaches range from preclinical and early-stage clinical development to late-stage clinical development and offer promise to shorten the traditional duration of therapy. It is being increasingly recognized that the interaction between intracellular bacteria and the immune system is not of the “all or nothing” type but is instead a “continuous struggle.” This realization has far-reaching implications for preventive and therapeutic strategies against intracellular bacterial infections. First, vaccination against intracellular bacteria has not yet been effected satisfactorily because of the involvement of several distinct T-cell subsets with different modes of stimulation and activity profiles. Second, chemotherapy has frequently proven to be suboptimal for the sterile eradication of bacteria hidden in cellular niches. A better understanding of the complex crosstalk between cytokines, T lymphocytes, macrophages, and infected host cells will no doubt directly promote the development of improved control measures.55

ACKNOWLEDGMENTS We are grateful to Mary Louise Grossman for excellent assistance and Diane Schad for the figures. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Kaufmann SHE, Rouse BT, Sacks D. The immune response to infectious diseases. Washington, D.C.: ASM Press; 2011. 2. Kaufmann SHE, van Helden PD. Handbook of tuberculosis: clinics diagnostics therapy epidemiology. 1st ed. Weinheim, Germany: Wiley-VCH; 2008. 3. WHO. Global tuberculosis report 2016. Geneva, Switzerland: WHO Press; 2016. 4. White C, Franco-Paredes C. Leprosy in the 21st century. Clin Microbiol Rev 2015;28:80–94. 5. Griffith DE, Aksamit T, Brown-Elliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175:367–416. 6. Sarfo FS, Phillips R, Wansbrough-Jones M, et al. Recent advances: role of mycolactone in the pathogenesis and monitoring of Mycobacterium ulcerans infection/Buruli ulcer disease. Cell Microbiol 2016;18:17–29. 7. WHO. Global Health Observatory (GHO). Data: Buruli ulcer. Geneva, Switzerland: WHO; 2016. 8. Crump JA, Mintz ED. Global trends in typhoid and paratyphoid Fever. Clin Infect Dis 2010;50:241–6.

CHAPTER 26  Host Defenses to Intracellular Bacteria 9. Pappas G, Papadimitriou P, Akritidis N, et al. The new global map of human brucellosis. Lancet Infect Dis 2006;6:91–9. 10. Pathela P, Blank S, Schillinger JA. Lymphogranuloma venereum: old pathogen, new story. Curr Infect Dis Rep 2007;9:143–50. 11. Limmathurotsakul D, Golding N, Dance DA, et al. Predicted global distribution of and burden of melioidosis. Nat Microbiol 2016;1: 15008. 12. Sjostedt A. Intracellular survival mechanisms of Francisella tularensis, a stealth pathogen. Microbes Infect 2006;8:561–7. 13. Dooling KL, Toews KA, Hicks LA, et al. Active Bacterial Core Surveillance for Legionellosis - United States, 2011-2013. MMWR Morb Mortal Wkly Rep 2015;64:1190–3. 14. Land JA, Van Bergen JE, Morre SA, et al. Epidemiology of Chlamydia trachomatis infection in women and the cost-effectiveness of screening. Hum Reprod Update 2010;16:189–204. 15. Wright HR, Turner A, Taylor HR. Trachoma. Lancet 2008;371: 1945–54. 16. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev 2005;18:719–56. 17. Winslow GM, Bitsaktsis C. Immunity to the ehrlichiae: new tools and recent developments. Curr Opin Infect Dis 2005;18:217–21. 18. Biswas S, Rolain JM. Bartonella infection: treatment and drug resistance. Future Microbiol 2010;5:1719–31. 19. Dorhoi A, Reece ST, Kaufmann SH. For better or for worse: the immune response against Mycobacterium tuberculosis balances pathology and protection. Immunol Rev 2011;240:235–51. 20. Kaufmann SHE, Dorhoi A. Molecular determinants in phagocyte-bacteria interactions. Immunity 2016;44:476–91. 21. Yin Q, Fu TM, Li J, et al. Structural biology of innate immunity. Annu Rev Immunol 2015;33:393–416. 22. Taylor PR, Martinez-Pomares L, Stacey M, et al. Macrophage receptors and immune recognition. Annu Rev Immunol 2005;23:901–44. 23. Netea MG. Training innate immunity: the changing concept of immunological memory in innate host defence. Eur J Clin Invest 2013;43:881–4. 24. Le Y, Zhou Y, Iribarren P, et al. Chemokines and chemokine receptors: their manifold roles in homeostasis and disease. Cell Mol Immunol 2004;1:95–104. 25. Cambier CJ, Takaki KK, Larson RP, et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature 2014;505:218–22. 26. Werner E. GTPases and reactive oxygen species: switches for killing and signaling. J Cell Sci 2004;117:143–53. 27. Bogdan C. Nitric oxide and the immune response. Nat Immunol 2001;2:907–16. 28. Lieberman J. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 2003;3:361–70. 29. Rudel T, Kepp O, Kozjak-Pavlovic V. Interactions between bacterial pathogens and mitochondrial cell death pathways. Nat Rev Microbiol 2010;8:693–705. 30. Brumell JH, Scidmore MA. Manipulation of rab GTPase function by intracellular bacterial pathogens. Microbiol Mol Biol Rev 2007;71: 636–52. 31. Salcedo SP, Holden DW. Bacterial interactions with the eukaryotic secretory pathway. Curr Opin Microbiol 2005;8:92–8. 32. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 2009;9:162–74. 33. Knaul JK, Jorg S, Oberbeck-Mueller D, et al. Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis. Am J Respir Crit Care Med 2014;190:1053–66.

389

34. Masaki T, Qu J, Cholewa-Waclaw J, et al. Reprogramming adult Schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection. Cell 2013;152:51–67. 35. Stavru F, Archambaud C, Cossart P. Cell biology and immunology of Listeria monocytogenes infections: novel insights. Immunol Rev 2011;240:160–84. 36. Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009;27:393–422. 37. van der Wel N, Hava D, Houben D, et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 2007;129:1287–98. 38. Iwakura Y, Ishigame H, Saijo S, et al. Functional specialization of interleukin-17 family members. Immunity 2011;34:149–62. 39. Lin Y, Ritchea S, Logar A, et al. Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis. Immunity 2009;31:799–810. 40. O’Shea JJ, Paul WE. Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 2010;327:1098–102. 41. Cox MA, Harrington LE, Zajac AJ. Cytokines and the inception of CD8 T cell responses. Trends Immunol 2011;32:180–6. 42. Walch M, Dotiwala F, Mulik S, et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell 2014;157:1309–23. 43. Bonneville M, O’Brien RL, Born WK. Gammadelta T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol 2010;10:467–78. 44. Ussher JE, Klenerman P, Willberg CB. Mucosal-associated invariant T-cells: new players in anti-bacterial immunity. Front Immunol 2014;5:450. 45. Napier RJ, Adams EJ, Gold MC, et al. The role of mucosal associated invariant T cells in antimicrobial immunity. Front Immunol 2015;6:344. 46. Corbett AJ, Eckle SB, Birkinshaw RW, et al. T-cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature 2014;509: 361–5. 47. Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol 2004;22:817–90. 48. Darmoise A, Teneberg S, Bouzonville L, et al. Lysosomal alpha-galactosidase controls the generation of self lipid antigens for natural killer T cells. Immunity 2010;33:216–28. 49. Lanzavecchia A, Sallusto F. Understanding the generation and function of memory T cell subsets. Curr Opin Immunol 2005;17:326–32. 50. Mueller SN, Gebhardt T, Carbone FR, et al. Memory T cell subsets, migration patterns, and tissue residence. Annu Rev Immunol 2013;31: 137–61. 51. Scott P. Immunologic memory in cutaneous leishmaniasis. Cell Microbiol 2005;7:1707–13. 52. Joller N, Weber SS, Oxenius A. Antibody-Fc receptor interactions in protection against intracellular pathogens. Eur J Immunol 2011;41: 889–97. 53. Neves P, Lampropoulou V, Calderon-Gomez E, et al. Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during Salmonella typhimurium infection. Immunity 2010;33:777–90. 54. Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nat Immunol 2005;6:353–60. 55. Kaufmann SH, Lange C, Rao M, et al. Progress in tuberculosis vaccine development and host-directed therapies-a state of the art review. Lancet Respir Med 2014;2:301–20.

CHAPTER 26  Host Defenses to Intracellular Bacteria

389.e1

MULTIPLE-CHOICE QUESTIONS 1. Which disease caused by intracellular bacterial infection results in the HIGHEST level of global mortality? A. Typhoid B. Tuberculosis C. Legionnaires’ disease

3. Which of these host cell processes is NOT harnessed for protection against intracellular bacterial infection? A. Autophagy B. Necrosis C. Apoptosis

2. Which host cytokine molecule is the MOST important in augmenting host cell defense mechanisms against intracellular bacteria? A. Interleukin (IL)-4 B. IL-17 C. Interferon (IFN)-γ

4. Which bacterial-derived ligand is presented to T cells via major histocompatibility complex (MHC) class I? A. Lipopolysaccharide B. Polypeptide C. RNA

27  Host Defenses to Extracellular Bacteria Marcos C. Schechter, Sarah W. Satola, David S. Stephens

The human host has developed protective mechanisms to interact with the multitude of bacterial species encountered in nature. These host defenses include nonspecific mechanisms of clearance, as well as innate and specific adaptive immune responses. Partly because of these mechanisms, the vast majority of bacterial species do not cause human disease. Many bacterial species have established symbiotic or commensal relationships with the human host and colonize skin and mucosal surfaces. These commensals are generally of low virulence except in individuals whose host defenses are compromised. Given the diversity of the microbial world, a relatively few pathogenic bacterial species or subpopulations of those species have evolved virulence factors or strategies that can overcome or circumvent intact human host defense mechanisms to cause localized or systemic disease. Important bacterial pathogens of clinical importance reside mostly extracellularly (Table 27.1). Examples are bacterial pathogens typically labeled extracellular, such as Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, and Bordetella pertussis, which are transmitted from one individual to another by close contact. Other “extracellular” bacterial pathogens, such as Clostridium spp. Vibrio cholerae, Shigella dysenteriae, enteropathogenic Escherichia coli, and Bacillus anthracis, are transmitted through food, water, animal, or other environmental contact. Staphylococcus aureus is an important extracellular pathogen for humans and can be acquired from other humans, from animals, or through environmental contact. Acquisition of these pathogenic bacteria may be transient, result in variable intervals of colonization, or rapidly cause localized or systemic disease. Extracellular bacterial pathogens can produce acute inflammatory and purulent infectious diseases, such as meningitis, septicemia, pneumonia, urethritis, pharyngitis, inflammatory diarrhea, cellulitis, and abscesses and/or produce disease by the release of toxins. Disease associated with some extracellular bacteria (e.g., Helicobacter pylori) results from chronic colonization. Susceptibility to extracellular bacterial pathogens is enhanced by hereditary, acquired, or age-related defects in innate or adaptive host defenses. Resistance to extracellular bacterial pathogens or their toxins can be accentuated by chemoprophylaxis, by vaccines, and by other immune modulation processes (e.g., passive immune globulin administration). Caution is urged in the interpretation of the term “extracellular.” The classification of bacteria as “extracellular” and “intracellular” is primarily based on observations in vitro and has been challenged by some authors, as some ”extracellular” bacterial species invade host cells as a part of their normal lifecycle and during steps in the disease process (e.g., S. aureus, S. pneumoniae, S. pyogenes, N. meningitidis).1

Conversely, bacteria typically classified as “intracellular” can have an extracellular component to their lifecycle (e.g., Mycobacterium tuberculosis in cavitary lesions). Host defense mechanisms against extracellular bacteria are a continuum. The innate and adaptive immune systems cooperate to protect the host from extracellular bacterial infections. The innate immune system senses bacteria through pattern recognition receptors (PRRs; Chapter 3). These receptors activate antimicrobial defenses and stimulate the adaptive response, all while balancing excessive immune and inflammatory responses with the need to protect against the infecting pathogen.2

CLINICAL PEARLS Distinguishing Clinical Characteristics of Infections With Extracellular Bacteria • • • • • •

Sterilizing immunity Colonization of mucosal surfaces often precedes disease Causes of pyogenic infections T-helper (Th)17 response critical in generating a neutrophilic response Antibodies are protective for some of the major pathogens Effective vaccines available for many of the major pathogens

CLEARANCE AND NONSPECIFIC HOST DEFENSES AT MUCOSAL EPITHELIAL SURFACES Bacteria first encounter a physical barrier, which comprises skin, mucus and mucosal surfaces, and the normal microbiota, as well as nonspecific factors, such as nutrient limitation (e.g., iron), and antimicrobial proteins or peptides. Intact skin and mucosal surfaces provide complex chemical and biological obstacles to extracellular bacteria and are an important line of defense preventing the invasion of these pathogens and their products.3 Humans commonly carry extracellular pathogens asymptomatically on skin and at respiratory and gastrointestinal (GI) mucosal surfaces. To cause disease, pathogens breach or disrupt epithelial barriers. Damage to epithelial barriers as a result of trauma; coinfections; drugs, such as those used in chemotherapy; environmental factors, such as smoking, allergies, or low humidity; and catheterization and intubation circumvent these barriers and allow bacteria access to subcutaneous tissues, blood vessels, and other normally sterile sites. Additionally, acquired and genetic diseases affecting epithelial barriers are also linked to increase in infections. The human epithelium has evolved to prevent colonization and invasion by pathogenic organisms.3 Skin is a relatively dry, acidic (pH 5–6) barrier that contains growth-inhibiting fatty acids and antimicrobial peptides (AMPs) (see below), characteristics that

391

392

Part THREE  Host Defenses to Infectious Agents

TABLE 27.1  Examples of Clinically Relevant Pathogenic Extracellular Bacteria Examples of Human Disease

Selected Mechanisms of Pathogenesis

Special Features Key to Host Infection

Examples of Susceptible Populations/Risk Factors

Staphylococcus aureus

Cellulitis, abscesses, bacteremia, endocarditis, toxic shock syndrome, osteomyelitis, pneumonia, wound infections

Protein A: promotes fibronectin binding PVL: cytotoxic α-toxin: membrane damage TSST-1: superantigen

Asymptomatic colonization, resistant to dehydration

Injection drug users, patients on hemodialysis, defects on Th17 response (Job’s syndrome), Surgical procedures and skin trauma

Streptococcus pneumoniae (Pneumococcus)

Pneumonia, otitis media, meningitis

Capsule: prevents phagocytosis, antigenic variation Pneumolysin: cytotoxic PspA & C: inhibition of complement Neuraminidase, hyaluronidase: spread and colonization IgA1 protease

Asymptomatic colonization, readily acquires new genes through transformation

Smokers, cerebrospinal fluid leak, asplenia, hypogammaglobulinemia, human immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS), unvaccinated children

Streptococcus pyogenes (group A Streptococcus)

Pharyngitis, cellulitis, erysipelas, toxic shock syndrome, necrotizing fasciitis, scarlet fever, rheumatic fever

Hyaluronic acid capsule, M-protein: prevents phagocytosis Streptolysin O & S: cytotoxic Streptococcal pyrogenic exotoxins C5a peptidase

High diversity of Mproteins, molecular mimicry of human antigens

School-age children, crowdedconditions (e.g., military barracks), injury to lymphatic system (e.g., surgical harvest of saphenous vein)

Streptococcus agalactiae (group B Streptococcus)

Neonatal sepsis, pneumonia and meningitis, perinatal infections, bacteremia

FbsA: fibrinogen receptor, promotes adherence Capsule β-hemolysin C5a peptidase β protein: downregulates complement

Asymptomatic colonization, acquisition by infants during birth

Neonates and infants (immunity dependent on passive transfer of maternal antibodies), diabetes mellitus

Neisseria meningitidis (Meningococcus)

Meningitis, bacteremia (purpura fulminans)

Capsular polysaccharide: promotes adherence and prevents phagocytosis Type IV pili: promote attachment to host cells LOS: analogues to LPS, activates TLR4 pathway IgA1 protease fHbp: downregulates the host alternative complement pathway

Molecular mimicry of human antigens, phase and antigenic variation, asymptomatic carriage

Terminal complement deficiencies, hypogammaglobulinemia

Neisseria gonorrhoeae (Gonococcus)

Urogenital infections, disseminated gonococcal infection, pharyngitis

Type IV pili: promote attachment to host cells Opa protein adhesion IgA1 protease LOS: analogs to LPS, activates TLR4 pathway

Phase and antigenic variation, molecular mimicry of human antigens

Terminal complement deficiencies, women during menstrual period (increases risk of dissemination)

Escherichia coli

Urinary tract infections, gastroenteritis, sepsis, neonatal meningitis

Capsular polysaccharide Tissue specific fimbriae Heat-labile enterotoxins: increases intestinal chloride secretion LPS: activation of TLR4

Antigenic heterogeneity of LPS and capsule

Bladder instrumentation, pregnancy

Pseudomonas aeruginosa

Ventilator-associated pneumonia, bronchiectasis

Pili and flagella: attachment to the host and formation of biofilms LPS Exotoxin A Lipases, lecithinases, elastase

Considerable adaptability to changes in environment, large genome size, biofilms

Orotracheal intubation, cystic fibrosis

Clostridium difficile

Colitis

Toxin B: cytotoxic Flagella

Endospore formation, asymptomatic carriage

Antibiotics and other disruptions of the microbiotia

Haemophilus influenzae

Otitis media, pneumonia, epiglottitis, bacteremia, meningitis

LPS with phosphorylcholine Pili: adherence Capsule High-molecular weight adhesins IgA1 protease

Phase variation of pili, asymptomatic carriage

Unvaccinated children, immunocompromised, sickle cell disease, smoking

Helicobacter pylori

Peptic ulcer disease

Urease: colonization of gastric mucosa Flagella: motile in gastric mucus CagA; bacteria-derived carcinogen

Polymorphism of CagA

Crowded living conditions, unreliable source of clean water, living with someone who has H. pylori

Bordetella pertussis

Whopping cough (children), chronic cough (adults)

Pertussis toxin: inhibits neutrophils, macrophages, lymphocytes Pertactin & filamentous hemagglutinin; mediates attachment

Antigenic variation of adhesins

Nonvaccinated adults and children, infants who have not completed vaccine series, adults and adolescents whose immunity has diminished

Species

TSST-1, toxic shock syndrome toxin 1; Psp, pneumococcal surface protein; LOS, lipooligosaccharide; fHbp, Factor H-binding protein; LPS, lipopolysaccharide; CapA, cytotoxinassociated gene A; Ig, immunoglobulin; TLR, Toll-like receptor; PVL, Panton-Valentine leukocidin.

CHAPTER 27  Host Defenses to Extracellular Bacteria

393

present in secretions, such as ABO blood group antigens. Cell adhesion and extracellular matrix molecules, such as fibronectin and proteoglycans, can also inhibit or enhance bacterial binding to epithelial surfaces. The Tamm-Horsfall glycoprotein, found in urine, can bind avidly to a variety of bacteria and facilitate clearance. Proteins, such as lactoferrin (Lf), present at mucosal surfaces, bind iron, an important requirement for bacterial growth. This action may reduce microbial proliferation, but some mucosal pathogens bind Lf and remove iron from the molecule for growth.

Normal Microbiota as Host Defense

FIG 27.1  Mucociliary Host Defense. Scanning electron micrograph of human upper respiratory mucosa showing the ciliated and nonciliated epithelial surface (×16000).41

are detrimental to many bacteria. The constant desquamation of stratified epithelial surface of skin helps in the removal of microorganisms.4 Nevertheless, a complex skin microbiome that can include bacterial pathogens has been identified, and this differs remarkably, depending on location in the body. Disruption of these physical barriers can augment pathogen tissue colonization and invasion. Infections by S. aureus and S. pyogenes, bacteria that can colonize skin, are often preceded by skin damage. Repeated trauma to skin (e.g., dialysis and intravenous drug use) also enhances skin colonization with pathogens, including that by S. aureus. Mucosal surfaces have additional nonspecific antibacterial defenses. The mucociliary blanket of the respiratory tract (Fig. 27.1) and the female urogenital tract (fallopian tube) move bacteria away from epithelial surfaces, as does the flushing of the urinary tract with urine, intestinal peristalsis, and the bathing of the conjunctiva with tears. Lysozyme is found in most mucosal secretions and lyses bacterial cell walls by splitting muramic acid β(1–4)-N-acetyl-glucosamine linkages. The acid pH of the stomach, intestinal peristalsis, and the antibacterial effect of proteolytic enzymes present in intestinal secretions are important GI tract host defenses against many pathogenic bacteria. The GI mucosa has a layer of mucus that acts as a physical shield to bacteria. Mucus is rich in mucin, glycoproteins that limit pathogen binding to other host molecules necessary for mucosal adhesion. Additionally, the mucus layer may function more than as a physical barrier by acting as a diffusion barrier to concentrate antimicrobial proteins at the appropriate epithelial cell surface. The glycocalyx, an extracellular layer of the apical surface of mucosal cells composed of carbohydrates, also protects cells against bacterial attachment. Bacterial attachment and colonization of mucosal surfaces can be inhibited by bacterial binding to human cellular antigens

The human microbiome is now recognized as a major host defense against bacterial pathogens by providing “colonization resistance,” maintaining a balance of commensals to pathogens, and by priming the immune system (Chapter 14).5 Altering or disrupting the normal microbiota by antibiotics facilitates the expansion of enteric pathogens as Clostridium difficile and Salmonella typhimurium or selection of antibiotic-resistant members of the microbiome. Similarly, changes in human physiology, for example, exposure of skin to elevated temperatures and humidity, chronic stress, host immune suppression, or active behavioral changes, such as smoking, can cause a commensal-to-pathogen switch. Recent studies have demonstrated that certain resident microbiota can resist pathogen colonization and infection. For example, matched volunteers were inoculated with Haemophilus ducreyi into the arms, and the subsequent infection either resolved or resulted in formation of abscesses; characterization of the skin microbiome before, during, and after the experimental inoculation showed that the microbiomes of those with pustule formation and of those with resolved infection were distinct and influenced the course of the H. ducreyi infection.6 The interaction of the microbiome with the immune system is also important for defense against extracellular pathogens. Normal microbiota facilitate a high level of priming of the immune system by maintaining high levels of major histocompatibility complex (MHC) class II molecule expression on macrophages and other antigen-presenting cells (APCs). PRRs (see below) are traditionally known to recognize microbial molecules during infection; however, ligands for PRRs are abundantly produced by the resident microbiota during normal colonization. The integrity of the intestinal epithelial layer is dependent on activation of Toll-like receptors (TLRs; see below) by normal microbiota.4 Stimulation of TLR-5 has been shown to increase resistance to E. faecium infection in a murine model. Activation of nucleotide oligomerization domain 1 (NOD1) receptors by gut resident microbiota is necessary for priming of the innate immune system. Additionally, resident microbiota produce such factors as bacteriocins, lantibiotics, and phenol-soluble modulin (PSM), which function in a similar manner to that of host-derived antimicrobial proteins and peptides (APPs; see below), suggesting an important host defense strategy against pathogen colonization.7 Importantly, members of the resident microbiota can cause disease, particularly with loss of epithelial integrity and translocation to a different host tissue.

Antimicrobial Peptides and Antimicrobial Proteins Pathogens colonizing or invading epithelial surfaces are confronted with APPs, which can be produced both by the host and the microbiota (Table 27.2).7 In addition to pathogen killing, APPs control host physiological functions, such as inflammation, angiogenesis, and wound healing. They also limit pathogen colonization and shape the composition of the host microbiome

394

Part THREE  Host Defenses to Infectious Agents

TABLE 27.2  Antimicrobial Proteins (AMPs) Against Extracellular Bacteria AMP

Tissue/Cell Sources

Mechanism of Action

Target Organisms

α-defensins

Small intestines, Paneth cells

β-defensins

Large intestines, skin, respiratory tract epithelial cells Large intestine, skin, lung, urogenital tract Skin, intestine, respiratory epithelia, placenta Skin, urogenital tract Abscesses/neutrophils Small intestines Neutrophils, epithelial cells

Membrane disruption; inhibits complement activation; chemoattracts dendritic cells Membrane disruption; lipid II binding

Gram-positive bacteria Gram-negative bacteria Gram-positive bacteria Gram-negative bacteria Gram-positive bacteria Gram-negative bacteria Gram-positive bacteria Gram-negative bacteria Escherichia coli Staphylococcus aureus Gram-positive bacteria Gram-negative bacteria

Cathelicidin (LL37) RNases Psoriasin (S100A7) Calprotectin (S100A8-A9) C-type lectins Bactericidal/permeabilityincreasing protein (BPI) Lysozyme

Membrane disruption Unknown Unknown Metal Chelation Peptidoglycan recognition Neutralizes lipopolysaccharide

Skin, body fluids, tears, intestinal Paneth cells Sweat glands

Degrades peptidoglycan

Peptidoglycan recognition proteins

Liver, intestines, skin, neutrophils

Activates bacterial two-component systems; targets peptidoglycan

Phospholipase A2

Tears, intestines

Hydrolysis of bacterial phospholipids

Dermcidin

Membrane disruption

TABLE 27.3  Immune Microbial Pattern

Recognition Molecules

Toll-like receptors (TLRs) Nucleotide-binding oligomerization domain (NOD), caterpillar proteins, peptidoglycan recognition proteins (PGRPs) RNA helicases/PkR Complement proteins: C1q, C1 inhibitor Antimicrobial peptides Collectins and surfactants C- and S-lectins: mannose-binding lectin, L-ficolin

and are both constitutively present and inducible by infection and injury.7 Humans produce two main classes of APPs: defensins and cathelicidins. Defensins, for example, are expressed in skin, intestines, and the respiratory tract and have activity against gram-positive and gram-negative bacteria. Interestingly, APPs expression can be altered by epithelial injury. Keratinocytes of inflamed psoriatic lesions produce increased levels of certain APPs, and patients with such lesions rarely have secondary bacterial infections. In contrast, keratinocytes from patients with atopic dermatitis have dampened APP production in response to inflammation, and colonization and superinfections of the skin by S. aureus are common. Not surprisingly, successful pathogens have developed several mechanisms to counteract APPs. Gonococci, for example, counteract APPs with efflux pumps.

RECOGNITION OF EXTRACELLULAR BACTERIA AND ACTIVATION OF THE IMMUNE SYSTEM Immune pattern recognition molecules (Table 27.3) are a major arm of the innate immune system and are released or expressed by a range of host cells, including lymphocytes, macrophages, or tissue histiocytes, dendritic cells (DCs), polymorphonuclear leukocytes or neutrophils (PMNs), and epithelial cells. The

Gram-positive bacteria Some gram-negative bacteria activity Gram-positive bacteria Gram-negative bacteria Especially active against grampositive bacteria Gram-negative bacteria Gram-positive bacteria

discovery and characterization of specific pattern recognition molecules (see below) has revolutionized our understanding of the initial specific events occurring between microbes and human cells. The identification and function of these molecules is rapidly expanding and include TLRs, NOD and caterpillar proteins, RNA helicases, complement proteins, antimicrobial peptides, collectins and surfactants, C-lectins, and S-lectins, such as mannose-binding lectin and L-ficolin.

Pattern Recognition Receptors Innate immune recognition relies on the detection of unique molecular structures found on microorganisms by host PRRs.8,9 TLRs and NOD-like receptors (NLRs) are the best studied PRRs (Table 27.4). TLRs (TLR1–11) (Chapter 3) are found on macrophages, neutrophils, and other host cells. These receptors recognize a variety of microbial ligands or pathogen-associated molecular patterns (PAMPs), including lipoproteins, lipopolysaccharide (LPS), flagellin, and nucleic acids produced by gram-negative and/or gram-positive bacteria. Alterations (polymorphisms) in TLRs (e.g., TLR4) and other pattern recognition molecules are associated with susceptibility or severity of specific infections (e.g., sepsis).10 TLR expression can be regulated by type I interferons (IFNs) and by microRNAs (miRNAs), and the dysregulation of TLRs can be involved in acute and chronic inflammatory diseases and cancer.11 The NLRs are a family of intracellular receptors, some of which function as PRRs.8 NOD1 and NOD2 are well characterized as PRRs to extracellular pathogens, such as S. flexneri. Importantly, in concert with TLR signaling, NLR can respond to a variety of PAMPs by forming the inflammasome complex. Inflammasome activation generates interleukin-18 (IL-18) and the active form of IL-1, an important step in the immune response to many bacteria. In addition to PAMPs, danger-associated molecular patterns (DAMPs), typically associated with activation of the innate immune system upon injury by noninfectious mechanisms, can be released both by the host and bacteria and result in amplification of the inflammatory response.

CHAPTER 27  Host Defenses to Extracellular Bacteria TABLE 27.4  Pattern Recognition

Receptors (PRRs) Recognize PathogenAssociated Molecular Patterns (PAMPs) From Various Bacteria PRRs

PAMPs

Microbes

Toll-like receptor (TLR)2/1 TLR2/6

Triacyl lipoproteins

Bacteria

Diacyl lipoproteins Lipoteichoic acid Peptidoglycan Porins Lipopolysaccharide (LPS) Flagellin

Mycoplasma Gram-positive bacteria Gram-positive bacteria Bacteria (Neisseria) Gram-negative bacteria

TLR2 TLR4 TLR5

TLR7/8 TLR9

RNA CpG-DNA DNA

TLR11 Nucleotidebinding oligomerization domain (NOD)1 NOD2

Not determined Meso-diaminopimelic acid

NOD-like receptor (NLR) P3 NLRP1 NLRP1b NLRC4

Muramyl dipeptide (MDP)

Whole pathogens Toxins, LPS, MDP, and RNA MDP Microbial toxin Flagellin

Flagellated bacteria (Helicobacter pylori, Salmonella) Group B Streptococcus Bacteria (Salmonella) Bacteria (Staphylococcus at low MOI) Uropathogenic bacteria H. pylori, Bacillus spp., Campylobacter jejuni, Pseudomonas aeruginosa Streptococcus pneumoniae, Staphylococcus aureus, Salmonella typhimurium S. aureus Bacteria Bacteria Bacillus anthracis P. aeruginosa

MOI, multiplicity of infection.

Complement Complement, a series of more than 20 proteins, is activated by microbial surfaces (alternative complement [AP] cascade) or via antibody or by the mannose-binding lectin system (Chapter 21). Complement activation leads to microbial lysis and the release of opsonins and chemoattractant molecules for phagocytic cells.13 The classical complement pathway (CP) can be initiated either by antibody binding to cell surface epitopes or by antibodyindependent autocatalytic activation of C1 to form C1q. Initiation of the APs by bacterial products or mannose-binding protein leads to the direct deposition of the C3b complex on the bacterial surface. Complement activation results in generation of opsonins (as C3b), anaphylatoxins (as C3a), and activation of the late components of the complement pathway, which results in the formation of a membrane attack complex (MAC). Gram-positive extracellular pathogens resist the bacteriolytic action of the MAC as a result of a thick peptidoglycan layer, which impedes the insertion of the MAC C5b-9 complex. Gram-negative bacteria can resist the MAC through structural alterations in their LPS (the possession of O antigen keeps the MAC at a distance from the bacterial surface) or by masking or deleting the epitope(s) responsible for binding bactericidal antibody.

Dendritic Cells DCs sample live bacteria at the mucosal surface, traffic to mucosal lymphoid tissue, and induce B cells to produce bacteria-specific

395

immunoglobulins.4 In most tissues, DCs are at low level of activation and are immature, but upon activation, they take up and process antigen. DCs are rich in PRRs (e.g., TLRs), and microbe–PRR interaction has a key role in shaping the T-cell response.14 For example, TLR5 stimulation by bacterial flagellin can induce a Th17 response and B-cell immunoglobulin A (IgA) production. Skin contains a major supply of tissue DCs (Langerhans cells), and their involvement in combating skin and soft tissue infections must be considered along with their function and contribution to stimulating immunity during vaccination. Limited information is available regarding the role of DCs in host resistance to extracellular bacteria, but some studies have examined the interaction between bacteria and DCs. For instance, Unkmeir et al. studied the interaction of serogroup B meningococci with DCs.12 Infection of DCs by meningococci resulted in a significant and rapid production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, and IL-8 through a lipooligosaccharide (LOS)–dependent mechanism.12 Murine studies provide some insight into the mechanisms extracellular bacteria use to avert phagocytosis by DCs. For example, S. suis polysaccharide capsule reduces bacterial adhesion to the DC plasma membrane.15 Once bacteria are internalized, they must resist degradation in the DC autophagolysosomes.

KEY CONCEPTS Host Defenses and Immune Response at Epithelial Surfaces to Extracellular Bacteria • Clearance and nonspecific host defenses at skin and mucosal surfaces • Epithelial barriers • Antibacterial factors (fatty acids, antimicrobial peptides, lysozyme, phospholipase A2) • Mucociliary activity • Normal microbiota • Adherence blocking molecules • Specific immune defenses at mucosal surfaces • Innate immune mechanism • Immunoglobulins • Phagocytosis at mucosal surfaces • Mucosa-associated lymphoid tissue (MALT), gut-associated lymphoreticular tissue (GALT), bronchus-associated lymphoid tissue (BALT)

Macrophages Phagocytic cells, macrophages, and PMNs are also present at mucosal surfaces (Fig. 27.2). These cells express PRRs and migrate to mucosal surfaces by chemotaxis and diapedesis between epithelial cells. Macrophages are also encountered after crossing the epithelial barrier. Specialized epithelial M cells of mucosal surfaces are key sites for antigen sampling, including viruses, and bacteria and macrophages surround these sites.16 However, enteroinvasive pathogens, such as Shigella, can resist macrophages. Shigella induce macrophage apoptotic death by direct interaction of the bacterial protein IpaB with IL-1β-converting enzyme.

Polymorphonuclear Leukocytes In areas of epithelial inflammation, PMNs can be recruited to mucosal and skin surfaces. PMNs are more effective in the presence of specific immune defenses, such as antibody and complement components. PMNs express PRRs and have both oxygen-dependent and oxygen-independent mechanisms of killing (Chapter 3) (Fig. 27.3). Activated neutrophils can release granule proteins with direct antibacterial action (e.g., bactericidal/ permeability-increasing [BPI]) or degradative activity (e.g.,

396

Part THREE  Host Defenses to Infectious Agents deficiency, to name a few). Bacterial infections associated with phagocytic dysfunction are described elsewhere (Chapter 22).

Innate Lymphoid Cells

FIG 27.2  Bacterial Phagocytosis at Mucosal Surfaces. Transmission electron micrograph of phagocyte engulfing Neisseria meningitidis at a human respiratory epithelial mucosal surface (×19 000).41

Innate lymphoid cells (ILCs), a heterogeneous group of cells of the innate immune system, have lymphoid morphology but lack the capacity for rearrangement of the antigen receptors, a cardinal feature of the cells of the adaptive immune system.18 Conventional natural killer (NK) cells, better known for generating inflammatory cytokines and cytotoxic activity against malignant cells and cells infected by viruses, seem to have a role in the defense against bacterial pathogens. Murine study data suggest that conventional NK-cell activation by lung macrophages is protective against S. aureus pneumonia.19 A subset of ILCs, known as NK-like cells, produces IL-22 and has been found in mucosal sites, where these ILCs appear to have a protective activity against bacterial pathogens. IL-22 derived from these cells modulates AMP expression by epithelial cells. NK-like cells have minimal cytotoxic activity and are not strong producers of IFN-γ, core characteristics of conventional NK cells.20

Lymphocytes Carbohydrate capsule Antiphagocytic structures Secreted Leukocidin bacterial C5a peptidase products that lyse phagocytes or impede chemotaxis

Pilus

FIG 27.3  Bacterial Resistance to Polymorphonuclear Leukocytes (PMNs) in the Extracellular Environment. The two principal mechanisms of bacterial resistance to PMN killing. These consist of resistance to phagocytosis as a result of bacterial surface components (e.g., capsule or pili) and the action of extracellular proteins that can lyse PMNs (e.g., leukocidins) or decrease chemotaxis (e.g., C5a peptidase). Bacteria growing in biofilms may be more protected from PMNs than are bacteria growing in the planktonic state.

elastase) and chromatin containing the antibacterial histone H2A.17 These released compounds work together to form extracellular fibers, termed neutrophil extracellular traps (NETs), which can trap and kill gram-positive and gram-negative bacteria and degrade their virulence factors as well. NETs have been observed in instances of acute inflammation (experimental dysentery and spontaneous appendicitis) and provide a mechanism for reducing bacterial spread at sites of acute infection. The importance of PMNs in host defense against extracellular pathogens can best be highlighted by the increased frequency of bacteremias and other life-threatening infections in patients with neutropenia or those individuals with neutrophil deficits (e.g., chronic granulomatous disease, Chediak-Higashi syndrome, or specific granule

Th1 cells are characterized by IFN-γ and function to activate macrophages to phagocyte and kill pathogens. While this mechanism of pathogen elimination is primarily directed against pathogens with a predominant intracellular lifecycle, Th1 cells are relevant for typical extracellular bacteria as pneumococcus and S. aureus.21 The neutrophilic response to extracellular bacteria is primarily coordinated by Th17 cells.22 Animal models have suggested that the Th17 response is central for protection against a wide variety of gram-positive and gram-negative bacteria. For example, Th17 response has been shown to induce nasopharyngeal clearance of Pneumococcus in both animal models and in children. Differentiation toward the Th17 subtype appears to be favored by strong antigenic signals and broad activation of pathogen recognition receptors. IL-17 and IL-22, the signature interleukins (ILs) of the Th17 response, promote AMP secretion by epithelial cells, neutrophil migration, and epithelial integrity. The increased susceptibility of subjects with Job’s syndrome to S. aureus infections demonstrates the importance of the Th17 response in humans. One in five cells in the intestinal epithelium is a lymphocyte. Mucosa-associated lymphoreticular tissue (MALT) comprises intraepithelial lymphocytes (IELs), lamina propria lymphocytes, and lymphoid follicles (e.g., Peyer patches) and is sometimes divided into gut-associated (GALT), bronchial-associated (respiratory tract) (BALT), and genitourinary tract lymphoid tissues (Chapter 20).23 Lymphocytes are important for homeostatic regulation and the maintenance of immune response against microbes at mucosal surfaces, including “extracellular” bacteria. These cells express PRRs (e.g., TLRs), have constitutive cytotoxic activity, secrete chemokines and cytokines important in regulation and host defense, and act in concert with mucosal epithelial cells and exocrine glands. Innate T cells represent a heterogeneous group of cells that possess T-cell receptors (TCRs) and are restricted to MHC-like molecules.24,25 Unlike other T cells, innate T cells gain effector capacity before exiting the thymus and therefore can respond more readily to stimuli, including infections. This has led to the idea that innate T cells provide a bridge between the innate and adaptive immune systems during infections. Invariant natural killer T (iNKT) cells, a subset of innate T cells, are restricted to

CHAPTER 27  Host Defenses to Extracellular Bacteria

397

the MHC-like molecule CD1d. iNKT cells can be directly activated by bacterial pathogens as a result of binding of cell wall components of gram-negative bacteria to CD1d. Interestingly, cytokines secreted by APCs that have encountered bacterial pathogens can increase iNKT-cell accumulation of self-lipid antigen for CD1d activation. Mucosa-associated invariant T (MAIT) cells are restricted by MR1, a MHC-like receptor that binds molecules derived from the microbial riboflavin synthesis pathway.24,26 Both gram-positive and gram-negative bacteria have been shown to activate MAIT cells. Murine models of intraperitoneal inoculation of gram-negative bacteria suggest the MAIT cells are important for the early clearance of pathogens.25,26

Immunoglobulins Immunoglobulins (Igs), principally secretory IgA and IgG, are present at mucosal surfaces and in mucosal secretions. Important in the generation of these immunoglobulins at mucosal surfaces is the dissemination of IgA and IgG class–committed B- and T-helper (Th) cells with specificity to an antigen encountered and processed at one mucosal site to local and distant mucosal sites. Protective mucosal antibodies against bacteria may be derived from prior colonization, vaccines, or shared cross-reactive antigens on normal flora. Mucosal Igs may neutralize bacterial toxins, facilitate phagocytosis or bactericidal activity, inhibit bacterial adherence ligands, or sterically hinder other events necessary for bacterial colonization and invasion. Many extracellular bacterial pathogens (N. meningitidis, N. gonorrhoeae, H. influenzae, certain streptococci) colonize and/or infect mucosal surfaces where protective IgA1 antibodies could become available.27 These pathogens secrete an IgA1 protease that cleaves IgA1, thereby inactivating the molecule. IgA1 protease can also recognize other substrates, notably lysosomal-associated membrane protein 1 (LAMP-1), which are important in host defense. Bacterial infections associated with abnormal immunoglobulin production or function are summarized in Chapter 34.

MECHANISM OF IMMUNE EVASION AND DISEASE BY EXTRACELLULAR BACTERIA To colonize human epithelial and mucosal surfaces, bacteria must overcome the local host defense mechanisms described above. After navigating these defenses, adhesion to host cells is usually the first important step for bacterial pathogens (Fig. 27.4). Initial attachment of bacteria to human epithelial cells is, in part, mediated by pili, fimbriae, or other bacteria ligands or adhesins, and close adherence of bacteria to the human cell-surface receptors involves the cell wall, outer membrane proteins, LPS, and other bacterial surface structures. The attachment of bacteria to human epithelial cells prevents elimination of bacteria from the host. Attachment can also induce host cell pathways leading to cytoskeletal rearrangements, such as elongation and branching of the microvilli, the accumulation of actin, and calcium efflux, which facilitates close adherence and invasion of epithelial cells by normally “extracellular” bacteria, especially at sites with fluid movement. Strains of E. coli that successfully colonize the bladder and cause renal infection possess pili that allow adhesion to the renal epithelium.28,29 Type IV pili are fundamental for attachment of gonococci to the male reproductive tract and play a role on the attachment of meningococci to vascular endothelial cells.28,30 Meningococcal pili also facilitate twitching motility and microcolony formation, which allows the penetration of mucus and

FIG 27.4  Colonization and Adherence of Extracellular Bacteria at Mucosal Surfaces. Scanning electron micrograph of Neisseria meningitidis adherence and microcolony formation of a human upper respiratory mucosa (×16 250).41

provides initial attachment. The pneumococcal CbpA surface protein promotes mucosal adhesion and dissemination.29 Bacteria utilize several mechanisms to avert the host immune response to bacterial surface antigens (see Table 27.1). Phase variation of adhesins is a mechanism of immune evasion common to pathogenic Neisseria spp. Meningococcus, for example, utilizes phase variation of the adhesion protein Opa and type IV pili during the process of colonization of human upper respiratory mucosal surfaces.31 Sialylation of LPS, a potent inducer of host inflammatory response, is an example of bacterial “hiding” of surface antigens. For example, sialylation of lipooligosaccharide, a molecule analogous to LPS, in meningococci has been shown to increase resistance to CP and AP complement-mediated killing by decreasing the deposition of C3b and IgM on the cell surface, irrespective of capsular phenotype. Many pathogenic extracellular bacteria interact with components of the complement system to induce negative regulation of the complement pathway.29 Binding of human factor H (hfH) by meningococci factor H-binding protein (fHbp) downregulates the host AP and helps the organism to evade host innate immunity and is now included in the new serogroup B vaccines.32 Proteolytic degradation of IgA1 present in the urogenital and respiratory tracts is used to avert the action of the humoral system.33 The elaboration of superoxide dismutase and catalase can reduce the efficacy of oxygen (O2)–dependent killing of bacteria, but the high levels of O2 radicals that accumulate in PMNs probably overcome these bacterial enzymes, as evidenced by the susceptibility of S. aureus to intraleukocytic killing. Several extracellular bacteria possess polysaccharide-rich capsules that resist phagocytosis. Polysaccharide capsule antigens can mimic human

398

Part THREE  Host Defenses to Infectious Agents

antigens. Antigen mimicry can lead to autoantibodies, as in the case of rheumatic fever and glomerulonephritis after S. pyogenes infection. Antigen mimicry can also dampen the immune response to bacterial antigens, as in the case of serogroup B Meningococcus. Other microbial surface structures, such as the pili of the gonococcus, can “stiff-arm” neutrophils, keeping them at a distance. A number of pyogenic bacteria (e.g., S. aureus) secrete leukocidins, which lyse phagocytes. Other pathogens (e.g., group A streptococci) inhibit chemotaxis of neutrophils through the elaboration of enzymes (e.g., C5a peptidase) that proteolytically cleave chemotactic signals. Some bacteria possess mechanisms to prevent opsonization by changing surface antigens.30 Many bacteria form biofilms, which shield these microorganisms from host defense molecules and antibiotics.34 Leukocytes that invade S. aureus biofilms exhibit impaired phagocytosis and decreased ability to kill bacteria. In addition, biofilm matrices can protect bacteria from antibody-mediated phagocytosis. As previously noted, many “extracellular” bacteria have an intracellular component to their lifecycle. The intracellular environment provides protection from proteins of the complement system, Igs, and nonspecific barriers to infection present in the epithelia.28 The entry of bacteria into epithelial cells provides access to nutrients and protection from host defenses, allows protected multiplication, and leads to shedding of organisms back to the mucosal surface, to facilitate transmission and further spread of the infection on the epithelium. Attachment can also initiate epithelial cell apoptosis or toxin-mediated cell death and lead to the breakdown of the epithelial barrier.

HOST RISK FACTORS FOR LOCAL AND SYSTEMIC INVASION BY EXTRACELLULAR PATHOGENS Bacteria that breach mucosal and skin barriers and reach submucosal tissues of sites, such as pulmonary alveoli or the middle ear and/or the bloodstream, induce immune responses, including cytokine release, phagocytosis, complement activation, antibody release or production, and other local or systemic induction of the inflammatory cascade (Fig. 27.5). The survival of bacteria following colonization of the epithelium and access to the bloodstream depends on the integrity of the host immune response (including variability caused by genetic polymorphisms) and on the ability of the bacteria to resist this host immune response. Host factors that increase the risk for the development of systemic disease as a result of extracellular bacteria include polymorphisms in innate immune mechanisms, the absence of bactericidal or opsonizing antibodies, deficiencies in the complement pathways, and an absence of or reduction in neutrophil function or levels (see Table 27.1). Complement deficiencies, either congenital or acquired, increase the risk for invasive bacterial diseases (Chapter 21). Because C3 plays a critical role in the complement cascade, congenital C3 deficiency or conditions that reduce C3 (e.g., systemic lupus erythematosus, cirrhosis, nephritis, C3 nephritic factor) increase the risk for invasive disease due to pyogenic bacteria, such as S. pneumoniae and N. meningitidis. Mannosebinding lectin (MBL) is a plasma opsonin that initiates complement activation. MBL gene polymorphisms are found in children with meningococcal and pneumococcal sepsis. Properdin deficiency, leading to defective AP killing, is also associated with severe and recurrent meningococcal infections. Terminal complement deficiencies (C5–C8) are also associated with recurrent

LPS, PG monomers, DNA repeats Lipoproteins, Teichoic acid Microbial toxins, other microbial components (Superantigens) Pattern recognition receptors (e.g., TLRs) Cytokine stimulation Coagulopathy Kinin stimulation Prostaglandins Leukotrienes PAF Fibrin deposition DIC

TNF-a IL-1 INF-γ IL-6, IL-8 IL-10

Complement activation C5a C3a Leukocyte chemotaxis Inflammation Nitric oxide

Generalized endothelial damage Vascular leak Tissue edema Vasodilation Leukocyte activation Bleeding Temperature dysregulation (e.g. fever) Tachycardia, hyperventilation Hypotension (↑CO, ↓SVR) Pallor, peripheral vasoconstriction Cutaneous signs Multiorgan failure (ARDS, renal failure) Altered mental status Shock Death

FIG 27.5  Inflammatory cascade initiated during sepsis.

invasive bloodstream meningococcal and gonococcal infections, indicating an important role for insertion of the complement MAC in the bactericidal activity of human serum against pathogenic Neisseria. In adults, 10–20% of invasive meningococcal disease has been associated with a defect in the complement system. In infants, antibacterial activity wanes as levels of passively transferred maternal antibody fall. This waning of antibody is correlated with the highest incidence of several “extracellular” pyogenic bacterial diseases (caused by S. pneumoniae, N. meningitidis, H. influenzae type b) in young children. During childhood and adolescence, levels of bactericidal antibodies rise and rates of these diseases decline. Specific antibodies are acquired through carriage and through cross-reacting epitopes on other commensal species. For example, cross-reactive antibodies to N. meningitidis are acquired by colonization with commensal Neisseria spp. (e.g., Neisseria lactamica) and unrelated bacteria (e.g., Enterococcus faecium, Bacillus pumilus, and E. coli). The lack of bactericidal antibodies against a strain recently acquired in the upper respiratory tract is an important risk factor for invasive meningococcal disease. In addition to defects in innate immunity, Igs, and complement deficiencies, human genetic polymorphisms are associated with an increased risk or severity of bacterial diseases. For example,

CHAPTER 27  Host Defenses to Extracellular Bacteria FcγIIa (CD32) receptor polymorphisms, Fcγ-receptor III (CD16), MBL, TLR4, tumor necrosis factor (TNF) promoter region polymorphisms, plasminogen activator and inhibitor expression, and hereditary differences in cytokine induction influence susceptibility to meningococcemia. Each of these polymorphisms can influence the course of invasive bacterial infection by influencing the response of the inflammatory cascade.

THERAPEUTIC PRINCIPLES Sepsis • Early effective antibiotic therapy associated with improved outcomes • Lack of “source control” (e.g., removal of infected lines, drainage of abscesses) linked to poor outcomes even in the presence of effective antibiotics • Intensive and supportive care, management of fluid, electrolytes, and respiratory function • Insulin for glucose control—unknown mechanism of protection; neutrophils have impaired function in the presence of hyperglycemia, insulin can have antiapoptotic effects

DELETERIOUS HOST RESPONSES Inflammation and Autoimmunity The host immune response can be the leading cause of tissue injury in the acute phase of an infection. Brain edema and infarcts, which are devastating consequences of pyogenic meningitis, occur as a result of the host inflammatory response. Corticosteroids are currently recommended as an adjunctive therapy to pneumococcal meningitis, an acute pyogenic infection. The use of small molecules targeting specific immunological pathways is an area of ongoing research. As acute infections are initially characterized by an inflammatory response followed by an antiinflammatory response, the timing of use of these compounds is of the utmost importance.35,36 Infections can lead to autoimmune diseases. Molecular mimicry between a bacterial antigen and a host protein is a mechanism of generation of autoantibodies. Examples include rheumatic fever and glomerulonephritis after S. pyogenes infections, reactive arthritis following Chlamydia trachomatis urethritis, and the Guillain-Barré syndrome following Campylobacter jejuni enteritis. Molecular mimicry can also limit selection of epitopes for vaccine development.

Sepsis Septicemia remains a leading cause of death in the United States and accounts for several billion dollars in health care expenditure.35,36 Both gram-negative and gram-positive bacteria can rapidly multiply in the bloodstream and trigger sepsis and septic shock (Table 27.5). Septic shock is a result of an initial and widespread systemic proinflammatory response, resulting in hypotension, organ failure, and death. The later phase of sepsis is also characterized by an antiinflammatory response. Although survival of patients with the acute phase of sepsis has improved, advances on treatment and prevention of death, which can be secondary to nosocomial infections, have been slower. These secondary infections are often caused by less virulent organisms, likely as a result of “immunoparalysis” (as a result of an exaggerated antiinflammatory reaction) and also breaching of the physical barriers to infection as a result of invasive medical procedures (e.g., intravenous lines, intubation, and bladder catheterization). The systemic inflammatory cascade of sepsis is initiated by recognition of PAMPs by PRR, both in extracellular

399

TABLE 27.5  Examples of Extracellular

Bacteria Causing Sepsis by Presumed Site of Infection Lung Abdomen Urinary tract Soft tissue Intravenous line Other

Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa Escherichia coli, mixed anaerobic infections E. coli, Klebsiella spp., Enterobacter spp., Enterococcus spp. Streptococcus pyogenes, Staphylococcus aureus, polymicrobial S. aureus, Candida spp. Neisseria meningitides

(e.g., LPS) and intracellular environments (e.g., DNA fragments) (see Table 27.3) The severity of sepsis is also influenced by polymorphic alleles of genes involved in the inflammatory cascade.35

KEY CONCEPTS Definitions of Sepsis and Septic Shock (Third International Consensus Definitions for Sepsis and Septic Shock) 42 • Sepsis—life-threatening organ dysfunction causes by dysregulated host response to infections (including nonbacterial pathogens) • Septic shock—subset of patients with sepsis with increased risk of death due to profound circulatory, cellular, and metabolic abnormalities • SIRS (systemic inflammatory response syndrome)—nonspecific response to infectious and noninfectious insults; current guidelines recommend use of qSOFA (Quick Sequential Organ Failure Assessment Score) score in lieu of SIRS criteria, given its higher predictive value for sepsis

The morbidity and mortality of bacteremia and sepsis have been directly correlated with the initial levels of proinflammatory cytokines and the amount of circulating bacterial components. Indeed, the severity of gram-negative sepsis has been equated with high levels of endotoxin, increased levels of cytokines, and excessive activation of the AP. Disseminated intravascular coagulation, which often accompanies gram-negative sepsis, is caused by excessive activation of the coagulation cascade and downregulation of the fibrinolytic system associated with high levels of LPS. Levels of natural anticoagulants in the vasculature, such as antithrombin and protein C, are often low in gram-negative sepsis. The onset and severity of disseminated intravascular coagulation may be influenced by genetic polymorphisms in plasminogen activation or inhibition. The generalized, altered vascular endothelial lining facilitates thrombosis and thrombocytosis. Although much remains to be learned about the mechanisms by which gram-negative and gram-positive bacteria and microbial products trigger sepsis, significant advances have been made recently, particularly with endotoxin-mediated sepsis. Advances during the past decade include the identification of certain LPS–host protein interactions that result in delivery of LPS to host cell receptors and gene activation events that result in elevated expression of a diverse array of proinflammatory and antiinflammatory mediators (Fig. 27.6). For example, TLR4 signaling requires an accessory protein, myeloid differentiation protein-2 (MD-2), which binds directly to endotoxin.37 The key point, however, is that the LPS engagement

400

Part THREE  Host Defenses to Infectious Agents Macrophage

Gram-negative bacteria Release of cytokines

Cytokine gene expression

LPS

Release of LPS in vesicles or by the action of bacteriolytic agents

Nucleus

mCD14

Mobilization of transcription factors

LPS oligosaccharide Signal transduction

mCD14 MD-2

Lipid A LBP

sCD14 LBP

LPS Lipopolysaccharide LBP LPS-binding protein sCD14 Soluble CD14

TLR-4 sCD14

mCD14 Membrane-bound CD14 TLR-4 Toll-like receptor 4

FIG 27.6  Lipopolysaccharide (LPS) Triggering of Cytokine Production by Macrophages. Steps known or presumed to be necessary for LPS triggering of proinflammatory cytokines.

of MD-2/TLR4 on host cells, particularly macrophages, triggers intracellular signaling events that ultimately, through NF-κB and other pathways, result in cytokine gene activation and production of cytokines (TNF-α, IL-1, IL-6, IL-8, IFNs). Other TLRs (e.g., TLR2) play a critical role in the recognition of lipoproteins, and the recognition of these components is a likely a key determinant in the development of septic shock seen with gram-positive infections. TLR5 recognizes bacterial flagella, and such recognition is of importance in the host response to motile bacteria. Some human pathogens (e.g., H. pylori) produce flagellin molecules that do not engage TLR5. TLR9 has been shown to recognize bacterial DNA CpG dinucleotides.14 Taken together, the clinical syndrome of septic shock represents a series of interactions of bacterial products in the vascular space with pattern recognition molecules on serum proteins (Lipopolysaccharide-binding protein [LBP] and soluble CD14) and with host cell receptors (MD-2/TLR4 and TLR2, other TLRs) leading to signaling events and release of transcriptional factors that modulate cytokine gene expression. These events also trigger other events in the inflammatory cascade, leading to activation of the coagulation, complement, and kinin pathways. Superantigens can activate large pools of non–antigen-specific T cells by binding to MHC class II molecules and the TCR Vβ region outside the peptidebinding domain. The result is a cytokine storm with clinical manifestations of sepsis. Early and effective antimicrobial therapy is the primary goal in the treatment of sepsis. In contrast to the initial phase of sepsis characterized by the release of TNF-α, IL-1, IL-6, and IFN-γ, an antiinflammatory response may predominate during the latter phase. The clinical failures of antiinflammatory therapeutic mediators (antiendotoxin antibodies, TNF-α, antagonists of IL-1, or platelet-activating factor) in sepsis suggests that this hypoinflammatory state encountered in many patients at presentation could be an additional target of immune modulation.

CLINICAL RELEVANCE Signs of Septicemia • Shaking chills, spiking fevers, or hypothermia (12 000 cells/mm3 or 10% immature (band) forms • Oliguria (90% Th2 response. As noted above, bacterial toxins as conjugates can be used to enhance immunogenicity. In addition, saponin adjuvants, liposomes, CpG DNA repeats, and TLR agonists are in use or under development as adjuvants. Monophosphoryl lipid A (MPL), long known to be an effective adjuvant in animal models, is now approved for human use, as an oil-in-water emulsion containing

401

squalene. Cytokines, such as IL-1, IL-2, IL-12, IL-18, and granulocyte macrophage–colony-stimulating factor (GM-CSF), also modify and enhance immune responses to vaccines. IL-12, for example, induces strong Th1 shifts, and GM-CSF is a comigrating signal for DCs and stimulates antigen processing and presentation. Antigen recognition and processing in macrophages is critical to determining T-cell responses and can be manipulated by selected adjuvants. Immune modulation is being evaluated not only for the enhancement of bacterial vaccines but also as adjunct therapy for serious bacterial infections, such as sepsis. Vaccines and specific immunotherapeutic approaches, such as cytokines, may also find use against chronic tissue-damaging inflammatory reactions created by persistent extracellular bacteria (e.g., Helicobacter) and autoimmune reactions that may be induced by cross-reactive bacterial antigens (e.g., C. jejuni and Guillain-Barré syndrome).

TRANSLATIONAL RESEARCH OPPORTUNITIES An important challenge for the next decade will be to take the rapidly expanding basic discoveries in innate immunity, systems biology, and response to bacterial antigens into clinical applications. The design and use of bacterial vaccines through the assessment of innate immune molecular signatures after vaccination, both for general use and for subpopulations of nonresponders, is one example. A second is the continued development of small-molecule inhibitors or enhancers that specifically target innate immune pathways to modulate bacterial immune responses. A third is the control of mucosal immune responses to prevent or eliminate colonization by bacterial pathogens. A fourth is the understanding of role of the microbiome in shaping the immune response to pathogens and vaccines and its therapeutic potential for both infections and noninfectious diseases. Fecal microbiota transplantation for C. difficile colitis is an early example of therapeutic use of the microbiome. Finally, the development of new therapies for acute bacterial sepsis may be based on improved understanding and control of the immune responses in sepsis.36

ON THE HORIZON • Tailoring vaccine design based on assessment of innate immune molecular signatures • Small molecule inhibitors or enhancers specifically targeting innate immune pathways • Identification of immune responses that prevent or eliminate mucosal bacterial pathogen colonization • Development of new therapies modulating immune response in sepsis • Defining microbial community and metagenome changes after antibiotic treatment • Managing disease based on the human microbiome

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Silva MT. Classical labeling of bacterial pathogens according to their lifestyle in the host: inconsistencies and alternatives. Front Microbiol 2012;3:71. 2. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature 2007;449:819–26.

402

Part THREE  Host Defenses to Infectious Agents

3. Moens E, Veldhoen M. Epithelial barrier biology: good fences make good neighbours. Immunology 2012;135:1–8. 4. Nestle FO, Di Meglio P, Qin JZ, et al. Skin immune sentinels in health and disease. Nat Rev Immunol 2009;9:679–91. 5. Eloe-Fadrosh EA, Rasko DA. The human microbiome: from symbiosis to pathogenesis. Annu Rev Med 2013;64:145–63. 6. van Rensburg JJ, Lin H, Gao X, et al. The human skin microbiome associates with the outcome of and is influenced by bacterial infection. MBio 2015;6:e01315–15. 7. Gallo RL, Hooper LV. Epithelial antimicrobial defence of the skin and intestine. Nat Rev Immunol 2012;12:503–16. 8. Kumar S, Ingle H, Prasad DV, et al. Recognition of bacterial infection by innate immune sensors. Crit Rev Microbiol 2013;39:229–46. 9. Olive C. Pattern recognition receptors: sentinels in innate immunity and targets of new vaccine adjuvants. Expert Rev Vaccines 2012;11:237–56. 10. Beutler B, Jiang Z, Georgel P, et al. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu Rev Immunol 2006;24:353–89. 11. Quinn SR, O’Neill LA. A trio of microRNAs that control Toll-like receptor signalling. Int Immunol 2011;23:421–5. 12. Unkmeir A, Kämmerer U, Stade A, et al. Lipooligosaccharide and polysaccharide capsule: virulence factors of Neisseria meningitidis that determine meningococcal interaction with human dendritic cells. Infect Immun 2002;70:2454–62. 13. Ricklin D, Hajishengallis G, Yang K, et al. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 2010;11:785–97. 14. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010;11:373–84. 15. Papadopoulos A, Gorvel JP. Subversion of mouse dendritic cell subset function by bacterial pathogens. Microb Pathog 2015;89:140–9. 16. Sansonetti PJ, Phalipon A. M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin Immunol 1999;11:193–203. 17. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532–5. 18. Spits H, Artis D, Colonna M, et al. Innate lymphoid cells—a proposal for uniform nomenclature. Nat Rev Immunol 2013;13:145–9. 19. Small CL, McCormick S, Gill N, et al. NK cells play a critical protective role in host defense against acute extracellular Staphylococcus aureus bacterial infection in the lung. J Immunol 2008;180:5558–68. 20. Fuchs A, Colonna M. Natural killer (NK) and NK-like cells at mucosal epithelia: mediators of antimicrobial defense and maintenance of tissue integrity. Eur J Microbio Immunol (Bp) 2011;1:257–66. 21. Cole J, Aberdein J, Jubrail J, et al. The role of macrophages in the innate immune response to Streptococcus pneumoniae and Staphylococcus aureus: mechanisms and contrasts. Adv Microb Physiol 2014;65:125–202. 22. Peck A, Mellins ED. Precarious balance: Th17 cells in host defense. Infect Immun 2010;78:32–8.

23. Yoshikai Y. The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense. Immunol Res 1999;20:219–35. 24. Gao Y, Williams AP. Role of innate T cells in anti-bacterial immunity. Front Immunol 2015;6:302. 25. Gold MC, Lewinsohn DM. Co-dependents: MR1-restricted MAIT cells and their antimicrobial function. Nat Rev Microbiol 2013;11:14–19. 26. Napier RJ, Adams EJ, Gold MC, et al. The role of mucosal associated invariant T cells in antimicrobial immunity. Front Immunol 2015;6:344. 27. Mulks MH, Plaut AG. IgA protease production as a characteristic distinguishing pathogenic from harmless neisseriaceae. N Engl J Med 1978;299:973–6. 28. Ribet D, Cossart P. How bacterial pathogens colonize their hosts and invade deeper tissues. Microbes Infect 2015;17:173–83. 29. Pan X, Yang Y, Zhang JR. Molecular basis of host specificity in human pathogenic bacteria. Emerg Microbes Infect 2014;3:e23. 30. Criss AK, Seifert HS. A bacterial siren song: intimate interactions between Neisseria and neutrophils. Nat Rev Microbiol 2012;10:178–90. 31. Rouphael NG, Stephens DS. Neisseria meningitidis: biology, microbiology, and epidemiology. Methods Mol Biol 2012;799:1–20. 32. McNeil LK, Zagursky RJ, Lin SL, et al. Role of factor H binding protein in Neisseria meningitidis virulence and its potential as a vaccine candidate to broadly protect against meningococcal disease. Microbiol Mol Biol Rev 2013;77:234–52. 33. Mahalingam S, Lidbury BA. Antibody-dependent enhancement of infection: bacteria do it too. Trends Immunol 2003;24:465–7. 34. Roilides E, Simitsopoulou M, Katragkou A, et al. How Biofilms evade host defenses. Microbiol Spectr 2015;3(3). 35. Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. New Engl J Med 2002;348:138–50. 36. Leentjens J, Kox M, van der Hoeven JG, et al. Immunotherapy for the adjunctive treatment of sepsis: from immunosuppression to immunostimulation. Time for a paradigm change? Am J Respir Crit Care Med 2013;187:1287–93. 37. Needham BD, Trent MS. Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis. Nat Rev Microbiol 2013;11: 467–81. 38. Kumar P, Chen K, Kolls JK. Th17 cell based vaccines in mucosal immunity. Curr Opin Immunol 2013;25:373–80. 39. Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol 2012;12:592–605. 40. Nakaya HI, Pulendran B. Vaccinology in the era of high-throughput biology. Philos Trans R Soc Lond B Biol Sci 2015;370(1671). 41. Stephens DS, Hoffman LH, McGee ZA. Interaction of Neisseria Meningitidis with human nasopharyngeal mucosa—attachment and entry into columnar epithelial cells. J Infect Dis 1983;148:369–76. 42. Singer M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016;315:801–10.

CHAPTER 27  Host Defenses to Extracellular Bacteria

402.e1

MULTIPLE-CHOICE QUESTIONS 1. With regard to the use of corticosteroids as adjunctive therapy for acute infections: A. Corticosteroids are immunosuppressive and should be avoided during acute infections. B. Corticosteroids are generally recommended for the treatment of sepsis. C. Corticosteroids are never contraindicated during acute infections. D. Corticosteroids are beneficial in certain types of acute infection, presumably because of their antiinflammatory effect. 2. A 18-year-old college freshman presents to the hospital with meningococcal meningitis. Upon review of his medical history, you learn that he had disseminated gonococcal infection at the age of 16 years. Should you consider investigation for an immunodeficiency? If so, which one? Should you offer any prophylactic therapies for this patient? A. No investigation for an immunodeficiency is warranted. Meningococcal vaccination should be offered. B. These infections suggest a T-helper (Th)17 deficiency, and there is no approved prophylactic therapy for this disorder.

C. This patient should have diagnostic workup for terminal complement deficiencies. Meningococcal vaccination should be offered. D. This patient should have diagnostic workup for terminal complement deficiencies. If confirmed, stem cell transplantation can be offered. 3. The normal microbiota maintains a balance with the host by avoiding activation of pathogen recognition receptors. Pathogen recognition receptors are activated by pathogenic bacteria only. A. False. Colonizers and pathogens have pathogen-associated molecular patterns, and both can activate pattern recognition receptors. B. True. Colonizers are devoid of pathogen-associated molecular patterns. C. True. Colonizers have pathogen-associated molecular patterns but do not activate pathogen recognition receptors. D. False. Colonizers and pathogens have pathogen-associated molecular patterns, and both can activate pattern recognition receptors, but colonizers only do so when invading host tissues.

28  Host Defenses to Spirochetes Nicolás Navasa, Erol Fikrig, Juan Anguita

Spirochetes constitute a unique group of bacteria that inhabit many different environments, such as soil, arthropods, and mammals. These microorganisms cause numerous human illnesses, including syphilis and Lyme disease. Spirochetes share a typical spiral shape and a distinctive flat-wave morphology. Cellular dimensions vary over a wide range, with a diameter between 0.09 to 0.75 µm and lengths that range from 3 to 500 µm. They are motile organisms with a multilayered outer membrane that encapsulates a peptidoglycan layer surrounding their inner membrane. Motility is enabled by the presence of endoflagella located in the periplasmic space. These axial filaments are arranged in a bipolar fashion and extend toward the opposite end of the cell (Fig. 28.1). The viability of the organism is dependent on an intact outer membrane, which can be damaged by variations in osmolarity, antibodies, or complement resulting in the loss of intracellular components and ultimately death of the bacterium. Several spirochetal species are able to induce disease in mammals (Table 28.1). Lyme disease was first discovered in 1976 as an illness affecting a cluster of children in Lyme, Connecticut. Several manifestations of the disease, however, had been known for over a century in parts of Europe. In 1982, the agent of Lyme disease was identified as Borrelia burgdorferi.1 In contrast, Treponema pallidum subspecies pallidum is the causative agent of venereal syphilis, a disease that has been recognized for over 5 centuries, although its agent was not identified until 1905.2 The genomes of B. burgdorferi and T. pallidum have been sequenced.3,4 Despite similar ancestry and morphological features, these spirochetes have striking differences at the genetic level, which may account for the differences in their lifecycles, environmental adaptations, and the diseases they cause. Both B. burgdorferi and T. pallidum have relatively small genomes compared with other microorganisms. B. burgdorferi has one of the most complex genomes known among prokaryotes, with a single linear chromosome and 21 plasmids, the largest number of plasmids of any characterized prokaryote. Of the 21 plasmids, nine are circular and 12 are linear. Furthermore, less than 10% of B. burgdorferi plasmid-coding regions are found in other microorganisms, including spirochetes, which underscore the uniqueness of this spirochete among microorganisms. Unlike B. burgdorferi, T. pallidum contains a single circular chromosome with no plasmids. Yet, 476 open reading frames (ORFs) in T. pallidum have orthologous genes in B. burgdorferi, and almost 60 of these orthologous genes encode proteins of unknown biological function that are specific to spirochetes. In contrast to other spirochetes, B. burgdorferi and T. pallidum do not contain lipopolysaccharide (LPS). Lipoproteins

are the major immunogens of B. burgdorferi and most likely T. pallidum, and thus they are their dominant proinflammatory agonists. Five percent of the chromosomal ORFs and 14.5–17% of the functionally complete ORFs contained in the plasmids of B. burgdorferi encode putative lipoproteins. Interestingly, only 2.1% of the T. pallidum ORFs encode putative lipoproteins. The abundant lipoprotein coding potential of B. burgdorferi suggests that these molecules may be important for the survival of the spirochete. In fact, as a result of environmental changes, expression of several B. burgdorferi lipoproteins changes throughout the lifecycle of the spirochete.5 For instance, the lipoprotein outer surface protein (Osp) A is expressed at high levels in the gut of the unfed tick, but upon feeding OspA expression is downregulated, and the expression of OspC increases 90-fold.5 It is thought that many Osps have adhesive functions, and it has been shown that OspA is involved in the attachment of B. burgdorferi to the gut of the tick through an interaction with the tick receptor TROSPA.6 Furthermore, OspC may be necessary for the migration of B. burgdorferi from the gut of the tick to the salivary glands and for its survival in the mammalian host. Changes in lipoprotein expression are primarily regulated by the alternative sigma factors, RpoS and RpoN7 and other unknown mechanisms.

CLINICAL MANIFESTATIONS Lyme Disease B. burgdorferi sensu stricto (B. burgdorferi) is the etiological agent of Lyme disease in the United States and other parts of the world, whereas B. afzelii and B. garinii are agents of Lyme disease that are restricted to Europe and Asia. Infection with Lyme disease– causing spirochetes has also been observed in Japan, Russia, and China. In the United States, transmission of the spirochete is through the hard-bodied ticks of the Ixodes complex, mainly I. scapularis and I. pacificus, whereas I. ricinus is responsible for the transmission of the spirochete in much of Eurasia. According to the Centers for Disease Control and Prevention (CDC), Lyme disease is the most common tickborne disease in the United States and the fifth most common nationally notifiable disease.8 The disease is concentrated in the North Eastern seaboard, with further presence in the northern Midwest and the West of the Continental United States. In Europe, diagnosed Lyme disease accounts for more than 65 000 cases annually, which tend to concentrate in countries of Central and Eastern Europe. However, the disease is present to different degrees throughout much of Europe. In both the United States and Europe, it is widely believed

403

Part THREE  Host Defenses to Infectious Agents

20-30µm

0.2-0.3µm

404

A

Outer membrane Inner membrane

Periplasmic flagella

Protoplasm

B

FIG 28.1  Borrelia burgdorferi structure is characterized by a distinctive flat-wave morphology consisting of approximately 18 bends and a length of 20–30 µm (A). A cross-section of this spirochete reveals the endoflagella, which are responsible for the unique morphology and motility of this organism (B).

that the disease is underreported and that the suspected real number of cases may be much higher. An early hallmark of infection is the appearance of a skin rash, known as erythema migrans (Fig. 28.2), which appears at the inoculation site, often during the first week of infection (stage I Lyme disease), as a result of local inflammatory responses. Other symptoms secondary to the local inflammatory responses occurring during stage I can affect more distal sites and may include fever, headache, malaise, myalgia, and/or arthralgia. Hematogenous dissemination of the spirochete is stage II Lyme disease and results in colonization of different tissues and/or organs and presentation of a range of symptoms, such as conduction system abnormalities, meningitis, and acute arthritis. Joint inflammation appears in 60% of untreated individuals in the United States and predominately affects large joints, especially the synovium of the knee. Some untreated individuals develop stage III Lyme disease, which is generally characterized by prolonged infection with the spirochete. Late-stage symptoms may include chronic arthritis, neuroborreliosis, or cutaneous lesions, such as acrodermatitis chronica atrophicans. The range of symptoms that appear upon infection in Europe and the United States vary in relative terms: arthritis and carditis are

TABLE 28.1  Major Diseases Caused by Spirochetes* Disease

Agents

Distribution

Transmission

Symptoms

Lyme disease

Borrelia burgdorferi B. garinii B. afzelii B. andersonii B. japonica B. lusitaniae B. valaisiana B. mayonii B. miyamotoi B. hermsii B. turicatae B. parkeri B. mazzotti B. venezuelensis B. duttonii B. crocidurae B. persica B. hispanica B. latyschewii B. caucasia Treponema pallidum pallidum

North America, Europe Asia, Europe Asia, Europe North America Japan Southern Europe Europe, Ireland, UK North America North America Western USA Southwestern USA, Mexico Western USA Central America Central America Sub-Saharan Africa North Africa, Middle East Middle East, Central Asia Iberian peninsula, North Africa Iran, Iraq, Eastern Europe Iraq, Eastern Europe Worldwide

Tick engorgement

Development of a skin rash known as erythema migrans, accompanied by other symptoms, such as malaise, myalgia, and/or arthralgia. Symptoms can progress to include carditis and arthritis. Persistent infection can result in chronic arthritis, neuroborreliosis, or cutaneous symptoms (acrodermatitis chronica atrophicans).

Tick engorgement

Clinical manifestations of infection include highdensity spirochetemia, high fever, myalgias, and arthralgias and can even include cerebral hemorrhage and fatality.

Sexual contact

Endemic syphilis or Bejel syphilis

T. pallidum endemicum

Eastern Mediterranean region, West Africa

Nonsexual skin contact

Yaws

T. pertenue

Humid equatorial countries

Pinta

T. carateum

Mexico, Central America, South America

Nonsexual skin contact Nonsexual skin contact

Leptospirosis

Leptospira interrogans

Worldwide

Disease progresses from a primary lesion (chancre) to a secondary eruption and then to a latent period, and if left untreated, tertiary symptoms may appear. Symptoms begin with a slimy patch inside the mouth, followed by blisters on the trunk and limbs. Bone infection in the legs soon develops, and in the later stages, lumps may appear in the nose and on the soft palate of the mouth. Destructive lesions of the skin and bones, which is rarely fatal but can be debilitating. Dark-colored skin lesions found on those areas of the body that are exposed to sunlight. Eventually, the skin lesions become discolored. Symptoms include fever, headache, chills, nausea and vomiting, eye inflammation, and muscle aches. In more severe cases, the illness can result in liver damage and kidney failure.

Relapsing fever

Venereal syphilis

Urine from an infected animal

*Spirochetes are the causative agents of many diseases, which can have social as well as lasting health-related consequences.

CHAPTER 28  Host Defenses to Spirochetes

FIG 28.2  Erythema migrans caused by infection with Borrelia burgdorferi, the Lyme disease agent. (Courtesy of Gary Wormser, MD.)

more commonly found in the United States, whereas patients in Europe tend to show higher incidence of skin and nervous system involvement. This may be attributed to the heterogeneity of the B. burgdorferi sensu lato genospecies that cause the disease. In the United States, in the large majority of cases, B. burgdorferi sensu stricto is involved, whereas in Europe infections by B. afzelii and B. garinii predominate. Diagnosis A detailed clinical history and comprehensive physical examination are critical for the accurate diagnosis of Lyme disease. Appropriate laboratory testing, however, is a valuable diagnostic aid. A two-tiered approach is standard for the serodiagnosis of Lyme disease. Using this approach, serum is first tested for the presence of B. burgdorferi–specific antibodies by using a sensitive method, such as enzyme-linked immunosorbent assay (ELISA) or immunofluorescent assay (IFA). Sera testing negative for antibodies generally need not be tested further; however, those found to be positive or equivocal are further evaluated by the more specific immunoblotting for immunoglobulin M (IgM) and IgG antibodies. The detection of at least two of three specific bands in IgM or five of 10 specific bands in IgG is considered positive. Patients with early Lyme disease often report to a physician during the first few days of infection, at which time a detectable humoral response may not have developed. Consequently, the two-tiered approach is considered highly sensitive during the later stages of the disease (>90%) and less sensitive during very early infection. Furthermore, a positive serological test, particularly IgG, is evidence of exposure to B. burgdorferi, but it does not necessarily indicate active infection. All serological tests must then be evaluated in conjunction with a clinical assessment by the attending physician. Other diagnostic methods, such as culture and polymerase chain reaction (PCR) detection of B. burgdorferi, may be very useful to detect active infection, particularly of skin, joints, and the central nervous system (CNS). Culture, however, is generally limited to research laboratories, and the sensitivity and specificity of PCR can vary greatly among testing centers.

405

Treatment During early stages of Lyme disease, such as that during which erythema migrans is present, oral administration of doxycycline (100 mg twice daily) or amoxicillin (500 mg three times a day) for approximately 2 weeks is recommended. Doxycycline has the advantage of being effective against Anaplasma phagocytophilum, which may also be transmitted by ticks. In areas where B. burgdorferi infection is prevalent, some experts recommend antibiotic therapy for individuals who served as hosts to I. scapularis ticks that were attached longer than 40–48 hours—the time required for transmission of the spirochete. It is extremely difficult, however, to consistently make accurate determinations of the species of tick and the degree of engorgement. Furthermore, randomized double-blind clinical trials involving individuals who were bitten by I. scapularis ticks led to the conclusion that antibiotic treatment of all individuals who had vector ticks removed is probably not warranted.

Venereal Syphilis Infection with the agent of syphilis, T. pallidum subspecies pallidum, occurs worldwide. T. pallidum is an obligate human parasite that is almost exclusively transmitted when contact with infectious exudates from lesions of the skin and mucous membranes of infected individuals occurs. Clinically, this treponemal infection is first characterized by the formation of a hardened and painless ulcer at the initial site of infection. This primary lesion, called a chancre, forms after invasion of the bloodstream by the spirochete. Four to 6 weeks after infection, the edges of the chancre roll inward and upward and, in most cases, a secondary eruption appears, often accompanied by a rash on the palms of the hands and the soles of the feet. The secondary manifestations resolve within weeks to a year after the development of a vigorous cell-mediated immune response. Long periods of latency followed by late lesions of skin, bone, and viscera, as well as the cardiovascular system and the CNS, can occur despite clearance of the majority of the treponemes, which coincides with the resolution of the primary syphilitic lesion. Diagnosis Much like Lyme disease, the diagnosis for syphilis is based on the clinical presentations of the disease and serological tests. In addition, dark-field microscopy can be used for the identification of T. pallidum in the serous exudates of the chancre. This approach, however, is limited by the number of live treponemes in the exudates and by the presence of nonpathological treponemes in oral and anal lesions; as such, negative examinations on three independent days are required before a lesion is considered negative for T. pallidum. Infection with T. pallidum leads to the production of nonspecific antibodies, which is the basis for other diagnostic tests, such as the traditional nontreponemal serological tests, including the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR) tests. Because these tests are nonspecific, false-positive reactions can occur as a result of pregnancy, autoimmune disorders, or infections. Therefore treponemalspecific tests, which detect antibodies to various antigens of T. pallidum, are often used to confirm the results of a nonspecific test. Interestingly, treponemal-specific tests are just as sensitive as nontreponemal tests; however, they are much more difficult and expensive to perform, which limits their use. These tests include, but are not limited to, the enzyme immunoassay (EIA)

406

Part THREE  Host Defenses to Infectious Agents

test for T. pallidum-specific IgG, T. pallidum hemagglutination test (TPHA), fluorescent treponemal antibody-absorption test (FTA-ABS), and ELISA. Treatment Susceptibility to infection with T. pallidum is universal, although only 30% of exposures with lesions teeming with the spirochete result in infection. Infection results in gradual development of immunity against T. pallidum and often against heterologous treponemes as well. However, treatment with long-acting penicillin subverts the development of immunity against T. pallidum. Administration of 2.4 million units in a single intramuscular dose the day that the primary, secondary, or latent syphilis is diagnosed is effective at killing the spirochetes. For people who are allergic to penicillin, there are alternative treatments, such as doxycycline.

HOST DEFENSES TO B. burgdorferi KEY CONCEPTS Protective Versus Pathological Responses to Borrelia burgdorferi The early immune response to B. burgdorferi is necessary to control spirochetal burden; however, by itself, it is not sufficient to resolve infection. Phagocytosis is a key element of the innate immune response, which is involved in the elimination of the bacteria while also contributing to the proinflammatory output of macrophages The T cell–mediated response appears to be involved in pathology arising from infection. A T cell–independent B-cell response is sufficient to resolve infection with B. burgdorferi.

B. burgdorferi virulence is attributed, in part, to the evolution of the spirochete’s sophisticated tactics to evade killing mechanisms during all stages of the immune response: the first stage, beginning with transmission into the host via tick engorgement, at which time the spirochete is exposed to serum complement and cellular immunity; and the second and third stages, that is, hematogenous dissemination to and colonization of peripheral sites, at which time the host is producing specific antibodies. Both the innate and adaptive immune responses elicited by B. burgdorferi are discussed, with the supposition that these responses are required for efficient bacterial clearance, while acknowledging that unnecessarily prolonged or intense responses may contribute to pathology arising from infection. Indeed, predisposition to infection could be the result of one or more monogenic traits that confer primary immunodeficiencies; whether or not this is the case remains to be determined, but human studies have shown that responses to B. burgdorferi are diminished in individuals with specific mutations in or diminished expression of innate immune cell receptors (nucleotide-binding oligomerization domain-2 [NOD2] and Toll-like receptor-1 [TLR1]).

Innate Immune Responses The initial recognition of pathogens with cells of the host relies on a complex interplay between pathogen recognition receptors (PRRs) and bacterial constituents, and this initiates a cascade of responses leading to the upregulation of chemokines and cytokines, adhesion molecules, and other effector molecules (Chapter 3). This response is often initiated by endothelial and/

or epithelial cells, resulting in the recruitment of different types of innate immune cells and their activation. Each PRR recognizes a specific structure that is present in a group or groups of microorganisms and that distinguishes them from the more specific recognition of antigens by the T- and B-cell receptors. The recognition of patterns instead of specific antigens provides the innate immune system with a rapid way to respond to infecting organisms until the more specific response mediated by T and B cells develops. A distinct innate cellular infiltration profile has been observed in B. burgdorferi–infected tissues. Based on studies using mice, the inflamed heart appears to predominately comprise macrophages, with smaller amounts of T and invariant natural killer T cells (iNKT cells). However, neutrophils are the primary innate immune cell found in the joints. In this tissue, resident myeloid cells act as the initiators of type I interferon (IFN) production upon encounter with B. burgdorferi, whereas endothelial cells and joint fibroblasts expressing adhesion/ activation markers amplify the response and serve as the major source of diseasepromoting chemokines. This tissue-specific tropism complicates our understanding of the host defense against the spirochete, but this suggests that macrophages are more efficient at preventing successful colonization of B. burgdorferi because carditis is a less frequent manifestation of Lyme disease. These early defense responses also have a role in determining the type, strength, and duration of the more specific adaptive immune response, beyond their direct bacterial-killing capability. Early Pathogen Recognition The interaction of B. burgdorferi lipoproteins with complexes formed by TLRs 1 and 2 initiates a series of signaling cascades that results in the production of proinflammatory cytokines (IL-1β, tumor necrosis factor [TNF], interleukin [IL]-12, and IL-18, among others), chemokines (IL-8, monocyte chemoattractant protein [MCP]-1, keratinocyte chemoattractant [KC]), metalloproteinases, adhesion molecules (E-selectin, vascular cell adhesion molecule-1 [VCAM-1] and intercellular adhesion molecule-1 [ICAM-1]),9 and type I IFNs. These interactions are critical for generating an effective spirochete-clearing inflammatory response, as demonstrated by experiments using mice deficient for TLR1, TLR2, or their adaptor molecule, MyD88. These mice had significant increases in B. burgdorferi burdens after infection. Moreover, humans with decreased TLR1 expression are hyporesponsive to the original OspA-based Lyme disease vaccine, and macrophages from these subjects also have diminished inflammatory responses to the lipoprotein. Besides TLR1/ TLR2 complexes, other members of this family of receptors participate in, and amplify, the inflammatory response to B. burgdorferi. These include TLR5, TLR7, TLR8, and TLR9, which recognize different bacterial constituents. Importantly, the development of inflammatory arthritis in the absence of TLRs or MyD88 was the first suggestion that alternative pathways trigger the inflammatory response to the spirochete. Other PRRs that have been shown to be involved in the response to the spirochete include NOD-like receptors. For the full inflammatory response to take shape upon recognition of B. burgdorferi, most of the interactions of B. burgdorferi constituents and PRRs occur within the phagolysosome. Therefore phagocytosis is a hallmark of the innate immune cell recognition of the spirochete. The interaction of several cell types with the spirochetes also involves several integrins belonging to the β1, β2, and β3 groups. Integrins are involved in the adhesion of cells to a variety of

CHAPTER 28  Host Defenses to Spirochetes Professional antigen-presenting cell Phagocytic receptor

Endosomal PRRs IL-1β IL-12 IL-18

TLR 1/2

IFNγ

MyD88

TCR Signaling

CD4+ T cell

Type I IFN TNF

Surface PRRs (i.e., integins) Endothelial cell

IL-8, MCP-1, KC

FIG 28.3  The interaction of Borrelia burgdorferi with pattern recognition receptors (PRRs) in innate immune cells and endothelial cells mediates the inflammatory response to the spirochete. Phagocytosis induces Toll-like receptor (TLR)–driven proinflammatory cytokine production as well as antigen presentation by professional antigen-presenting cells (APCs), which leads to the activation of CD4 effector T cells, marked by the production of IFN-γ. Likewise, PRR- and tumor necrosis factor (TNF) receptor signaling lead to the upregulation of chemokines by endothelial cells. Overall, these responses lead to increased activation and recruitment of innate immune cells in sites of infection.

ligands and mediate essential cellular processes, including attachment and cell migration. Some integrins have also been associated with the phagocytosis of microorganisms. For the most part, study of the interaction between the spirochete and integrins has focused on their role aiding the adhesion of B. burgdorferi to cells and the colonization of tissues and as receptors that contribute to signals that induce the production of proinflammatory factors (Fig. 28.3). Phagocytic Cell Recruitment and Spirochetal Clearance The recruitment of phagocytic cells and other cell types into sites of infection is mediated by the production of chemokines, increased vascular permeability, and upregulated expression of cell adhesion molecules in endothelial cells. B. burgdorferi induces the upregulation of these factors in different cell types. Chemokine production at sites of pathology in disease-susceptible C3H/HeJ mice and disease-resistant C57BL/6 J mice shows that inflammation is related to increased production of neutrophil and monocyte–macrophage chemokines, KC and MCP-1, respectively. In patients, the production of chemokines, especially IL-8, during the initial response to B. burgdorferi correlates well with the onset of symptoms known to occur during the early stages of infection, and this suggests that their production is increased during the beginning of the infection to recruit phagocytic cells, which are involved in the initial clearance of the spirochete.

407

Little is known about the molecular events or receptors that mediate B. burgdorferi phagocytosis despite the importance of this cardinal mechanism of pathogen clearance. MyD88-mediated signals substantially mediate the phagocytosis of B. burgdorferi. However, MyD88-mediated phagocytosis occurs independently of any known B. burgdorferi-recognizing TLRs. The mechanism of MyD88-mediated phagocytic uptake is therefore unclear. Furthermore, the analysis of MyD88-deficient macrophages shows that although reduced, phagocytosis is not absent in these cells. Therefore the uptake of B. burgdorferi by phagocytic cells seems to be mediated by more than one receptor with MyD88-dependent and MyD88-independent components. Complement receptor 3 (CR3) is a β2 integrin, which, in cooperation with the GPIanchored CD14, acts as a phagocytic receptor for B. burgdorferi independently of TLR or MyD88-induced signals.10 Phagocytosis plays a major role in the pathogenesis of Lyme disease, not only through the control of bacterial numbers but also through modulation of the potency and quality of proinflammatory cytokine induction. Indeed, as opposed to MyD88-mediated phagocytosis, which is proinflammatory, the internalization of B. burgdorferi by CR3 tempers the inflammatory response of macrophages. Therefore the presence of alternative phagocytic mechanisms has nonredundant physiological consequences during infection with the spirochete.

Complement The complement system is a key component of the innate immune system (Chapter 21). It comprises a collection of serum proteins and cell surface receptors that are involved in the early response to pathogens, including B. burgdorferi.11 Destruction of microorganisms via complement involves the formation of a pore in the microbial cell membrane by the membrane attack complex (MAC), which results in the lysis of the organism. Three different pathways elicit complement activation: the classical (antigen/ antibody–mediated) pathway (CP), the lectin pathway, and the alternative (pathogen surface) pathway (AP). These pathways converge at the level of C3 convertase, a protease that cleaves complement component C3 into C3a and C3b. As a result, C3b can (i) bind to the surface of the bacteria and facilitate internalization of the spirochete via opsonization; or (ii) it can bind C3 convertase and facilitate the deposition of downstream components onto the surface of the spirochete resulting in the formation of MAC and lysis of the cell. B. burgdorferi activates the CP and AP of the complement cascade.12 Moreover, the activation of complement has been associated with dramatic decreases in spirochetal numbers in different tissues of infected mice, indicating the importance of the complement system early in B. burgdorferi infection. The members of the B. burgdorferi sensu lato group, which includes B. burgdorferi sensu stricto, B. garinii, and B. afzelii, have evolved a variety of mechanisms enabling them to escape complement-mediated lysis, including the expression of complement regulator-acquiring surface proteins (CRASPs). Of these CRASPs, the Erp (OspEF-related protein) family of outer membrane proteins serve as binding sites for the complement inhibitor factor H and factor H–like protein 1 (FHL-1).13 The interaction of factor H with these proteins recruits a protease (factor I) that cleaves and inactivates the complement serum proteins, C3b and C4b. Cleavage of these two complement proteins prevents the deposition of downstream components onto the surface of the spirochete, thereby halting the formation of the membrane attack complex. B. burgdorferi also express a CD59-like

408

Part THREE  Host Defenses to Infectious Agents

molecule on the outer membrane that can inactivate MAC and prevent complement-mediated lysis.14 It has been speculated that most borreliae are able to evade complement-mediated lysis, and recently, a novel protein expressed by B. hermsii (a relapsing fever spirochete) has been discovered that affords protection to spirochete by inactivating C3b.15 More recently, it has been shown that B. burgdorferi expresses a protein, BBK32, on its surface, which prevents activation of the CP by blocking activation of the C1 complement complex.16

Adaptive Immune Responses

T Cell–Mediated Responses Upon antigen presentation by macrophages, dendritic cells (DCs), or B cells, naïve CD4 T cells are activated and differentiate into effector T cells. Effector CD4 T cells are classified on the basis of their cytokine production profile, which determines their mode of action and downstream effects, and include Th1 (IFNγ-producing), T-helper [Th]2 (IL-4, IL-5, IL-13), Th17 (IL-17), or regulatory T cells (Tregs; IL-10) (Chapter 16). Interaction of B. burgdorferi antigen with TLRs induces the production of IL-12, which drives the differentiation of CD4 T cells into Th1 effector cells. Th1 cells are regulators of the cell-mediated inflammatory reactions, which are characterized by macrophage activation, including phagocytosis, or the induction of opsonizing IgG antibodies. Although IFNγ and Th1 CD4 T cells have been shown to be protective during cardiac inflammation with B. burgdorferi, joint inflammation is independent of this effector cell type in mice. However, in patients with Lyme disease, Th1 cells dominate in the synovial fluid, and the severity of arthritis directly correlates with increased levels of Th1 cells in the synovium.17 Whether neutrophilic infiltration during joint infection with the spirochete is influenced by Th effector cells that more directly affect this cell type, such as Th17 cells (through the production of IL-17) remains to be elucidated. B Cell–Mediated Responses Antibodies are specific and powerful effector molecules of the adaptive immune response (Chapter 15). Once antibodies bind to their specific foreign antigen, they confer protection to the host by using a variety of effector mechanisms: antibodies contribute to adaptive immunity by neutralizing microbes or their products, activation of complement, and opsonization, which leads to phagocytosis of microorganisms. The activation of B cells and their differentiation into antibody-secreting plasma cells often requires an interaction with Th cells, and this interaction controls isotype switching as well as somatic hypermutation. However, in response to B. burgdorferi, T cell–independent humoral responses also confer protection to the host. Thus mice deficient in T cells, CD40L, and MHC class II infected with B. burgdorferi mount a protective antibody response, which, upon passive serum transfer, affords protection to severe combined immune deficient (SCID) mice from homologous challenge. However, mice that lack both B and T cells developed severe arthritis and carditis in response to infection with B. burgdorferi. The role of antibodies in controlling B. burgdorferi infection may be more important during the hematogenous dissemination phase, when they are easily accessible, than after the spirochetes have colonized tissues. B. burgdorferi is able to evade the humoral response by interacting with the extracellular matrix of the mammalian host via attachment to decorin, a major component of the extracellular matrix. B. burgdorferi attaches to decorin

with a ligand-binding lipoprotein known as decorin-binding protein A (DbpA). A deficiency in decorin reduces the incidence of Lyme arthritis in mice, suggesting that the interaction of DbpA with the extracellular matrix provides a protective niche for B. burgdorferi, preventing humoral-mediated bacterial killing. Once the spirochete is in the joints and the heart, its clearance may be more dependent on cellular responses (e.g., macrophages in the heart) than on antibodies. In fact, the lack of IFNγ-mediated activation of macrophages has profound consequences on murine cardiac inflammation, even in the presence of strong antibody responses. Furthermore, the bacterial clearance potential of infected mouse sera administered in newly infected mice is lost when administered 4–8 days after infection, potentially the result of the colonization of tissues into which antibodies are less able to penetrate. B. burgdorferi can also avoid clearance by antibodies through antigenic diversity. B. burgdorferi differentially expresses outer membrane antigens under pressure from the immune response, which might contribute to the ability of the spirochetes to persist in the host. A mechanism that is potentially essential for spirochetal immune escape is the recombination that takes place at the vls locus,18 located near the right telomere of the linear plasmid lp28-1. B. burgdorferi is also able to evade antibody responses during transmission from the tick by attachment to the tick salivary protein, Salp15, which interacts with the lipoprotein, OspC, and protects the spirochete from antibody-mediated killing.19

HOST DEFENSES TO T. pallidum KEY CONCEPTS Protective Versus Pathological Responses to Treponema pallidum The role of the early innate response to T. pallidum is still poorly understood because immunogenic cell surface proteins able to activate pattern recognition receptors remain to be found. It is likely that the cell-mediated immune response to T. pallidum is involved in the development of pathology following infection with the spirochete. Likewise, this response seems to be involved in the resolution of infection with T. pallidum. The protection that the humoral response affords to the host is unclear. Furthermore, no definite antigens have been isolated from the spirochete because of the inability to cultivate this organism in vitro.

T. pallidum is known colloquially as the “stealth pathogen” because of its denuded outer membrane, which comprises mostly nonimmunogenic transmembrane proteins, whereas the highly immunogenic lipoproteins are contained within the periplasmic space.20 This molecular architecture, coupled with the ability to generate antigenic variants, is responsible for the treponeme’s remarkable ability to cause persistent infection with relatively few organisms.21 We have yet to elucidate many of the specific immunological phenomena that occur as a result of infection with the treponeme and that are inherent to the clinical manifestations of the disease. Our limited understanding of immunological phenomena during infection mainly results from our inability to culture these organisms in vitro and to reproducibly infect an animal model. Consequently, our understanding of the immune responses to this pathogen is not nearly as detailed as our knowledge of those elicited in response to infection with B. burgdorferi.

CHAPTER 28  Host Defenses to Spirochetes Innate Immune Responses Similar to B. burgdorferi lipoproteins, treponemal lipoproteins appear to be the major proinflammatory agonists during treponemal infection through engagement with their cognate receptors, TLR1/2 and CD14. The inflammatory milieu established by treponemal lipoproteins is a principal driving force for immune cell recruitment to T. pallidum–infected tissues. The importance of this immune system compartment during the response to T. pallidum is further supported by the systemic upregulation of innate immune cells during treponemal dissemination and by demonstration that macrophages are principal effectors of treponemal clearance during infection.22 Early Pathogen Recognition Because of their location in the skin, DCs (i.e., Langerhans cells), likely mediate the initial responses to treponemes at the primary syphilitic lesion. Because of the denuded outer membrane of T. pallidum, extremely high treponeme to DC ratios (500 : 1 and higher) are required for observable levels of phagocytosis in vitro. Thus unhindered replication of spirochetes at the initial site of infection probably underlies the vigorous cell-mediated immune response, as well as chancre appearance, which follows antigen presentation to and differentiation and activation of CD4 T cells. Phagocytic Cell Recruitment and Spirochetal Clearance It is not until after the recruitment of macrophages and their activation via CD4 T cell–derived IFN-γ, that the majority of spirochetes are killed and resolution of syphilitic lesions occurs. Activated macrophages readily phagocytose antibody-opsonized treponemes through Fc receptor–mediated uptake. Recently, it has been shown that MyD88 mediates the opsonophagocytosis of T. pallidum by macrophages without affecting the production of opsonic antibodies. As result, MyD88-deficent mice infected with T. pallidum showed a defect in bacterial clearance that resulted in worse inflammation. These results reveal a previously unsuspected link between MyD88 signaling and FcR-mediated phagocytosis. However, as with DCs, uptake via a direct PAMP– PRR interaction does not occur readily, and because of treponemal antigenic variation, not all spirochetes will be eliminated via opsonophagocytosis despite the vigorous inflammatory response. The relatively few remaining spirochetes are able to cause persistent infection.

Complement The immunoprotection afforded by human syphilitic serum is, in large part, a result of the activation of the complement cascade by bactericidal antibodies and spontaneous hydrolysis of C3. Thus like B. burgdorferi, T. pallidum activates the CP and AP of the complement cascade, and there is a considerable amount of evidence for an important role of these pathways in syphilitic lesion resolution during human infection with treponemes. In contrast to B. burgdorferi, there is no evidence indicating that T. pallidum has evolved mechanisms to evade complementdependent killing, suggesting that these complement pathways have a larger role in controlling treponemal infection than in the case of B. burgdorferi. During experimental syphilis, immunization with purified outer membrane vesicles (OMVs) isolated from T. pallidum results in complement-dependent bactericidal activity.23 More recently, immunization with OMVs led to the isolation of a

409

bactericidal monoclonal antibody (mAb), M131, which provides partial protection to experimental syphilis. M131 binds to a phosphorylcholine surface epitope of T. pallidum and is the first demonstration of such an antigen on the surface of the spirochete.24

Adaptive Immune Responses

T Cell–Mediated Responses The infiltration of T cells (CD4 and CD8) and macrophages into the primary and secondary syphilitic lesion facilitates local clearance of the majority of treponemes and lesion resolution via a vigorous cell-mediated immune response characteristic of a delayed-type hypersensitivity or Th1 response.25,26 CD4 T cells are the principal T-cell subset found in the lesions and are believed to promote macrophage activation and subsequent treponeme clearance through IFN-γ secretion. The role of CD8 cells is not clear, but throughout the course of primary lesion development during experimental syphilis, their proportion related to CD4 cells increases. Once the treponemes are cleared and the lesion heals, a latency stage ensues, which is characterized by long-lasting, protective T-cell memory to T. pallidum antigens. B Cell–Mediated Responses Many functional activities of human syphilitic serum originate from B-cell responses to T. pallidum. Infection with T. pallidum invokes a humoral immune response early in the course of infection, which strengthens as the number of recognizable antigens increases during the progression of infection. A polymorphic gene family of T. pallidum, T. pallidum repeat (tpr), has been recently identified as related to the major surface protein (msp) genes of T. denticola.27 A single member of the tpr family, tprK, serves as an antigen for opsonizing antibodies, suggesting that this protein is a surface antigen of T. pallidum. However, there are only inferential indications that surface antigens of T. pallidum exist. So, although candidate T. pallidum surface proteins have been advanced on the basis of porin activity or homology with a surface protein of T. denticola, there is no direct evidence identifying specific surface antigens on T. pallidum. Furthermore, like most pathogenic organisms, T. pallidum has evolved various mechanisms to escape host killing. TprK is an immunogen with seven discrete variable regions differing among isolates of the spirochete.27 In fact, the antibody response to T. pallidum is directed against these variable regions, which leads to immune selection of new TprK variants; thus antigenic variation is involved in the reinfection of hosts despite robust immune responses.27,28

Translational Research ON THE HORIZON Efficient diagnostic methods that are required to rapidly identify infection and procure antimicrobial treatments Development of new generation vaccines for Lyme disease, including those that target the tick vector and that can prevent other tick-borne infections Attention to communities at risk for contracting syphilis with investments in prophylaxis and efficient treatments

The challenge in the next 5–10 years is to develop effective diagnostic methods for both Lyme disease and syphilis and to devise new preventive measures. For Lyme disease, advances in genetic manipulation, as well as other means to study structure/

410

Part THREE  Host Defenses to Infectious Agents

function of key components of the spirochete, could lead to development of single or combination vaccines. Further, the identification of host factors that mediate a protective response and those that contribute to inflammation may result in a better understanding of the disease. Overall, the identification of the elements that mediate specific tissue tropisms for the bacterium and their interaction with local/infiltrating cellular components can permit the design of targeted therapies in conjunction with antimicrobial treatments. A significant effort is being made to find new antigenic determinants that can be the base for a vaccine to prevent infection with B. burgdorferi. While the search for spirochetal antigens continue, a new wave of research has focused on finding antigenic determinants that can prevent the efficient attachment of the tick vector to the mammalian host, which, in turn, could dampen the ability of the arthropod to transmit B. burgdorferi and perhaps other pathogens. For syphilis, the study of the microorganism is hampered by the difficulties associated with its culture and manipulation. Although advances have been made in our understanding of the pathology associated with infection, a significant challenge in the near future is to fully understand this pathogen. An area in which advances can be made in the next decade consists of better education of populations that are at risk of acquiring the infection. Because these populations are usually associated with low-income communities, a significant investment needs to be made to enable persons at risk to better evade the transmission of the pathogen, through education, better diagnostics, and rapid antimicrobial treatment.

CONCLUSIONS Spirochetes are a phylogenetically ancient and distinct group of microorganisms. Because of their propensity to cause diseases in humans, B. burgdorferi and T. pallidum are the two most-well-studied spirochetes. However, the inability to culture T. pallidum in vitro has made researching this spirochete very difficult, and as a consequence, the immune response that follows infection with this spirochete remains poorly understood. The etiological agents of Lyme disease and syphilis are similar in that they have relatively small genomes, surviving only in association with a host and eliciting inflammatory disease, but genomic comparison clearly shows that T. pallidum and B. burgdorferi are not closely related. It seems likely that despite some similarities, these spirochetes evolved independently from a more complex ancestor, resulting in differences in their lifecycles, environmental adaptations, and the pathology associated with their infection. Therefore it is not surprising to learn of the differences in the host immune responses to B. burgdorferi and T. pallidum. Both the host response to these spirochetes and their infectivity determine the extent of pathology following infection. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Burgdorfer W, Barbour AG, Hayes SF, et al. Lyme disease-a tick-borne spirochetosis? Science 1982;216:1317–19.

2. Krause RM. Metchnikoff and syphilis research during a decade of discovery, 1900-1910. Development of an animal model and a preventive treatment set the stage for progress. ASM News 1996;62:307–10. 3. Fraser CM, Casjens S, Huang WM, et al. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 1997; 390:580–6. 4. Fraser CM, Norris SJ, Weinstock GM, et al. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 1998;281:375–88. 5. Anguita J, Hedrick M, Fikrig E. Adaptation of Borrelia burgdorferi in the tick and the mammalian host. FEMS Microbiol Rev 2003;27:493–504. 6. Pal U, Li X, Wang T, et al. TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi. Cell 2004;119:457–68. 7. Hubner A, Yang X, Nolen DM, et al. Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway. Proc Natl Acad Sci USA 2001;98:12724–9. 8. Center for Disease Control and Prevention. 2016. Available at: https:// www.cdc.gov/lyme/stats/index.html. 9. Guerau-de-Arellano M, Huber BT. Chemokines and Toll-like receptors in Lyme disease pathogenesis. Trends Mol Med 2005;11:114–20. 10. Hawley KL, Olson CM Jr, Iglesias-Pedraz JM, et al. CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi. Proc Natl Acad Sci USA 2012;109:1228–32. 11. Lawrenz MB, Wooten RM, Zachary JF, et al. Effect of complement component C3 deficiency on experimental Lyme borreliosis in mice. Infect Immun 2003;71:4432–40. 12. Kochi SK, Johnson RC. Role of immunoglobulin G in killing of Borrelia burgdorferi by the classical complement pathway. Infect Immun 1988;56:314–21. 13. Madar M, Bencurova E, Mlynarcik P, et al. Exploitation of complement regulatory proteins by Borrelia and Francisella. Mol Biosyst 2015;11:1684–95. 14. Pausa M, Pellis V, Cinco M, et al. Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule. J Immunol 2003;170:3214–22. 15. Hovis KM, McDowell JV, Griffin L, et al. Identification and characterization of a linear-plasmid-encoded factor H-binding protein (FhbA) of the relapsing fever spirochete Borrelia hermsii. J Bacteriol 2004;186:2612–18. 16. Garcia BL, Zhi H, Wager B, et al. Borrelia burgdorferi BBK32 inhibits the classical pathway by blocking activation of the C1 complement complex. PLoS Pathog 2016;12:e1005404. 17. Gross DM, Steere AC, Huber BT. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis. J Immunol 1998;160:1022–8. 18. Zhang JR, Hardham JM, Barbour AG, et al. Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 1997;89:275–85. 19. Ramamoorthi N, Narasimhan S, Pal U, et al. The Lyme disease agent exploits a tick protein to infect the mammalian host. Nature 2005;436:573–7. 20. Cox DL, Chang P, McDowall AW, et al. The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum. Infect Immun 1992;60:1076–83. 21. Salazar JC, Hazlett KR, Radolf JD. The immune response to infection with Treponema pallidum, the stealth pathogen. Microbes Infect 2002;4:1133–40. 22. Salazar JC, Cruz AR, Pope CD, et al. Treponema pallidum elicits innate and adaptive cellular immune responses in skin and blood during secondary syphilis: a flow-cytometric analysis. J Infect Dis 2007;195:879–87. 23. Blanco DR, Champion CI, Lewinski MA, et al. Immunization with Treponema pallidum outer membrane vesicles induces high-titer complement-dependent treponemicidal activity and aggregation of T. pallidum rare outer membrane proteins (TROMPs). J Immunol 1999;163(5):2741–6.

CHAPTER 28  Host Defenses to Spirochetes 24. Blanco DR, Champion CI, Dooley A, et al. A monoclonal antibody that conveys in vitro killing and partial protection in experimental syphilis binds a phosphorylcholine surface epitope of Treponema pallidum. Infect Immun 2005;73:3083–95. 25. McBroom RL, Styles AR, Chiu MJ, et al. Secondary syphilis in persons infected with and not infected with HIV-1: a comparative immunohistologic study. Am J Dermatopathol 1999;21:432–41. 26. Van Voorhis WC, Barrett LK, Koelle DM, et al. Primary and secondary syphilis lesions contain mRNA for Th1 cytokines. J Infect Dis 1996;173:491–5.

411

27. Centurion-Lara A, Castro C, Barrett L, et al. Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response. J Exp Med 1999;189:647–56. 28. Giacani L, Molini BJ, Kim EY, et al. Antigenic variation in Treponema pallidum: TprK sequence diversity accumulates in response to immune pressure during experimental syphilis. J Immunol 2010;184:3822–9.

CHAPTER 28  Host Defenses to Spirochetes

411.e1

MULTIPLE-CHOICE QUESTIONS 1. The major immunogens of Borrelia burgdorferi and Treponema pallidum are: A. Lipopolysaccharide molecules present on the outer membrane B. Sugar coated-proteins that decorate their surface C. A group of lipoproteins that constitute Toll-like receptor (TLR)1/TLR2 ligands D. Both spirochetes, which are considered “stealth pathogens” because of their lack of immunogens 2. The phagocytosis of spirochetes: A. Allows the elimination of the bacteria before they have a chance to induce a proinflammatory response B. Allows the interaction of pathogen recognition receptors with their ligands within the phagolysosome t modulate the inflammatory response C. Depends exclusively on the presence of opsonins that target the bacteria to professional antigen-presenting cells D. Occurs only on tissues where the pathogens reside, exclusively upon activation of the adaptive immune system

3. The generation of a protective antibody response against B. burgdorferi: A. Occurs even in the absence of T-cell responses B. Is targeted exclusively to variable regions of the vlsE protein C. Is always accompanied by a T-helper type 2 response D. Occurs within the first 3 days of infection and is the basis for the successful diagnosis of patients 4. T-cell responses against T. pallidum: A. Are always weak because of the low level of infection in patients B. Induce a type I delayed hypersensitivity response that helps the elimination of the bacteria C. Are always composed of CD4 T cells that exclusively aid the development of antibody responses D. Can be used as diagnostic and prognostic tools for the disease

29  Host Defenses to Fungal Pathogens Allison K. Lord, Jatin M. Vyas

Advances in modern medicine have significantly improved the prognosis and quality of life for patients with a wide spectrum of diseases, including many forms of cancer. However, opportunistic fungal pathogens have exploited these modern immunosuppressive and invasive medical interventions. Invasive fungal infections (IFIs) represent a significant cause of morbidity and mortality among immunocompromised patients. Patients at highest risk for IFIs include solid organ or hematopoietic stem cell transplant (HSCT) recipients on intensive immunosuppressive regimens, patients with hematological malignancies, and other patients that are immunocompromised as a result of various clinical conditions and treatments (e.g., human immunodeficiency virus/acquired immunodeficiency syndrome [HIV/AIDS], advanced age, recent surgery, etc.) (Fig. 29.1).1,2 The clinical consequences of these pathogenic fungi include superficial disease, allergic disease, and IFIs.

KEY CONCEPTS Invasive Fungal Infections • Invasive fungal infections are systemic life-threatening infections that are estimated to cause 1.5 million deaths annually. • Aspergillus, Candida, Cryptococcus, and Pneumocystis are opportunistic fungi that cause the majority of invasive fungal infections. • Risk factors for invasive fungal infections include bone marrow or solid organ transplantation, intensive immunosuppressive regimens, hematological malignancies, HIV/AIDS, advanced age, recent surgery, and other clinical conditions and treatments that cause immunosuppression.

Superficial fungal infections of the skin and nails affect 25% of the global population (≈1.7 billion people) and give rise to various conditions, including athlete’s foot and ringworm of the scalp. Fungi also commonly cause mucosal infections of the oral and genital tracts; vulvovaginal candidiasis occurs at least once in 50–75% of women in their childbearing years.3,4 Superficial fungal infections of the skin and mucosa can become chronic, but they are rarely life threatening. IFIs occur when fungal pathogens invade the bloodstream, resulting in a systemic life-threatening infection that affects multiple organs. IFIs are estimated to cause 1.5 million deaths annually, with four genera accounting for most of the infections: Cryptococcus, Candida, Aspergillus, and Pneumocystis. Mortality resulting from infections with these fungal organisms exceeds that caused by tuberculosis or malaria.4 As a result, IFIs have emerged as an escalating and worrisome clinical problem. There

is an urgent need to define the mechanisms of host defense against fungal pathogens with the goal of developing novel therapeutics as well as improving diagnostic and preventative procedures. One of the greatest limiting factors in the treatment of IFIs is lack of effective and prompt diagnostic tools. Although Candida is capable of growing in conventional blood cultures, most other invasive fungal organisms, including Cryptococcus, Aspergillus, and Histoplasma, are typically not recovered by adding blood to growth media. There has been a heavy reliance on clinical diagnosis and a few biomarkers. Fungal cell wall carbohydrates can be detected in the bloodstream of some patients. β-1,3 glucan, galactomannan, and glucuronoxylomannan (GXM) are three carbohydrates that can be analyzed clinically. β-1,3 glucan is a common fungal cell wall carbohydrate found in Candida albicans and many other fungi (with the notable exception of Cryptococcus neoformans). Galactomannan is typically associated with Aspergillus fumigatus. This fungal biomarker can be measured in blood and bronchoalveolar lavage fluid. GXM is an abundant fungalderived carbohydrate produced by C. neoformans. GXM can also be found on the surface of Candida gattii, a closely related fungal organism that can cause disease in immunocompetent individuals. Although GXM appears to be a useful biomarker for Cryptococcus, its widespread use is limited by technical and economic issues. Finally, a urine test for histoplasmosis has been established. Again, the antigen for this test is a polysaccharide derived from the cell wall of this fungal organism. Many of these biomarkers suffer from poor sensitivity (galactomannan) and, in some cases, poor specificity (e.g., testing urine for Histoplasma antigen). Current therapeutic approaches for IFIs include the prompt institution of antifungal agents (including polyenes, azoles, and echinocandins) and even more important, the reversal of underlying host immune defects, such as neutropenia or high doses of immunosuppressive therapy. The first-line therapy for the treatment of invasive aspergillosis (IA) is voriconazole, an extended-spectrum azole. Despite its in vitro efficacy, voriconazole only demonstrated a survival rate at 12 weeks of 70.8% compared with 57.9% in the amphotericin B group.5 Although voriconazole has become the gold standard for treatment, its high mortality rate indicates that the host immune response plays a critical role in determining host outcome. Despite clinical vigilance, prevention, diagnosis, and treatment of IFIs remain a significant challenge because of lack of rapid and reliable diagnostic methods and limited treatment options. Discovery of novel diagnostic tools and antifungal therapies is crucial to ameliorating this public health burden.

413

414

Part three  Host Defenses to Infectious Agents Immunotherapy for autoimmune diseases

Immunotherapy for cancer

Cancer and chemotherapy

Intensive care unit treatment

Parenteral nutrition Catheterization

Immunosuppressive therapy

Neonates and elderly Organ/bone marrow transplant

HIV / AIDS

Invasive fungal infections

Hematologic malignancy

Broad-spectrum antibiotics Surgery Inherited immunodeficiencies

FIG 29.1  Major risk factors for developing invasive fungal infections. (Adapted from KarkowskaKuleta J, Kozik A. Cell wall proteome of pathogenic fungi. Acta Biochim Pol 2015; 62: 339–51.)

CLINICALLY RELEVANT FUNGAL ORGANISMS Although it is estimated that there are in excess of 5 million distinct species of fungi, only a handful are considered significant to human health: Aspergillus spp., Candida spp., and Cryptococcus spp. cause the majority of IFIs in the United States and Europe. Invasive infections caused by these pathogens in immunocompromised patients are serious and often life threatening.

Aspergillus fumigatus Aspergillus fumigatus is a ubiquitous airborne mold found in soil, air, food, and decaying organic material. Humans routinely inhale A. fumigatus conidia, but the microorganism is rapidly eliminated by the innate immune system in immunocompetent individuals. In immunocompromised patients, A. fumigatus can cause IA, a severe and usually fatal infection. It is estimated that IA occurs in 200 000 patients annually.4 In cases of widespread infection, IA mortality rates approach 90%, with favorable responses to antifungal therapy observed in 40 µm in length. For both C. albicans and A. fumigatus, this dimorphism is a virulence factor as organisms that fail to do so are avirulent.

THE INNATE IMMUNE RESPONSE TO FUNGAL PATHOGENS Once a fungal pathogen has invaded the host system, innate immune cells, such as macrophages/monocytes, neutrophils, and dendritic cells (DCs), phagocytose and degrade the organism (Chapter 3). In immunocompetent individuals, physical barriers (e.g., epithelial cells, mucous, skin) are effective at preventing infection. In the respiratory tract, macrophages routinely neutralize fungal pathogens that make their way to the alveoli. Understanding the rules that govern the recognition and response to these fungal pathogens provide important insights into how these organisms cause disease in immunocompromised individuals. Recognition of fungal pathogens by innate immune cells is mediated by pattern recognition receptors (PRRs). Engagement of PRRs and PAMPs triggers a cascade of molecular events that coordinates phagocytosis and degradation of the pathogen. Additionally, release of cytokines, reactive oxygen species (ROS) production, and presentation of fungal antigens to the adaptive immune system aids in controlling infection.

416

Part three  Host Defenses to Infectious Agents

KEY CONCEPTS Innate Immunity to Fungal Pathogens • Fungi express highly conserved pathogen-associated molecular patterns (PAMPs) that are recognized by pathogen recognition receptors (PRRs) expressed on host phagocytes. • Dectin-1 is a C-type lectin receptor (CLR) that acts as a PRR to recognize β-1,3 glucan expressed on the cell walls of Candida, Aspergillus, and other fungal pathogens. • Toll-like receptors (TLRs) recognize fungal cell wall components and nucleic acids. • Engagement of PRRs and PAMPs initiates signaling that coordinates secretion of cytokines, reactive oxygen species (ROS) production, and presentation of fungal antigens to the adaptive immune system to facilitate elimination of the pathogen.

Recently engulfed pathogens become enclosed in membrane delineated compartments called phagosomes, which traffic toward the lysosome, as directed by modifications to the phagosomal membrane proteins and changes to the intraphagosomal environment. Proteins recruited to the phagosomal membrane are specific to its contents. Phagolysosomes intersect with class II major histocompatibility complex (MHC) molecules and permit loading of pathogen-specific peptides. T cells are then activated in an antigen-specific manner to augment the immune response and generate long-term immunity.

Role of Neutrophils Neutrophils are the most critical cell in the host defense against fungal pathogens. Patients with neutropenia or acquired defects in neutrophil-mediated killing are at higher risk of developing IFIs, including IA. Although phagocytosis is a critical feature in controlling infection, other mechanisms also limit the spread of infection and serve to kill invading organisms. Neutrophils rely on multiple mechanisms for killing, including granule-dependent killing, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–dependent killing, and neutrophil extracellular trap (NET) formation (Chapter 22). Phagocytosis of fungal organisms by neutrophils triggers production of antimicrobial ROS. Generation of ROS by the NADPH oxidase complex is a result of engagement of surface receptors, including dectin-1, and signaling adaptors, including caspase recruitment domain family member 9 (CARD9) and Syk. Loss of ROS production has a clear phenotype in Aspergillus infections. Patients with chronic granulomatous disease (CGD) fail to control Aspergillus hyphae, which leads to invasion and metastatic spread of disease. In contrast, conidial forms of Aspergillus do not require ROS. The data for Candida infections are less compelling. There appears to be modest reduction in killing of serum-opsonized C. albicans, but killing of unopsonized organisms is unimpaired. In addition to cytotoxic granules and ROS production, neutrophils are capable of NET formation. This relatively newly discovered cytotoxic mechanism delivers a web-like structure composed of chromatin and histones. For Candida, it appears that NET formation contributes to fungal killing.17 However, in Aspergillus, data from studies do not support a clear role for NETs.

Role of Macrophages Macrophages also play a critical role in neutralizing fungal organisms. These phagocytic cells are much longer lived than

neutrophils. Macrophages are recruited to the site of infection by chemotaxis. They are also responsible for patrolling interfaces with the environment, including the lung and the GI tract. In contrast to neutrophils, macrophages express class II MHC and can activate T cells. Upon activation by fungi through PRRs and PAMPs, macrophages produce potent inflammatory cytokines, including tumor necrosis factor α (TNF)-α and IL-6. Engagement of the inflammasome within these cells leads to copious production of IL-1β. Macrophages receive assistance from activated T cells that secrete interferon-γ (IFN-γ), which can activate a host of genes to improve the antifungal response from macrophages.

Role of Dendritic Cells In contrast to macrophages, DCs can stimulate naïve T cells. Moreover, DCs are capable of taking up both yeast and conidia. DC subsets are characterized by expression of specific surface markers. Plasmacytoid DCs (pDCs) expressing specific surface markers phagocytose A. fumigatus conidia and spread over hyphae. pDCs incite an immune response by release of proinflammatory cytokines, including IFN-α and TNF-α. pDCs are typically found in the spleen but will migrate to the lungs following challenge by A. fumigatus conidia. Depletion of pDCs has been shown to result in increased mortality, indicating a nonredundant role for pDCs in host defense against A. fumigatus. C. albicans epithelial infections appear to recruit pDCs. These cells reorganize and become more concentrated in the T-cell zones of lymph nodes. Draining lymph nodes of mice infected with Candida versus control mice showed that pDCs were the predominant DC subset after vaginal infection. pDCs are involved in the induction of Th1 responses to C. albicans vaginal infection.

PATTERN RECOGNITION RECEPTORS Recognition of fungal pathogens is mediated by PRRs expressed by innate immune cells, including DCs and myeloid cells (e.g., macrophages, monocytes, and neutrophils). The interaction between PRRs and PAMPs expressed on the fungal cell wall triggers downstream signaling, which elaborates the host immune response and facilitates elimination of the pathogen. The major PRRs involved in antifungal immunity are Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). Other relevant PRRs include nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs), and retinoic acid-inducible gene I (RIG-I)–like receptors (RLRs).

Toll-Like Receptor Toll-like receptors (TLRs) are transmembrane receptors that recognize a broad range of microbial ligands, including fungal and bacterial cell wall components, bacterial and viral nucleic acids, and bacterial lipoproteins (Fig. 29.2) (Chapter 3). TLR1, -2, -4, -5, -6, and -10 are expressed on the cell surface, whereas TLR3, -7, -8, and -9 are expressed on intracellular membranes. Intracellular TLRs recognize nucleic acids derived from fungi, bacteria, and viruses and signal through MyD88 or TRIF. MyD88 signaling triggered by TLRs is essential for host fungal defense; mice lacking MyD88 are more susceptible to IFIs. However, humans with MyD88 signaling defects do not have increased incidence of fungal infections. To date, several TLRs have been implicated in fungal immunity in mice, including TLR2, TLR4, TLR7, and TLR9.18 The importance of TLRs has been demonstrated in human biology. In humans, polymorphisms in TLR1, TLR3, TLR4, and TLR6 are associated with increased susceptibility to IFIs (especially IA).19

CHAPTER 29  Host Defenses to Fungal Pathogens Fungi

C. albicans A. fumigatus C. neoformans

C. albicans A. fumigatus C. neoformans

PAMPs

GXM Mannan (O-linked)

GXM PLM (phospholipomannans)

TLR2

TLR

Gene expression output

A. fumigatus RNA

Candida spp. A. fumigatus C. neoformans DNA

417

Candida spp. RNA

TLR6

Plasma membrane

TLR4

IFNγ IL-12

Endosome

IFNδ IL-12 TNFα TGF-β IL-10 IL-23

TLR3

TLR9 TLR7

IFNβ

IL-6 IL-10 IFNβ IL-12

IFNβ IL-12

FIG 29.2  Toll-Like Receptors (TLRs) and Fungal Immunity. Surface and endosomal TLRs recognize fungal pathogen-associated molecular patterns (PAMPs) resulting in downstream signaling that promotes production of cytokines. (Adapted from Bourgeois C, Kuchler K. Fungal pathogens-a sweet and sour treat for Toll-like receptors. Front Cell Infect Microbiol 2012;2:142.)

In contrast, TLR9 polymorphisms are more tightly associated with allergic bronchopulmonary aspergillosis. Surface-disposed TLR2 recognizes β-glucans of several fungal species (C. albicans, A. fumigatus, and C. neoformans). TLR2 also interacts with phospolipomannans and linear β-1,2-oligomannoside structures found on the cell wall of C. albicans. TLR2 can heterodimerize with TLR1 or TLR6 to recognize GXM, a cell wall component of C. neoformans. TLR2 is critical for early recruitment and the killing abilities of neutrophils.19 TLR2-deficient mice infected with Pneumocystis jiroveci display more severe symptoms, increased fungal burden, and less TNF-α production. TLR4 recognizes O-linked mannan expressed on the cell wall of C. albicans and through downstream signaling, stimulates production of TNF-α. Mice deficient in TLR4 do not appear to have increased susceptibility to disseminated candidiasis compared with wild-type mice. In contrast, killing of Aspergillus is impaired in TLR4−/− mice in a corneal inflammation model, although recruitment of immune cells to the infection, remains unaffected. TLR9 recognizes intracellular microbial ligands, such as CpG-rich fungal DNA. Dectin-1–dependent Syk activation triggers

phagosomal acidification, which permits cleaved TLR9 to traffic to the phagosomes. Moreover, dectin-1 regulates TLR9-dependent changes in gene expression.20 Interestingly, TLR9 deficient macrophages have increased fungicidal activity and production of antiinflammatory cytokines. Thus TLR9 appears to modulate the inflammatory response by downregulating cytokine production.21

C-Type Lectin Receptor C-type lectin receptors (CLRs) comprise a diverse family of proteins characterized by a conserved C-type lectin domain (CTLD) that recognizes a variety of ligands. CLRs that are recognized to play a role in fungal immunity include dectin-1, dectin-2, mannose receptor, Mincle, and dendritic cell–specific intercellular adhesion molecule–grabbing nonintegrin (DC-SIGN) (Fig. 29.3). However, dectin-1 is the best-characterized CLR associated with fungal immunity. Dectin-1 is a type II transmembrane protein expressed primarily on myeloid cells. It is considered the major mammalian cell surface receptor for β-1,3 glucan and β-1,6 glucan, carbohydrates widely expressed on the cell wall of many fungal organisms. Furthermore, dectin-1

418

Part three  Host Defenses to Infectious Agents

Fungi

*C. albicans *C. glabrata *Coccidioides spp. *H. capsulatum *P. jirovecii

*C. albicans *C. glabrata *Paracoccidioides brasiliensis

*C. albicans *P. brasiliensis

*Candida spp. *Malassezia spp. *Saccharomyces spp.

*Candida spp. *A. fumigatus *Chrysosporium tropicum

PAMP

β-glucan

α-mannan

β-mannan

?

N-mannan

CLR

Dectin-1

Dectin-2

Galectin-3

MINCLE

DC-SIGN

TNFα IL-6 IL-10 IL-1β IL-23

IL-6 IL-10 IL-12

Plasma membrane

Gene expression output

TNFα IL-1β IL-23 IL-2 IL-10 IL-6 IL-12

TNFα IL-1β IL-23 IL-6 IL-10

IL-1β IL-6 IL-10 TNFα TGFβ

FIG 29.3  C-Type Lectin Receptors (CLRs) and Fungal Immunity. CLRs recognize fungal PAMPs and trigger downstream signaling that leads to cytokine production.

promotes acidification and maturation of phagosomes.22 Patients with mutations in dectin-1 are more susceptible to IFIs, which demonstrates the essential role of dectin-1 in mediating fungal immunity.23 Upon dectin-1 binding to β-1,3 glucan, a tyrosine residue within the cytoplasmic hemi–immunoreceptor tyrosine-based activation motif (ITAM) domain of dectin-1 is phosphorylated by Src family kinases, which leads to the recruitment and activation of Syk. Syk triggers the activation of the caspase recruitment domain family member 9–Bcl-10– mucosa-associated lymphoreticular tissue 1 (MALT1) complex, inducing nuclear factor (NF)-κB and production of cytokines, including IL-1β, IL-6, IL-10, IL-112, IL-23, and TNF-α. Dectin-1 can signal through a Syk-independent pathway via activation of the serine/threonine kinase, Raf-1, that phosphorylates p65, a subunit of NF-κB.24 Cytokines produced in response to canonical (via Syk) and noncanonical (via Raf-1) NF-κB signaling are essential for the activation of Th1 and Th17 cells (Fig. 29.4). Syk also triggers recruitment and activation of NADPH oxidase, which stimulates production of ROS, elaboration of proinflammatory cytokines, and ultimately fungal killing. Syk appears to be a master controller for phagosomal maturation and recruitment of light chain 3 (LC3), a protein associated with autophagy.

Collaboration Between TLRs and CLRs Although most cognate ligands found on the fungal cell wall are carbohydrates that preferentially trigger CLRs, experimental evidence indicates that CLRs can collaborate with TLRs on the cell surface to coordinate cytokine production. Dectin-1 can collaborate with TLR2 and TLR4 to induce synergistically cytokines, including TNF-α, IL-10, and IL-23. This collaboration has been used clinically for management of clinical disease. Fonsecaea pedrosoi is the fungus that causes chromoblastomycosis, a skin infection that is difficult to treat. The CLR Mincle recognizes this pathogen. If costimulation of both CLR and TLR pathways are not engaged, the inflammatory response is defective. Exogenous administration of imiquimod, purified TLR7 ligand, facilitated pathogen clearance in mouse models and humans. This signaling requires both Syk/CARD9 and MyD88. Imiquimod is a synthetic compound with potent antiviral and antitumor activity that stimulates the innate immune system through TLR7 activation. When applied to the lesions of four patients with chromoblastomycosis, there was a rapid resolution of the infection.25 This provides proof of concept that multiple signaling pathways are necessary for optimal activation of the innate immune system against fungal pathogens.

419

CHAPTER 29  Host Defenses to Fungal Pathogens Fungus

Dectin-1

Dectin-1 Neutrophil recruitment

Plasma membrane

IL-17A IL-17F IL-2 2

Syk Th17

CARD9 Raf-1

NFAT

BCl-10

NADPH oxidase

MALT1

6,

ILLC3 recruitment to phagosome

ROS NF-κβ

β

F TG

Th2 IL-4

IL-4 IL-5 IL-13 etc.

NRLP3

caspase1 pro-IL1β NFKβ

NFAT

IL1β IL-1β IL-23 IL-6

Cytokines

IL-10 IL-12 TNFα

IL-12

IL-2 IL-10

Cytokines

Th1

IFNδ etc.

Nucleus Macrophage activation

FIG 29.4  Dectin-1 Signaling in Response to Fungal Pathogens. Upon recognition of fungal pathogens, dectin-1 signaling triggers secretion of cytokines, production of reactive oxygen species (ROS), and activation of the adaptive immune system to facilitate elimination of the pathogen.

CARD9

THE INFLAMMASOME

Mutations in the CARD9 adaptor molecule, downstream of dectin-1, are associated with increased susceptibility to invasive Candida infections. Patients with CARD9 deficiencies have decreased Th17 cells, reduced chemokine/cytokine production, and impaired neutrophil activation.26 Numerous patients have been identified to have mutations in CARD9 that lead to fungal infections. The vast majority of inborn errors in immunity that predispose to fungal disease typically also lead to development of nonfungal infections. Patients with gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) or autosomal dominant STAT3 also suffer from mycobacterial and other bacterial infections. In contrast, mutations of CARD9 appear to affect susceptibility to fungal infections exclusively. This observation makes CARD9 a key regulator in antifungal immunity. Other genes associated with increased risk of fungal infections typically predispose patients to increased mucocutaneous or invasive disease. In contrast, CARD9 deficiency predisposes to both mucosal and systemic fungal diseases.

IL-1β activation occurs when its precursor pro–IL-1β is cleaved by caspase-1. IL-1β is critical for neutrophil recruitment and for induction of the Th17 response. Activation of the inflammasome regulates IL-1β, IL-18, IL-17, and IFN-γ. NLR family domain containing protein 3 (NLRP3) is required for activation of caspase 1. Caspase 8 may also play a role in regulating activation of IL-1β.26 Syk-dependent production of ROS activates the NLRP3 inflammasome, which coordinates with NF-κB to promote production of IL-1β.27 Mice lacking the inflammasome or IL-1β are highly susceptible to disseminated candidiasis.28 In addition to ROS, lysosomal rupture, release of cathepsins, and efflux of potassium can lead to activation of the inflammasome. Many components of the fungal cell wall can trigger inflammasome activation in vitro. However, in these experiments, cells were pretreated with adenosine triphosphate (ATP) or lipopolysaccharide (LPS) to provide an extra signal. When whole live fungal organisms are used, pretreatment with ATP or LPS is not required. Mice lacking NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), or

420

Part three  Host Defenses to Infectious Agents

caspase 1 demonstrate increased susceptibility to C. albicans infection. The mechanism appears to be a reduction in neutrophil influx. However, in these mouse models, early neutrophil influx does not appear to be dependent on an intact inflammasome; rather, it is required for sustained neutrophil influx.

MEMORY OF INNATE IMMUNE CELLS Historically, the innate immune response was considered a rapid and nonspecific attack against invading pathogens. The widely accepted dogma was that innate immune cells confer no immunological memory, unlike adaptive immune cells that respond more slowly but execute a specific attack based on memory. An increasing body of evidence suggests that the innate immune system can, in fact, form memory, challenging the historical dogma. For example, innate immune memory has been demonstrated in organisms that lack the adaptive immune system, such as plants and invertebrates.29 Several studies have demonstrated increased responsiveness of innate immune cells to secondary encounters with pathogens.30 Immunological memory of innate immune cells has been described in the context of C. albicans infection; in mice, preexposure to a nonlethal infection or purified β-1,3 glucan confers protection from a secondary lethal infection in the absence of functional B cells and T cells. Epigenetic modifications serve as the mechanism by which innate immune cells generate immunological memory.31 There are numerous lingering questions about innate immune memory, including the duration of this response, its ability to provide protection from other microbes, and whether this type of response can be elicited by vaccination.

LINKING THE INNATE IMMUNE RESPONSE TO ADAPTIVE IMMUNE RESPONSE A secondary, but critical, role of the innate immune system is to amplify the immune response by activating and recruiting cells of the adaptive immune system, including T and B lymphocytes. The innate immune signaling events that trigger activation of adaptive immunity are complex and may be pathogen dependent. This amplification of the immune response is partially achieved by secretion of cytokines and ROS that recruit immune cells to the site of infection. Additionally, antigen-presenting cells (APCs) of the innate immune system use class II MHC molecules on their surface to present pathogen-derived antigens to I CD4 T cells, resulting in differentiation into effector or regulatory T cells (Tregs). DCs provide the critical link between the innate and adaptive arms of the immune system. DCs are capable of recognizing fungal pathogens similar to macrophages. Endowed with both TLRs and CLRs, DCs are activated by components of the fungal cell wall. Subsets of DCs provide specialized function. pDCs produce IFN-α in response to ligands that engage endosomal TLRs. Although pDCs play a key role in viral infections, their role in fungal infections is not fully understood. pDCs are able to recognize A. fumigatus through engagement of TLR9. When A. fumigatus and pDCs are coincubated in vitro, growth of the fungus is inhibited, indicating that these cells are capable of controlling growth of this mold. A subset of pDCs secretes IL-6 and IL-23, the latter of which can serve to prime antigen-specific Th17 lymphocytes. CD8+ DCs possess the unique capacity to cross-present antigens. Cross-presentation of Histoplasma capsulatum antigens

has been demonstrated. Extracellular H. capsulatum are taken up by DCs and are then capable of triggering CD8 T cells through antigen-specific peptide-loaded class I MHC molecules. Monocyte-derived DCs have a nonredundant role in antifungal immunity, through the induction of Th1 cells. CCR2−/− mice show skewed Th2 responses and poorly controlled H. capsulatum infection compared with wild-type control mice. Priming of Th1 lymphocytes require CCR2+ monocyte-derived inflammatory DCs in mouse models of A. fumigatus or C. neoformans infection. These data indicate a critical role for DCs to connect the innate immune response to drive T-cell responses and coordinate the adaptive immune response.

THE ADAPTIVE IMMUNE RESPONSE TO FUNGAL PATHOGENS The primary function of the adaptive immune system is to launch a highly specific attack to destroy invading pathogens. The adaptive immune system also provides long-term protection by creating “immunological memory” following an initial response to a pathogen. The two major branches of the adaptive immune system are cell-mediated immunity (CMI) and humoral immunity. B cells and T cells carry out this adaptive immune response. CMI by T cells is critical for the host response to fungal pathogens. There are two major classes of T cells that orchestrate CMI in response to invading pathogens: CD4 T cells and CD8 T cells. Antigen presentation by DCs activates T cells. T-cell activation is also driven by cytokines secreted from macrophages and DCs. In turn, activated T cells secrete cytokines that elaborate the immune response.

KEY CONCEPTS Adaptive Immunity to Fungal Pathogens • T-cell immunity is critical for fungal host defenses. Patient with advanced HIV infection and AIDS are at heightened risk for C. neoformans infection. • T-helper 1 (Th1) and Th17 T cell subsets are the most important T cells for control of fungal infection • Th2 cells mediate fungal asthma and hyperreactivity. • Dectin-1 signaling is required for Th17 immunity. • Humoral immunity may play a role in antifungal defense but appears to play a supportive role.

CD4 T Cells CD4 T cells are major players in host fungal immunity. The critical role for CD4 T cells became overwhelmingly apparent during the AIDS epidemic. Patients with HIV/AIDS experience loss of CD4 T cells and are extremely susceptible to opportunistic fungal pathogens, especially C. neoformans infection. Additionally, subsets of CD4 T cells, including Th1, Th2, and Th17 cells, have specialized roles in fungal defense (Chapter 16). Th1 Cells Th1 cells are a subset of CD4 T cells that play a critical role in controlling fungal infections. After exposure to fungal pathogens, APCs secrete IL-12, which is critical to the maintenance and expansion of Th1 lymphocytes. Mutations in the IL-12 signaling pathway predispose mammals to Candida, Cryptococcus, and Coccidiomycosis infections. Th1 lymphocytes produce copious amounts of IFN-γ, TNF-α, and granulocyte macrophage–colony

CHAPTER 29  Host Defenses to Fungal Pathogens stimulating factor (GM-CSF), all of which play a critical role in antifungal defense. Although IFN-γ has multiple effects on different cell types, this cytokine induces several genes in macrophages that potently modify the distribution of proteins on the phagosome. The net result of these effects is enhanced intracellular killing. IFN-γ–treated macrophages have enhanced phagocytosis and are more efficient in antigen presentation. TNF-α has similar effects similar to those of IFN-γ and enhances the function of macrophages. Patients with mutations in the TNF-α receptor or those who are treated with TNF-α are at heightened risk for fungal infections, including A. fumigatus. GM-CSF appears to increase ROS production in neutrophils and macrophages. Two lines of evidence in humans suggest an important role for Th1 cells in antifungal defense. Patients with fever and neutropenia with documented IFIs, including candidemia, have improved resolution of infection independent of the effect of GM-CSF on neutrophil mobilization. Patients with protein alveolar proteinosis, which is a disease related to anti– GM-CSF autoantibodies, have increased risk of IFIs.

CLINICAL RELEVANCE Opportunistic Fungal Pathogens Cause Invasive Fungal Infections in Immunocompromised Patients • Advances in clinical medicine provide treatment modalities that bring new promise to once-deadly diseases. During the course of these treatments, many patients develop significant deficiencies in their immune system that permit the development of opportunistic infections. • Invasive fungal infections (IFIs) caused by Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans are considered the most serious infections that affect immunocompromised patients. Despite effective fungicidal therapies, such as voriconazole and amphotericin, mortality in these patients exceeds 25%. • These data indicate that a robust immune response is required for clinical efficacy. Patients with defects in innate immunity are at highest risk despite the fact that they typically have functional circulating lymphocytes. • There is a dire clinical need to develop novel therapeutics for IFIs to address the significant public health burden caused by opportunistic fungal pathogens.

Th2 Cells In contrast to Th1 cells, Th2 lymphocytes appear to be defense against most fungal infections. Th2 lymphocytes secrete IL-4, IL-5, and IL-13. The effects of these cytokines on macrophages diminish the antifungal response. IL-4 drives macrophage differentiation toward the alternatively activated phenotype. In mice exposed to either Aspergillus or Cryptococcus in the respiratory tract, Th2 skewing drives an allergic phenotype with airway hyperresponsiveness (AHR) and goblet hyperplasia. There is one notable exception to the deleterious effect of Th2 cells on invasive fungal disease—P. jiroveci. Th2 responses seem to be protective in this infection. Mouse studies suggest that alternatively activated macrophages are critical for clearance of this fungal pathogen. Recent studies have identified a nonredundant role for Th17 lymphocytes, suggesting that Th2 responses are necessary, but not sufficient, for clearance of P. jiroveci. Th17 Cells Th17 cells are a subset of CD4 T cells that have emerged as major contributors to host defense against fungal pathogens.

421

These lymphocytes play a critical and nonredundant role in the control of IFIs. Th17 cells are characterized by production of cytokines, IL-17A, IL-17F, and IL-22. Differentiation of this T-cell lineage requires stage-specific stimulation by cytokines and activation of specific transcription factors. Transforming growth factor-β (TGF-β) and IL-6 prime the initial differentiation of CD4 T cells to Th17 cells. IL-23 is required for both maintenance and expansion of these cells.32 Activation of STAT3 by IL-6 regulates transcription of retinoid orphan receptor-γt (ROR-γt), the master transcription factor controlling Th17 lineage commitment. Several studies have confirmed the importance of Th17 cells and IL-17/IL-23 signaling for antifungal immunity.33 Mice deficient in IL-17 receptor fail to generate functional Th17 cells and are highly susceptible to both systemic and mucocutaneous candidiasis. Additionally, IL-23–deficient animals develop progressively worse mucocutaneous disease. Likewise, in mouse models of vulvovaginal C. albicans infection, impairment of Th17 cell function led to increased fungal burden. Interestingly, fungal pathogens have devised mechanisms to subvert this component of the immune system. Cell surface receptors found on Candida and Aspergillus bind IL-17 and cause increased hyphal growth and transcriptional changes within these fungal organisms that are thought to combat the immune response.

CD8 T Cells Although CD8 T cells are critical for immune defense against viral pathogens and tumors, their contribution to fungal immunity is incompletely understood. They may provide functional redundancy to confer protection in the absence of CD4 T cells. These cells will be important in the development of fungal vaccines, but further studies are required to delineate if they have any nonredundant role in antifungal immunity.

B Cells Early data dismissed the role of humoral immunity during fungal infection. However, clinical observations have suggested that antibodies contribute to fungal host defense. Patients with B-cell defects, including X-linked hyper-IgM syndrome and hypogammaglobulinemia, demonstrate increased susceptibility to cryptococcosis. Additionally, pediatric patients with selective IgG2 deficiency are at heightened risk for IFIs and other bacterial infections that rely on antipolysaccharide antibody formation. Antibodies observed in normal individuals are typically against fungal cell wall components and have the capacity to inflict negative changes within the fungal cell. These include inducing transcriptional changes that are deleterious to the microbe. Antibodies can work in conjunction with complement and phagocytic cells to facilitate damage of the cell wall and trigger uptake by cells, respectively. Antibodies also have the capacity to trigger transcriptional changes that increase pathogen virulence, thereby exacerbating disease. Pleiotropic effects have made it difficult to resolve the net value of humoral immunity to IFIs.

Natural Killer Cells Natural killer (NK) cells exert an antifungal response, but the rules that govern this response have only been elucidated recently. NK cell–mediated killing requires direct contact with the fungus, which leads to release of perforin. The NK cell receptor p30 is required for NK cell-fungal conjugate formation, pI3K signaling, and perforin release. p30 was previously identified as an activating receptor against tumor cells. In patients with

422

Part three  Host Defenses to Infectious Agents

AIDS, who are highly susceptible to C. neoformans infection, p30 expression is decreased on NK cells leading to defective perforin release and reduced microbicidal activity. IL-12 restored p30 expression and fungal killing in a mouse model of C. neoformans infection by NK cells. These data indicate a direct role for NK cells against Cryptococcus through direct recognition by a cell surface receptor. Evidence for the role of NK cells against invasive fungal pathogens has been expanded to show that they play a nonredundant role in clearing C. glabrata infections. The human NK receptor p46 and its mouse orthologue natural cytotoxicity triggering receptor 1 (NCR1) bind C. glabrata. In vitro, NK cells lacking NCR1 show reduced capacity to kill C. glabrata. Indeed, NCR1−/− mice have increased susceptibility to C. glabrata infection. The ligands for p46/NCR1 are discrete members of a family of proteins called EPA (epithelial adhesion), which are glycan-binding lectins and permit attachment of fungal cells to host cells.

GENETIC SUSCEPTIBILITIES TO INVASIVE FUNGAL INFECTIONS Although certain clinical diagnoses and medical interventions are associated with susceptibility to fungal infections, not all patients in these high-risk groups become infected. Moreover, individuals lacking traditional risk factors (e.g., immunosuppression, HIV, etc.) can develop chronic or invasive fungal infections, suggesting that additional factors confer susceptibility. Primary immunodeficiencies (PIDs) include a group of hereditary immune disorders that render patients more susceptible to infection. Recent studies of PIDs have provided the opportunity to define some of the genetic and molecular mechanisms that contribute to infection. A list of genes associated with susceptibility to fungal infection are summarized in Table 29.1.

Chronic granulomatous disease (CGD) is a well-known example of a genetic disorder associated with increased susceptibility to invasive bacterial and fungal infections, especially invasive aspergillosis. CGD is caused by mutations in genes encoding subunits of the NADPH complex (e.g., CYBA, CYBB NCF1, NCF2, NCF4), which results in defective ROS production, a critical step in the microbial killing process.34,35 Recent work has shown that deficiencies in ROS production resulting from a mutation in a subunit of the NADPH complex also result in defective autophagy. These defects prevent autophagy-dependent inhibition of IL-1β and increased inflammasome activation. Blocking of IL-1 protects mice with CGD from invasive aspergillosis and colitis and restores autophagic function in monocytes derived from patients with CGD.36 Chronic mucocutaneous candidiasis (CMC) is an immune disorder characterized by persistent or recurrent candidal infections of the skin, nails, and mucous membranes. Primarily, CMC appears to be associated with defects in IL-17 signaling. A direct role for IL-17 signaling in the protection against CMC was demonstrated in two families with deficiencies in the IL-17 receptor A (IL-17RA) and the cytokine IL-17F.37 As mentioned previously, mutations in dectin-1 cause increased susceptibility to CMC as a result of impaired IL-1β production and a reduced frequency of Th17 cells.38 Similarly, patients with CARD9 deficiencies have increased susceptibility to invasive fungal infections resulting from decreased Th17 cells, reduced chemokine/cytokine production, and impaired neutrophil killing.39 CMC is also common in patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1), an autosomal recessive disorder caused by defects in the autoimmune regulator (AIRE) gene. In these patients, CMC results from the generation of autoantibodies against IL-17 and IL-22.40 Hyper-IgE syndrome (HIES) is an autosomal dominant or recessive immune disorder

TABLE 29.1  Genes Associated With Immunity to Fungi in Humans Gene Product

Disease

Fungal Pathogen

Immunological Phenotype

ACT1 AIRE CARD9

CMCa CMC; Autoimmune Polyendocrinopathy Syndrome-1 CMC; disseminated candidiasis; Candida meningoencephalitis; deep dermatophytosis; subcutaneous phaeohyphomycosis; invasive Exophiala infection CGDb

Candida Candida Candida, Trichophyton, Phialophora, Exophiala

Impaired interleukin-17 (IL-17) signaling. Autoantibodies against IL-17 and IL-22 Defective dectin-1 signaling and reduced frequency of T-helper 17 (Th17) cells Impaired neutrophil killing

Candida, Aspergillus

Nicotinamide adenine dinucleotide phosphate (NADPH) complex deficiency causing impaired reactive oxygen species (ROS) production

CMC

Candida

STAT3

CMC; HIESc CMC CMC Candidiasis CMC, cutaneous fusariosis, disseminated coccidioidomycosis, histoplasmosis, mucormycosis, and Penicillium marneffei infection CMC; HIES

Candida Candida Candida Candida Candida, Fusarium, Coccidioides, Histoplasma, Penicillium marneffei, Apophysomyces Candida

TYK2

CMC; HIES

Candida

Impaired IL-1β and IL-17 production and reduced frequency of Th17 cells Defective activation of Th17 cells Defective IL-17 signaling Defective IL-17 signaling Absent IL-17A/-17F–producing T cells Defective production of IL-17, IL-22, and interferon-γ (IFN-γ) and reduced frequency of Th17 cells Impaired Th17 differentiation and reduced frequency of Th17 cells Defective IL-23 signaling and reduced frequency of Th17 cells

CYBA CYBB NCF1 NCF2 NCF4 Dectin-1 DOCK8 IL-17F IL-17RA RORC STAT1

a

Chronic mucocutaneous candidiasis. Chronic granulomatous disease. Hyper–immunoglobulin E syndrome.

b c

CHAPTER 29  Host Defenses to Fungal Pathogens characterized by high serum IgE, eczema, and CMC. HIES is caused by mutations in multiple genes, including STAT3, Tyk2, and DOCK8.41 Patients with STAT3 mutations have impaired Th17 differentiation, which results in reduced number of Th17 cells and IL-17 production.42 Autosomal dominant CMC is caused by GOF mutations in STAT1, which lead to decreased IL-17, IL-22, and IFN-γ production and defective Th1 and Th17 responses.43-45 In contrast, STAT1 deficiencies confer susceptibility to viral and mycobacterial infections. RORC loss-of-function mutations have also been reported in humans with candidiasis; RORC isoforms ROR-γ and ROR-γT are critical for T-cell development and function.46 Thus the absence of IL-17A/-17F–producing T cells likely accounts for the candidiasis in these patients. The identification of genes associated with PIDs revealed several important pathways in host immunity to fungal infections. Collectively, genes associated with PIDs highlight the protective and critical role of IL-17 signaling against invasive fungal infections.

ON THE HORIZON • The study of fungal immunology has led to a fundamental understanding of the role of C-type lectin receptors (CLRs) in immunology. Innate immunology plays a critical and nonredundant role in the defense against invasive fungal infections. • When patients lack neutrophils or develop defects in neutrophil function, only a subset of patients develop invasive fungal infection (e.g., A. fumigatus infection). • These observations suggest that genetic defects in the immune system likely contribute to increased risk of infection. • Identification of genetic mutations that are associated with increased risk of fungal disease may permit the development of genomic risk stratification for high-risk patients. • To this end, rational use of antifungal prophylaxis may decrease the burden of fungal disease.

SUMMARY To date, more than 5 million distinct species of fungi have been identified, but only a few are relevant to human health. Aspergillus and Cryptococcus are ubiquitous in the environment, and their spores are routinely inhaled by humans; Candida spp. colonize human skin, mucosae, and GI tract and, under normal conditions, do not cause disease. Normally, the immune system is able to clear and/or limit these pathogens, but in immunocompromised individuals, these fungi can cause serious and often life-threatening infections. Using both TLRs and CLRs, the innate immune system plays a critical role in clearing the invading pathogens. Indeed, patients with defects in either innate immune cells (e.g., neutropenia) or signaling through TLRs and CLRs have an increased risk for diseases caused by invasive fungal organisms. Adaptive immunity is also critical, as evidenced by patients with advanced HIV disease who are at heightened risk for C. neoformans disease. In general, Th1 and Th17 responses are protective, whereas Th2 responses skew the immune response to allergic and hyperactive responses. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

423

REFERENCES 1. Ruping MJ, Vehreschild JJ, Cornely OA. Patients at high risk of invasive fungal infections: when and how to treat. Drugs 2008;68(14): 1941–62. 2. Azie N, Neofytos D, Pfaller M, et al. The PATH (Prospective Antifungal Therapy) Alliance(R) registry and invasive fungal infections: update 2012. Diagn Microbiol Infect Dis 2012;73(4):293–300. 3. Sobel JD. Vulvovaginal candidosis. Lancet 2007;369(9577):1961–71. 4. Brown GD, Denning DW, Gow NA, et al. Hidden killers: human fungal infections. Sci Transl Med 2012;4(165):165rv13. 5. Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 2002;347(6):408–15. 6. Garcia-Vidal C, Viasus D, Carratala J. Pathogenesis of invasive fungal infections. Curr Opin Infect Dis 2013;26(3):270–6. 7. Vazquez JA, Miceli MH, Alangaden G. Invasive fungal infections in transplant recipients. Ther Adv Infect Dis 2013;1(3):85–105. 8. Cornely OA, Gachot B, Akan H, et al. Epidemiology and outcome of fungemia in a cancer Cohort of the Infectious Diseases Group (IDG) of the European Organization for Research and Treatment of Cancer (EORTC 65031). Clin Infect Dis 2015;61(3):324–31. 9. Kullberg BJ, Arendrup MC. Invasive Candidiasis. N Engl J Med 2015;373(15):1445–56. 10. van Woerden HC, Gregory C, Brown R, et al. Differences in fungi present in induced sputum samples from asthma patients and non-atopic controls: a community based case control study. BMC Infect Dis 2013;13:69. 11. Weindl G, Wagener J, Schaller M. Epithelial cells and innate antifungal defense. J Dent Res 2010;89(7):666–75. 12. Naglik JR, Moyes DL, Wachtler B, et al. Candida albicans interactions with epithelial cells and mucosal immunity. Microbes Infect 2011;13(12-13):963–76. 13. Liu Y, Zhao G, Lin J, et al. The role of Syk signaling in antifungal innate immunity of human corneal epithelial cells. BMC Ophthalmol 2015;15:55. 14. Moyes DL, Runglall M, Murciano C, et al. A biphasic innate immune MAPK response discriminates between the yeast and hyphal forms of Candida albicans in epithelial cells. Cell Host Microbe 2010;8(3): 225–35. 15. Kim YG, Udayanga KG, Totsuka N, et al. Gut dysbiosis promotes M2 macrophage polarization and allergic airway inflammation via fungi-induced PGE(2). Cell Host Microbe 2014;15(1):95–102. 16. Nosanchuk JD, Stark RE, Casadevall A. Fungal Melanin: What do We Know About Structure? Front Microbiol 2015;6:1463. 17. Urban CF, Reichard U, Brinkmann V, et al. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 2006;8(4):668–76. 18. Plato A, Hardison SE, Brown GD. Pattern recognition receptors in antifungal immunity. Semin Immunopathol 2015;37(2):97–106. 19. Bourgeois C, Kuchler K. Fungal pathogens-a sweet and sour treat for toll-like receptors. Front Cell Infect Microbiol 2012;2:142. 20. Khan NS, Kasperkovitz PV, Timmons AK, et al. Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing beta-1,3 Glucan. J Immunol 2016;196(5):2249–61. 21. Kasperkovitz PV, Khan NS, Tam JM, et al. Toll-like receptor 9 modulates macrophage antifungal effector function during innate recognition of Candida albicans and Saccharomyces cerevisiae. Infect Immun 2011;79(12):4858–67. 22. Mansour MK, Tam JM, Khan NS, et al. Dectin-1 activation controls maturation of beta-1,3-glucan-containing phagosomes. J Biol Chem 2013;288(22):16043–54. 23. Ferwerda G, Netea MG, Joosten LA, et al. The role of Toll-like receptors and C-type lectins for vaccination against Candida albicans. Vaccine 2010;28(3):614–22. 24. Gringhuis SI, den Dunnen J, Litjens M, et al. Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-κB activation through Raf-1 and Syk. Nat Immunol 2009;10(2):203–13.

424

Part three  Host Defenses to Infectious Agents

25. de Sousa Mda G, Belda W Jr, Spina R, et al. Topical application of imiquimod as a treatment for chromoblastomycosis. Clin Infect Dis 2014;58(12):1734–7. 26. Underhill DM, Pearlman E. Immune interactions with pathogenic and commensal fungi: a two-way street. Immunity 2015;43(5):845–58. 27. van de Veerdonk FL, Joosten LA, Netea MG. The interplay between inflammasome activation and antifungal host defense. Immunol Rev 2015;265(1):172–80. 28. Caffrey AK, Obar JJ. Alarmin(g) the innate immune system to invasive fungal infections. Curr Opin Microbiol 2016;32:135–43. 29. Netea MG, Latz E, Mills KH, et al. Innate immune memory: a paradigm shift in understanding host defense. Nat Immunol 2015;16(7):675–9. 30. Netea MG, Joosten LA, Latz E, et al. Trained immunity: A program of innate immune memory in health and disease. Science 2016;352(6284):aaf1098. 31. Quintin J, Saeed S, Martens JH, et al. Candida albicans infection affords protection against reinfection via functional reprogramming of monocytes. Cell Host Microbe 2012;12(2):223–32. 32. Zuniga LA, Jain R, Haines C, et al. Th17 cell development: from the cradle to the grave. Immunol Rev 2013;252(1):78–88. 33. Hamad M. Innate and adaptive antifungal immune responses: partners on an equal footing. Mycoses 2012;55(3):205–17. 34. Clark RA, Malech HL, Gallin JI, et al. Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system. N Engl J Med 1989; 321(10):647–52. 35. Wang X, van de Veerdonk FL. When the Fight against Fungi Goes Wrong. PLoS Pathog 2016;12(2):e1005400. 36. de Luca A, Smeekens SP, Casagrande A, et al. IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans. Proc Natl Acad Sci USA 2014;111(9): 3526–31.

37. Puel A, Cypowyj S, Bustamante J, et al. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science 2011;332(6025):65–8. 38. Ferwerda B, Ferwerda G, Plantinga TS, et al. Human dectin-1 deficiency and mucocutaneous fungal infections. N Engl J Med 2009;361(18): 1760–7. 39. Drummond RA, Collar AL, Swamydas M, et al. CARD9-dependent neutrophil recruitment protects against fungal invasion of the central nervous system. PLoS Pathog 2015;11(12):e1005293. 40. Puel A, Doffinger R, Natividad A, et al. Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I. J Exp Med 2010; 207(2):291–7. 41. Gow NA, Netea MG. Medical mycology and fungal immunology: new research perspectives addressing a major world health challenge. Philos Trans R Soc Lond B Biol Sci 2016;371(1709):pii: 20150462. 42. Ma CS, Chew GY, Simpson N, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. J Exp Med 2008;205(7):1551–7. 43. Liu L, Okada S, Kong XF, et al. Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis. J Exp Med 2011;208(8):1635–48. 44. van de Veerdonk FL, Plantinga TS, Hoischen A, et al. STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis. N Engl J Med 2011;365(1):54–61. 45. Depner M, Fuchs S, Raabe J, et al. The extended clinical phenotype of 26 patients with chronic mucocutaneous candidiasis due to gain-of-function mutations in STAT1. J Clin Immunol 2016;36(1):73–84. 46. Okada S, Markle JG, Deenick EK, et al. Immunodeficiencies. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations. Science 2015;349(6248):606–13.

CHAPTER 29  Host Defenses to Fungal Pathogens

424.e1

MULTIPLE-CHOICE QUESTIONS 1. Loss of dectin-1 in patients leads to: A. Chronic mucocutaneous candidiasis B. Candidemia C. No clinical disease unless patient has neutropenia D. Increased risk of Cryptococcus neoformans disease 2. Which of the following carbohydrates is NOT found on the fungal cell wall? A. β-1,3 glucan B. Galactomannan

C. Glucuronoxylomannan D. Peptidoglycan 3. Which T-cell response typically makes fungal infection worse? A. T-helper 1 (Th1) B. Th2 C. Th17 D. CD8

30  Host Defenses to Protozoa Peter C. Melby, Robin Stephens, Sara M. Dann

Protozoal infections are an important cause of morbidity and mortality worldwide (Table 30.1). Protozoan pathogens exact their major toll in the tropics, but infection by these parasites remains a significant problem in developed countries because of travel to and emigration from developing countries, the susceptibility of patients with acquired immunodeficiency syndrome (AIDS) to opportunistic protozoans, and episodic transmission within communities.

KEY CONCEPTS Host Defense Against Protozoa • Interaction of the parasite with host cells induces an array of cytokines that stimulate the innate and adaptive immune responses to eliminate the pathogen, and/or cytokines that inhibit or downregulate the antiparasitical responses to enable the initiation of tissue parasitism. • The outcome of infection is determined by the balance between the infection-promoting and the host-protective cytokines and effector cells. Often there is a mixed response, resulting in a persistent infection. • A persistently infected host may develop clinical disease if there is a waning of the immune mechanisms (e.g., in acquired immunodeficiency syndrome [AIDS]) that are critical to the control of infection.

Protozoan pathogens make up a group of highly diverse organisms that utilize a wide array of mechanisms for pathogenesis and immune evasion. There are numerous host targets for the intracellular protozoan parasites, including erythrocytes (Plasmodium and Babesia), macrophages (Leishmania and Toxoplasma gondii), or multiple cell types (Trypanosoma cruzi). The luminal parasitical protozoan may be extracellular, such as amebae and the flagellates (Giardia and Trichomonas), or primarily intracellular, such as the coccidian parasite Cryptosporidium. The innate and adaptive immune systems respond in diverse ways to the blood and tissue and intestinal protozoan pathogens. Neutrophils, macrophages, and natural killer (NK) cells are the effector cells that mediate the innate response against the extracellular protozoan parasites. The NK cell–activated macrophage system is central to the innate response to intracellular parasites (Fig. 30.1) (Chapters 3, 17). The innate cytokine response activates phagocytes and is critical to the induction of the adaptive immune response via antigen presentation by dendritic cells (DCs). For the intracellular pathogens (e.g., Leishmania spp., T. cruzi, T. gondii), the early production of interleukin-12 (IL-12) and interferon-γ (IFN-γ) drives the differentiation of T cells to a protective T-helper 1 (Th1) phenotype. In most cases CD4 T cells play a primary role in adaptive cellular immunity, but CD8

T cells can be critically important through cytokine production (e.g., Plasmodium spp., T. cruzi, T. gondii) or direct cytotoxic activity (e.g., Cryptosporidium). For the parasites that have an extracellular stage (e.g., Plasmodium spp., Trypanosoma spp., Giardia, and Trichomonas), specific antibodies mediate acquired immunity. Intensive effort has been dedicated to the development of effective vaccines for protozoal diseases, but as of 2016, only the malaria circumsporozoite vaccine (RTS,S) has reached the stage of clinical use. The reader is referred to a number of excellent reviews of the potential vaccine candidates.1-4 A discussion of the immune responses to some of the individual protozoal pathogens follows.

Plasmodium spp. Pathogenesis Soon after Plasmodium spp. sporozoites are injected into the bloodstream by the Anopheles mosquito, they invade hepatocytes and undergo schizogony (asexual reproduction). A dormant form of P. vivax and P. ovale (hypnozoites) can reside within hepatocytes for months and then cause clinical bloodstream infection. Following schizogony, merozoites are released from hepatocytes into the bloodstream in a membrane-bound structure, known as merosomes. The merosomes rupture in blood, and free merozoites invade red blood cells (RBCs) to produce ring-stage parasites. These parasites mature into trophozoites, which again undergo schizogony, leading to rupture of the erythrocyte and the release of new invasive merozoites. Merozoites can also develop into sexual-stage gametocytes, which can be ingested by a feeding mosquito to continue the transmission cycle. The clinicopathological features of malaria are caused by intraerythrocytic infection and the associated immune response. The cyclical rupture of erythrocytes is associated with fever. The induction of a proinflammatory cytokine cascade plays a central role in the pathogenesis of P. falciparum malaria and its complications. Parasite antigens, particularly those having glycophosphatidyl inositol (GPI) membrane anchors, released during the rupture and reinvasion of RBCs, activate the innate immune response. The production of proinflammatory cytokines (IL-1, TNF, lymphotoxin, IL-12, and IFN-γ) leads to fever, expression of endothelial adhesion molecules, and cytoadherence. It is mediated, in part, by TLR2 and is MyD88-dependent.5 NK cells and memory T cells produce early IFN-γ, which contributes to the production of a pathologically high level of TNF. IL-10- and transforming growth factor-β (TGF-β)–mediated downregulation of the Th1 immune response and leukotriene (LT)/tumor necrosis

425

426

Part THREE  Host Defenses to Infectious Agents

TABLE 30.1  Worldwide Significance of the Major Protozoal Infections Parasite

Estimated Worldwide Cases (Annual Mortality)

Clinical Manifestations

Plasmodium spp.

400–490 million (P. falciparum: >2 million deaths/year, primarily children) 10–50 million people infected, 1.2 million new cases per year

Fever with potential complications of severe hemolysis, renal failure, pulmonary edema, cerebral involvement Asymptomatic infection; skin ulcers or nodules; destructive oropharyngeal lesions; visceral disease with fever, hepatosplenomegaly, cachexia, pancytopenia Asymptomatic infection; dysrhythmias or chronic heart failure; hypertrophy and dilation of the esophagus, colon Self-limited fever, hepatosplenomegaly; lymphadenopathy and encephalitis (reactivation in patients with acquired immunodeficiency syndrome [AIDS]); congenital infection, with fetal death, chorioretinitis, meningoencephalitis Asymptomatic infection, diarrhea, dysentery, or liver abscess Asymptomatic infection, chronic diarrhea

Leishmania spp.

Trypanosoma cruzi

24 million (60 000 deaths)

Toxoplasma gondii

Several hundred million people infected worldwide. 5–9% of healthy US adults are seropositive

Entamoeba histolytica Giardia lamblia

50 million (100 000 deaths) 200 million (most common in young children and immunocompromised persons) Prevalence 3–10% in patients with diarrhea in developing countries 170 million/year

Cryptosporidium parvum and C. hominis Trichomonas vaginalis Pathogen or soluble products

KEY CONCEPTS Immunopathogenesis of Severe Plasmodium falciparum Malaria

IFN-α/β IL-10 TGF-β PGE2

(+) Macrophage or dendritic cell

Self-limited diarrhea in immunocompetent persons, severe intestinal and biliary disease in patients with AIDS Asymptomatic infection, vaginal discharge, urethritis

IL-12 TNF-α IL-18 IL-1β

IFN-γ TNF-α

(-)

(+) NK cell IFN-γ TNF-α

(+) (+)

NO

RNI ROI Macrophage activated for intracellular killing

FIG 30.1  Macrophage, Natural Killer (NK) Cell, and Cytokine Interactions in the Innate Immune Response to Intracellular Protozoa. Exposure of macrophages or dendritic cells to a pathogen or microbial product can result in the release of cytokines and inflammatory mediators that may stimulate (+) or suppress (−) NK cell activation. Activated NK cells produce cytokines that can then activate macrophages for intracellular killing. It must be recognized that this diagram is oversimplified and that these cytokines, most notably interferon (IFN)-α/β, interleukin (IL)-10, transforming growth factor (TGF)-β, and IL-12, may be produced by other types of cells, such as epithelial cells or enterocytes. NO, nitric oxide; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates.

factor (TNF) production, which have a role in severe and cerebral malaria, act to limit the inflammatory response. Severe malaria includes severe anemia, respiratory distress, placental malaria, and cerebral malaria, with the latter causing up to 90% of deaths. In cerebral malaria, a combination of inflammation, cytoadhesion of parasites, and leukocytes, and vascular pathology leads to coma. Cytokines and endothelial cell production of nitric oxide (NO) contribute to inflammatory lesions in the brain. Patients may die as a result of severe edema and swelling of the brainstem. Severe anemia is the result of a combination of destruction of infected and uninfected RBCs and dyserythropoiesis. Both destruction of uninfected RBCs and malformation of RBCs in bone marrow are increased by inflammatory cytokines, as is the damage in placental malaria.

• Release of malarial antigens stimulates tumor necrosis factor (TNF), interleukin (IL)-1, and lymphotoxin production from innate immune cells. • TNF/leukotriene (LT) induce vascular leakage, hemorrhage, endothelial cell activation with expression of endothelial adhesion molecules, platelet activation and adhesion, and coagulation. • Inflammatory cytokines amplify severe anemia caused by loss of infected red blood cells (RBCs) by inducing dyserythropoiesis and phagocytosis of uninfected RBCs, which can be coated with parasite antigens. • Lethal outcomes for the fetus of infected women during pregnancy are caused by the local immune response induced by placenta-specific parasites.

Innate Immunity Complement-mediated lysis can occur at the sporozoite and merozoite stages, though parasites have evolved evasive mechanisms. While sporozoites rapidly transit from the skin to the liver, they can activate DCs at the site of inoculation or in the regional lymph node. Early activation of NK cells and IFN-γ are associated with better outcomes of infection. Although high levels of TNF are associated with severe malaria, physiological levels are protective through the activation of macrophages. γδ T-cell receptor (TCR)+ T cells that respond to phosphorylated nonpeptide antigens on live parasites have been demonstrated. DCs initially induce a strong adaptive immune response to the erythrocytic stage, which is tempered during prolonged infection.

Adaptive Immunity Partial immunity to Plasmodium spp. infection is acquired slowly following repeated exposure in endemic areas.6 In areas of intense perennial P. falciparum transmission, the density of parasitemia, morbidity, and the incidence of cerebral malaria and malariarelated deaths are highest in the early childhood years, declining thereafter. Naturally acquired immunity to repeated infections develops in young adults, with the exception of the morbidity associated with placental infections. Adaptive immunity to the preerythrocytic stage is primarily mediated through major histocompatibility complex (MHC)

CHAPTER 30  Host Defenses to Protozoa class I-restricted parasite-specific CD8 T cells and, to a lesser extent, CD4 T cells, via IFN-γ-induced NO-dependent killing of intrahepatocyte parasites.7 Potentially protective immune mechanisms against the preerythrocytic stage have largely been identified by study of mice vaccinated with irradiated sporozoites and challenged with murine Plasmodium spp. Irradiated sporozoite challenge also protects humans and is being scaled up for vaccine trials. Antisporozoite immunity requires the presence of high antibody titers and high numbers of T cells to block sporozoite invasion of the hepatocyte, which occurs in just a few cells within minutes of inoculation. Both antibody-dependent and cell-mediated immune mechanisms are active against the erythrocytic stage of infection. Both B cells and CD4 T cells are required for complete clearance of parasites.8 Adoptive transfer of human immune serum is protective for naïve individuals. Antibodies directed against merozoite surface proteins can inhibit invasion. A significant proportion of antibodies to infected erythrocytes are directed toward variant antigens, such as PfEMP-1 on the surface of the RBC. Immunoglobulin G1 (IgG1) and IgG3 isotype antibodies specific for parasite antigens exported to the surface of the RBC play a role in naturally acquired immunity by opsonizing infected cells for phagocytosis in the spleen. CD4 T cells are implicated in protection by experiments showing that MHC class II–restricted antigen presentation is required for reduction of parasitemia and pathology. Protective cellular immune responses (CD4 and CD8 T-cell proliferation, IFN-γ production, and NO synthesis) in the absence of detectable antibody responses were identified in naïve volunteers, who were protected by repeated exposure to low doses of blood-stage parasites. In addition to promoting phagocytosis, CD4 T cells are required to help B cells, especially by the production of IL-21. One of the most important roles for CD4 T cells is the regulation of the intense inflammatory response through production of antiinflammatory cytokines IL-10 and TGF-β.

Evasion of Host Immunity The malaria parasite uses several different mechanisms to evade the host immune response.9,10 Sporozoites and merozoites evade circulating antibody by rapidly entering hepatocytes or RBCs, respectively. Some sporozoite proteins enter the hepatocyte nucleus and influence the expression of a number of host genes, thereby favoring parasite survival. Mature RBCs do not express MHC molecules on their surface and so avoid recognition by T cells. The few parasite proteins that are expressed on the erythrocyte surface exist in multiple allelic forms to avoid quick recognition by the adaptive immune system. Many of the immunodominant antigens in Plasmodium spp. are proteins having extensive repeat sequences that vary over time. The large number of bloodborne antigens induces a plasmablast response generating a short-lived burst of low-affinity antibodies. Although there is an increase in atypical B cells, memory B cells and long-lived plasma cells are generated. It is unclear if poor immunity following a single infection is caused by ineffective, atypical B cells or misdirected antibody specificity.

Leishmania spp. Pathogenesis The intracellular Leishmania amastigote replicates within macrophages in the vertebrate host, and the extracellular promastigote develops within the insect vector. The female phlebotomine sand

427

fly becomes infected by ingesting amastigotes during a blood meal. In the sand fly gut, the amastigotes differentiate into infectious metacyclic promastigotes that infect the vertebrate host during the next blood meal. The surface lipophosphoglycan (LPG) plays a central role in the parasite’s entry and survival in host cells. Immunomodulatory factors present in the sand fly saliva may enhance the infectivity of the parasite. Once introduced into skin, the promastigotes are phagocytosed (through complement–complement receptor–mediated coiling phagocytosis) by neutrophils, DCs, and macrophages, where they transform to amastigotes and replicate within the acidic and hostile environment of the phagolysosome. Eventually, the phagocytes rupture and release amastigotes to infect other macrophages or a sand fly.

Innate Immunity Much of what we know of immunity in leishmaniasis comes from studies of inbred mouse strains, which demonstrate a genetically determined spectrum of innate and adaptive immune responses that shape the outcome of infection. The innate immune response to Leishmania is mediated by complement, NK cells, cytokines, and phagocytes.11 The production of IL-12 early in the course of infection by DCs leads to the early activation of NK cells and the production of IFN-γ. Chemokines (IP-10, MCP-1, and lymphotactin), as well as LPG–TLR interaction, can also promote the early NK-cell activation. Activated NK cells have been shown to be cytolytic for Leishmania-infected macrophages, but NK cell–derived IFN-γ plays a more prominent role in host defense by activating macrophages to kill the intracellular parasite through the generation of reactive oxygen intermediates (ROIs) or reactive nitrogen intermediates (RNIs). Parasite-induced, MyD88-dependent signaling through TLR2, TLR3, and TLR4 contributes to macrophage activation and NO production. Activated polymorphonuclear leukocytes (PMNs) can kill parasites through oxidative mechanisms, but the role of neutrophils in vivo depends on the timing of their recruitment and their interaction with other immune cells.12 An important study demonstrated that infiltrating neutrophils promote sand fly– transmitted infection, probably through modulation of macrophage function following engulfment of apoptotic parasitized neutrophils.13 Type 1 IFNs participate in the early induction of NO and control of parasite replication early in infection.

Adaptive Immunity Within an endemic area there is acquisition of immunity in the population over time. Retrospective epidemiological studies indicate that most individuals with prior (primary) infection (subclinical or healed) are immune to a subsequent clinical infection. Following primary infection, parasites persist for the life of the host and maintain long-term immunity. There is extensive evidence from experimental models that cellular immune mechanisms mediate adaptive resistance to Leishmania infection, and human studies have generally confirmed this. Antileishmanial antibodies, which are produced at a low level in localized cutaneous leishmaniasis (LCL) and at a very high level in visceral leishmaniasis (VL), play no role in protection. The general mechanisms of cellular immunity in leishmaniasis can be summarized (Fig. 30.2). Following infection in the skin, migratory dermal DCs phagocytose Leishmania and presumably transport the intracellular parasite to the regional lymph node, where they induce a T-cell response. Adaptive immunity is primarily mediated by parasite-induced production of IFN-γ by CD4

428

Part THREE  Host Defenses to Infectious Agents Leishmania promastigotes

NK

IL-12 DC

IFN-γ IFN-γ

IL-12

IFN-γ IFN-γ

Th1

IFN-γ IL-4 IL-10 IL-10 TNF-α TGF-β

Th2 IL-4 IL-10 IL-10 TGF-β PGE2

Activation RNI ROI

IFN-γ IL-10 TGF-β

Parasite killing Deactivation

Parasite replication

Macrophage

FIG 30.2  Immunity in Leishmaniasis. Exposure of dendritic cells to parasites or parasite antigens leads to the release of interleukin (IL)-12, which induces natural killer (NK) cells to produce interferon (IFN)-γ and drives the adaptive immune response toward a protective T-helper 1 (Th1) phenotype. IL-12 production by dendritic cells and IFN-γ production by NK and Th1 cells negatively regulates the Th2 response. IFN-γ activates macrophages to kill the intracellular pathogen. In genetically susceptible individuals, a counterregulatory Th2 cytokine response can suppress the Th1 response and impair classic macrophage activation, leading to parasite replication and uncontrolled infection. Counterprotective macrophage-derived cytokines can also inhibit the Th1 response, stimulate the Th2 response, and impair classical activation through an autocrine loop. Activating stimuli are shown by solid arrows, and deactivating stimuli are shown by dashed arrows.

T cells (Th1 subset). CD4 T cells are absolutely required, but immunity to cutaneous disease is also mediated by CD8 T cells via production of IFN-γ.14 Both CD4 and CD8 T cells are required for an effective defense against murine visceral L. donovani infection, but the precise role of CD8 T cells is unclear. The generation of the Th1 response is critically dependent on CD40CD40L-mediated IL-12 production and driven by NK cell–derived IFN-γ. IL-12 and STAT4 are required for the maintenance of immunity. Tumor necrosis factor-α (TNF-α) contributes to protective immunity by synergizing with IFN-γ to activate macrophages. Recently nuclear factor-kappa B (NF-κB) family members have been shown to regulate T-cell responses and immunity to L. major infection in mice. Two subpopulations of CD4 T cells mediate immunity induced by primary infection. Effector memory cells, which are short-lived and dependent on the persistence of antigen, rapidly respond to secondary infection by migrating to the infected tissue and generating effector cytokines. Central memory T (Tcm) cells, which can be maintained in the absence of persistent antigen, circulate throughout the lymphatic system and upon secondary challenge migrate to and proliferate in the draining lymph node,

gain the capacity to produce IFN-γ, and then migrate to the site of infection. Thus Tcm cells act as a reserve of antigen-reactive T cells that can expand and become effector T cells in response to secondary antigenic challenge. The generation of RNI by activated macrophages is the primary mechanism of parasite killing in the murine model. Although IFN-γ-induced production of NO may not be detectable in human macrophages, inhibition of nitric oxide synthase 2 (NOS2) was shown to impair killing of intracellular Leishmania. Several adaptive immune mechanisms promote parasite replication and disease.15 The progression of murine L. major infection has been correlated with the expansion of Th2 cells and the production of IL-4, IL-5, and IL-10. In susceptible mice, IL-4 production within the first day of infection was shown to downregulate IL-12 receptor β-chain expression and drive the response to a Th2 phenotype. However, other nonsusceptible mouse strains appear to be able to overcome an early IL-4 response and develop a resistant phenotype, and susceptibility to some L. major strains is not strictly mediated by IL-4 (IL-13 and/or IL-10 may have a prominent role). The cytotoxic activity of CD8 T cells may promote cutaneous inflammation and lesion pathology.14 The macrophage production of immune suppressive molecules, such as TGF-β or prostaglandin E2 (PGE2), may also contribute to susceptibility. Peripheral blood mononuclear cells (PBMCs) isolated from patients with localized cutaneous leishmaniasis demonstrate a Th1 response to Leishmania antigens, and in the cutaneous lesion, there is an exuberant Th1 and granulomatous response that mediates parasite killing and localized tissue damage, which usually leads to a scar. Patients with mucosal leishmaniasis (ML) exhibit vigorous cellular immune responses characterized by high levels of TNF-α and Th1 and Th17 cytokines; it is postulated that this hyperresponsive state contributes to the prominent tissue destruction of ML. Patients with diffuse cutaneous leishmaniasis (DCL) resemble the progressive infection caused by L. major in BALB/c mice in that there are minimal or absent Leishmania-specific lymphoproliferative responses, and predominant Th2 cytokine expression. During active VL in humans, there is a marked depression of Leishmania-specific lymphoproliferative and IFN-γ responses, contraction of circulating memory T cells, and an absence of delayed-type hypersensitivity (DTH) response to parasite antigens. This T-cell unresponsiveness appears to be mediated, at least in part, by a suppressive effect of IL-10 and low levels of IL-12. Successful treatment of active disease restores an antigen-specific Th1 response.

Evasion of Host Immunity The Leishmania parasite has numerous ways in which it adapts to and survives within the vertebrate host.16 In skin, the promastigotes may be phagocytosed by neutrophils and macrophages, which, unlike DCs, do not actively participate in T-cell priming. Furthermore, the clearance of apoptotic neutrophils is likely to make macrophages more permissive to infection.13 The parasite’s surface LPG (and, to a lesser extent, the surface protein gp63) plays an important role in the entry and survival of Leishmania in the mammalian host by conferring complement resistance and by facilitating the entry of complement-opsonized parasites into the macrophage without triggering a respiratory burst. Macrophage phagosome–endosome fusion and phagolysosomal biogenesis are also inhibited by parasite LPG. Leishmania-infected macrophages have diminished capacity to initiate and respond to a T-cell response, and the impaired

CHAPTER 30  Host Defenses to Protozoa

429

TABLE 30.2  Evidence for Autoimmune and Parasite-Induced Inflammatory Mechanisms in

Chronic Chagas Disease

Evidence for Autoimmune-Mediated Disease

Evidence for Parasite-Induced Inflammatory Disease

Inflammatory disease presents in tissues with few or no parasites on routine histopathology studies

Sensitive parasite detection techniques (polymerase chain reaction [PCR], immunohistochemistry) correlate with the presence of parasites (or parasite material) and severity of inflammatory disease Organs free of parasites (by sensitive parasite detection techniques) are also free of disease Absence of effective cellular immune response (in mice or humans) almost invariably exacerbates rather than reduces the parasite burden and disease In chronically infected mice, the destruction of a transplanted heart is dependent on parasite infiltrating the transplanted tissue Degree of disease in hearts transplanted into chronically infected mice correlates with level of parasite burden in transplanted tissue

Peculiar pattern of organ involvement (heart and gastrointestinal tract) in patients with chronic disease Long delay in the onset of chronic disease following infection; only a minority of infected persons develops disease Wide variability in the expression of disease among infected people Self-reactive antibodies and T cells demonstrable in infected people and in experimental animals. Level of antibodies to the ribosomal P protein (R13 peptide) and cardiac myosin (B13 antigen) correlate with cardiac disease Transient or limited disease reported in experimental models following lymphocyte transfer

antimicrobial effector activity provides a safe haven for the intracellular parasite.16 Infected macrophages have decreased synthesis of IL-1 and IL-12, and blunted IFN-γ-mediated activation through the disruption of signal transduction pathways involving JAK/STAT, protein kinase C, p38 MAPK, ERK, AP-1, and NF-κB. Signaling mediated by tyrosine phosphorylation is decreased by the rapid induction of the host phosphotyrosine phosphatase SHP-1. Conversely, there is increased synthesis of the immunosuppressive molecules IL-10, TGF-β, and PGE2. Parasite factors and IL-4/IL-13 enhance expression of arginase by infected macrophages. Arginase promotes infection through depletion of l-arginine, enhanced production of polyamines, and reduced NO production. IL-10 produced by CD4+CD25+ T regulatory cells (Tregs) has an essential role in parasite persistence. Recently, it was shown that metastasizing parasites that cause mucosal disease harbor a high burden of Leishmania RNA virus, which subverts the host immune response and promotes parasite persistence through activation of Toll-like receptor 3 (TLR3).17

Trypanosoma cruzi Pathogenesis T. cruzi is transmitted to the mammalian host when the infectious metacyclic trypomastigote, which is deposited on skin in the feces of the reduviid insect vector while it takes a blood meal, is scratched into the wound or transferred to a mucous membrane (e.g., the eyes). The trypomastigotes can infect almost any cell type and replicate as amastigotes in the cytoplasm. Eventually, the amastigotes transform back into trypomastigotes and rupture the cell to enter the bloodstream, from where they invade other cells or are picked up by another insect vector. Following primary infection, the parasites replicate locally and then disseminate through the bloodstream to a variety of tissues. Muscle and glial cells are the most frequently infected cells, and acute myocarditis or meningoencephalitis can develop. In most cases, however, primary infection occurs without clinical symptoms, and the infected individual may enter an indeterminate phase of asymptomatic seropositivity. Only 10–30% of chronically infected individuals will ultimately develop symptomatic Chagas disease, usually involving the heart or the gastrointestinal (GI) tract. Pathologically, there are few parasites observed in cardiac

Reduction of parasite burden by chemotherapy usually leads to decreased tissue inflammation and disease

tissue, but an intense chronic inflammatory infiltrate with fibrosis and loss of muscle fibers is evident. In the digestive tract, there is lymphohistiocytic infiltration of the myenteric plexuses, with reduction in the number of ganglion cells. The tissue damage of acute T. cruzi infection is the result of a direct effect of the parasite and an indirect effect of the acute inflammatory response. In chronic infection, the balance between immune-mediated parasite containment and host-damaging inflammation determines the course of disease. The pathological mechanisms related to chronic Chagas disease are controversial and certain to be multifactorial. Trypanosomal clonal variation and host genetic polymorphisms both contribute to tissue tropism, parasite persistence, and disease severity. Whether tissue damage is caused directly by parasites or indirectly through parasite-driven inflammatory or autoimmune mechanisms, parasite persistence is a significant driver of disease (Table 30.2).18,19 Autoimmunity could arise from molecular mimicry of self by parasite antigens or by the release of self molecules from damaged or dying host cells within the environment of an activated immune response. There is evidence for both of these autoimmune mechanisms. The production of IL-10 by T. cruzi–infected cells may downregulate the pathological cellular immune response.

Innate Immunity The early innate immune response to T. cruzi infection is mediated by NK cells, DCs, and macrophages.20 Macrophages and DCs exposed to T. cruzi trypomastigote antigens produce IL-12 and TNF, through a MyD88-dependent mechanism. MyD88-deficient mice had impaired inflammatory responses and host defense against T. cruzi. Immune activation through another protein involved in TLR2 signaling, the Toll/IL1R domain-containing adapter protein– inducing IFN-β (TRIF), promotes resistance through production of IFN-β and downstream expression of IFN-β inducible genes, such as the p47 guanosine triphosphatases (GTPase) IRG47. IL-12 activates NK cells to secrete IFN-γ, which synergizes with TNF to activate macrophages to control parasite replication. The generation of NO is the primary trypanocidal mechanism in murine macrophages. A number of trypomastigote antigens, including free glycosyl-phosphatidylinositol (GPI) anchors, glycoinositol phospholipids (GIPLs), GPI-linked glycoproteins, and GPI-mucins activate the innate immune response,

430

Part THREE  Host Defenses to Infectious Agents

in part through TLR2 and possibly TLR4. CpG motifs in the T. cruzi genome have also been shown to activate TLR9. Several other TLR-independent mechanisms of innate immunity, including the activation of nucleotide-binding oligomerization domain (NOD)–like receptors, have been identified.20

Adaptive Immunity The innate immune response, through production of IL-12, type I IFNS, and other proinflammatory mediators, is critically linked to the generation of an effective adaptive immune response. Antibodies contribute to immunity within the bloodstream through opsonization, complement activation, and antibodydependent cellular cytotoxicity. Several lines of evidence establish the importance of T cells in adaptive immunity to T. cruzi infection. Parasite-specific CD4 and CD8 T cells are activated during infection, and mice lacking CD4 or CD8 T cells have impaired parasite control. CD8 T cells with cytotoxic activity against T. cruzi–infected cells have been identified in infected mice, and these cells confer protection when passively transferred to naïve mice.21 In the early stage of infection, CD4 T cells are the predominant subset recruited to the myocardium, but activated CD8 T cells soon dominate the inflammatory process in cardiac tissue. IFN-γ and TNF production by parasite-specific CD8 T cells is more important than cytolytic activity in the control of infection.21 T. cruzi infection leads to a mixed Th1/Th2 cytokine response, and in general, the Th1/Th2 balance determines resistance or susceptibility. As noted earlier, IL-12/STAT4-dependent IFN-γ production by NK cells in early infection, and later by T cells, is critically important to protection. IL-4 does not appear to play a major role in susceptibility to T. cruzi infection, but IL-10 promotes parasite replication by inhibiting macrophage trypanocidal activity. IL-10 also plays a critical role in minimizing inflammation-mediated tissue pathology by regulating the Th1 and TNF responses. Polymorphisms in the IL-10 gene that lead to reduced IL-10 production are associated with increased severity of cardiomyopathy in patients with Chagas disease. Similarly, TGF-β has been shown to inhibit macrophage trypanocidal activity and increase parasitemia and mortality. In addition to these regulatory cytokines, the secretion of prostaglandins and NO, the induction of apoptosis of T and B cells, and the expansion of a myeloid suppressor cell population serve to control the intensity of the immune response.

Evasion of Host Immunity A significant part of the pathogenesis following T. cruzi infection is its dissemination through the bloodstream to many tissues. T. cruzi bloodstream trypomastigotes resist complement lysis via a complement regulatory protein (GP-160). This parasite protein is functionally similar to mammalian decay-accelerating factor in that it inhibits C3 convertase formation and activation of the alternate complement pathway. T. cruzi invades host cells, in particular cardiomyocytes, by subverting a host plasma membrane repair pathway that promotes parasite persistence and tissue tropism.22 The establishment of chronic infection by T. cruzi is favored by a generalized depression of T-cell responses. A number of different mechanisms may contribute to this, including low IL-2 production or IL-2 receptor expression; downregulation of components of the T cell–receptor complex; T-cell receptor dysfunction; apoptosis of T cells; defects in the processing and presenting of antigens in the MHC class II (but not the class I)

pathway, T-cell or macrophage suppressor activity, and PGE2 production. Within the foci of myocarditis, apoptosis of both parasites and host cells occurs. The phagocytosis of these apoptotic cells by macrophages leads to their acquisition of an M2 phenotype, which enables parasite replication and persistence. The rapid escape of the parasite from the phagosome into the cytoplasm through the action of acid-activated porins enables the organism to avoid phagolysosomal enzymatic destruction.

Toxoplasma gondii Pathogenesis Transmission occurs via the ingestion of oocysts, which are shed in the feces of felines or via tissue cysts present in undercooked meat. Following the oral ingestion of cysts, phagocytes recruited to the gut lumen facilitate transepithelial migration into the lamina propria. These phagocytes have been termed “Trojan Horses,” as they carry the parasite to new, unsuspecting host cells. Hostcell invasion starts with loose attachment facilitated by laminin. Intracellular tachyzoites replicate within a parasitophorous vacuole (PV) and ultimately leave the cell by using an active egress pathway. The released tachyzoites can disseminate to invade virtually any nucleated cell type, but mononuclear phagocytes are the preferred host cells. The toxoplasma SAG proteins mediate parasite attachment, and the transmembrane adhesins, MIC and AMA1, and RON family proteases play major roles in the myosin motor-driven process of host-cell invasion. Under pressure from the host immune response, tachyzoite replication is controlled and tissue cysts, containing slowly replicating bradyzoites, are formed. The tissue cysts persist as a chronic latent infection as long as the host immune function is intact. If the latently infected person is immunosuppressed, reactivation occurs, and tachyzoites are released to infect more cells. Because tissue cysts (bradyzoites) are found in proportionately larger numbers in the brain, reactivation of latent infection in the immunocompromised host most commonly manifests as encephalitis.

Innate Immunity Similar to immunity to Plasmodium and Leishmania, IL-12, IFN-γ, TNF-α, and NK cells contribute to the control of the early stages of T. gondii infection.23 CD8α+ DCs are the primary producers of IL-12, which is essential to host resistance. IL-12 is produced through a MyD88-dependent mechanism, at least in part, through the interaction of parasite-derived proteins and TLR2 and TLR11, and activation of CCR5.24 IL-12-dependent activation of NK cells leads to IFN-γ production, which, in turn, activates macrophages to limit parasite replication. T cells expressing the γδ receptor and neutrophils are also an early source of IFN-γ and TNF-α. Recruitment of inflammatory macrophages to the site of infection is crucial to control of parasite replication and dissemination. IFN-γ-induced macrophage activation is the key effector mechanism leading to parasite killing. In mice, the induction of immunity-related GTPases (IRG), which damage the parasitophorous vacuole membrane and kill T. gondii within the cytosol, is the primary macrophage effector mechanism. The generation of reactive nitrogen and oxygen intermediates, degradation of tryptophan, and the production of LTs have also been implicated in the control of T. gondii in human macrophages. However, humans lack the entire IRG family, as well as TLR11, and the mechanism(s) of early IFN-γ–mediated macrophage activation and parasite control in humans is unclear.

CHAPTER 30  Host Defenses to Protozoa Adaptive Immunity Although serum antibodies can be used in the diagnosis of T. gondii infection, the systemic antibody response does not play a role in adaptive immunity. Mucosal IgA, however, does provide resistance to oral infection with T. gondii cysts. CD4 and CD8 T cells are highly activated during infection and are essential for adaptive immunity. As such, patients with defects in T cell– mediated immune responses (e.g., patients with AIDS) are at risk for reactivation of latent infection. IL-12 from DCs drives the differentiation of T cells into type-1 CD4 and CD8 T cells that are essential to adaptive immunity. CD4 and CD8 T cells act synergistically to prevent cyst reactivation during chronic latent infection. Parasite-specific cytolytic T cells have been demonstrated; however, CD8 T cells mediate protection primarily through the generation of IFN-γ. IL-12 drives the generation of terminally differentiated CD8 effector T cells, which express the killer cell lectin-like receptor G1 (KLRG1) and high levels of granzyme B and IFN-γ. The combination of IL-7 and IL-15 is required for Tcm-cell differentiation. Antiinflammatory molecules, particularly IL-10 and IL-27 made by Th1 cells, play an important role in modulating the adaptive immune response and restricting host tissue damage.25

Evasion of Host Immunity T. gondii escapes early macrophage killing in a number of ways.25 Virulent parasites are protected by localization to the parasitophorous vacuole, which does not fuse with host cell lysosomes (probably because the PV membrane proteins are of parasite rather than host origin), and so the vacuole is not acidified to kill the parasite. The infected macrophage is also a suboptimal target for T cell–induced immunity because of reduced expression of MHC class II and costimulatory molecules. Infection also induces the production of counterregulatory molecules, such as IL-10, TGF-β, lipoxin A4. These not only downregulate a potentially pathological host inflammatory response but also inhibit Th1 induction and macrophage antimicrobial activity. In addition, T. gondii interferes with normal macrophage signaling. For example, infection inhibits DNA binding of signal transducer and activator of transcription 1 (STAT1) and NF-κB by interfering with chromatin structure, and promotes antiinflammatory pathways downstream of the suppressor of cytokine synthesis proteins. The parasite also has several virulence proteins, including ROP5 and ROP16, which bind to the parasitophorous vacuole and reduce accumulation of the IRGs. ROP proteins also activate host STAT3 and STAT6 driving an M2 macrophage phenotype that is permissive to infection.26

Entamoeba histolytica Pathogenesis E. histolytica causes asymptomatic intestinal colonization, acute diarrhea, dysentery, colitis, liver abscess, and, rarely, disseminated disease. Susceptibility to amebiasis is determined by the host’s nutritional status, intestinal microflora, genetics, and gender.27 E. histolytica cysts are ingested through consumption of food or water contaminated by feces. After excystation, the trophozoites degrade the colonic mucus barrier through secretion of proteases and glycosidases. The trophozoites adhere to the colonic epithelial cells by a galactose/N-acetylgalactosamine-inhibitable lectin (Gal/ GalNAc), which leads to NF-κB activation and proinflammatory cytokine release. Epithelial cells with adherent amebae undergo

431

microvilli shortening and apical separation that allows the parasite to penetrate between the epithelial cells and degrade connective tissue, resulting in ulceration of the mucosa and submucosa (Fig. 30.3). The trophozoites can lyse multiple cell types, including neutrophils, which release enzymes that further damage the tissue. The cytotoxic effects of amebae are mediated by a secreted cysteine protease, the Gal/GalNAc lectin, phospholipase A, and contactdependent cytolysis, in which an ion channel (amebapore) is inserted into the membrane of target cells. E. histolytica can induce apoptosis in mammalian cells by a caspase-dependent, TNF-α/ Fas–independent process. Amebic liver abscesses develop when trophozoites erode through the intestinal submucosa, enter the portal circulation, and disseminate to the liver.28 Comparison of the transcriptional profiles of virulent and nonvirulent Entamoeba species has identified novel virulence factors in E. histolytica.29

Innate Immunity Adherence of trophozoites to intestinal epithelial cells stimulates the release of a variety of proinflammatory mediators, including IL-1α, IL-1β, IL 6, IL-8, and TNF-α, which triggers the recruitment of neutrophils and macrophages to the site of invasion. Following activation by IFN-γ and TNF-α, neutrophils and macrophages become amebicidal through the release of reactive oxygen species and production of nitric oxide, respectively.28 Invariant natural killer T (iNKT) cells, which comprise about 30% of hepatic lymphocytes, are stimulated by amebic lipopeptidephosphoglycan (LPPG) to release IFN-γ. Production of IFN-γ reduces parasite burden and controls abscess formation.28

Adaptive Immunity Secretory IgA responses against E. histolytica have been well characterized and shown to correlate with protection against infection and disease. For this reason, vaccines designed to generate IgA antibodies against the Gal/Gal/NAc lectin have been highly efficacious in preventing experimental E. histolytica infection in mice and baboons.28 However, the role of antilectin IgG is unclear, and protection may be subclass-dependent. Cellmediated immunity is also critical for host defense against E. histolytica. IFN-γ–producing CD4 T cells provide protection through their ability to activate macrophages and neutrophils to the parasite. CD8 T cells mediate protection through the secretion of IL-17, a key player in the secretion of mucin and antimicrobial peptides, recruitment of neutrophils, and IgA transport across the epithelial barrier. Impairment of cell-mediated immunity results in parasite dissemination. Indeed, patients with human immunodeficiency virus infection and coinfected with E. histolytica have high rates of invasive amebiasis, liver abscesses, and seroconversion.29

Evasion of Host Immunity E. histolytica utilizes a number of strategies to circumvent the immune defenses of the host. It resists complement-mediated lysis during hematogenous spread by proteolytic degradation of C3a and C3b. In addition, the Gal/GalNAc lectin binds to C8 and C9, preventing assembly of the C5b-9 membrane attack complex. The cytolytic capability of E. histolytica affords protection from neutrophils, macrophages, and eosinophils, unless these cells are activated. Cytolysis by E. histolytica can occur via necrosis and apoptosis. Trophozoites also inhibit the macrophage respiratory burst and the production of IL-1 and TNF-α. A protective antibody response is subverted by the degradation of IgA and IgG by amebic cysteine proteases and by capping, ingesting, or

432

Part THREE  Host Defenses to Infectious Agents Pathogen

Innate immune response

Intestinal response

Trophozoite Complement

Giardia

Inflammatory mediators

Macrophage Sporozoite

Cryptosporidium

Eosinophil

IL-6, IL-8, IL-1 GM-CSF, GROα, prostaglandins

NK cell

ROI, RNI

Neutrophil

Trophozoite

Entamoeba

Villous atrophy and crypt hyperplasia Epithelial damage

Proteases Erosions and ulcerations

Enterocyte (Cryptosporidium)

Cytokines B- and T-lymphocyte activation Acquired immune response

Secretion Malabsorption Exudation

Host protection

Diarrhea

FIG 30.3  Immunopathogenesis of Intestinal Protozoal Pathogens. After adherence (Giardia and Entamoeba) or epithelial invasion (Entamoeba and Cryptosporidium), there is release of various inflammatory mediators from macrophages and neutrophils. This causes the activation of resident phagocytes and recruitment of phagocytes into the lamina propria. Enterocyte death can be due to direct action of the parasites or to immune-mediated damage from complement, cytotoxic lymphocytes, proteases, and reactive oxygen and nitrogen intermediates (ROI and RNI, respectively). The inflammatory mediators also act on enterocytes and the enteric nervous system, inducing the secretion of water and chloride. In response to enterocyte damage, under the influence of activated T lymphocytes, the crypts undergo hyperplasia, and the villi become shorter (villous atrophy). The immature hyperplastic cells have poor absorptive ability but retain secretory ability. Damage to the epithelium can cause leakage (exudation) from lymphatics and capillaries. Similar mechanisms are probably responsible for the diarrhea that occurs in infection with Cyclospora and Isospora. Isospora is unique in causing an eosinophilic infiltrate.

shedding ameba-specific antibodies.27 Amebic proteases can also cleave the Fc region so that interaction with host cell surface receptors is avoided. Another secreted product of E. histolytica, monocyte locomotion inhibition factor (MLIF), inhibits monocyte locomotion and the monocyte and neutrophil respiratory burst and NO production; enhances antiinflammatory cytokine and chemokine release from host cells; and alters adhesion molecule expression on macrophages. The suppression of host macrophage NO production by an array of trophozoite secretory products, including parasite-derived PGE2, is a major factor in the persistence of amebic liver abscesses. In chronic infection, E. histolytica promotes the development of Tregs that suppress the proliferation of responder T cells by releasing IL-10, TGF-β, and IL-35.28

Giardia lamblia Pathogenesis Recent studies of Giardia lamblia have identified eight genotypes, two of which infect humans (assemblages A and B).30 The severity of giardiasis ranges from asymptomatic carriage to

chronic watery diarrhea, epigastric pain, nausea, vomiting, and weight loss, depending on host factors and the virulence of the Giardia strain.30,31 Recent studies suggest even with treatment the parasite can elicit intestinal complications that persist for years. Younger age, malnutrition, and immunodeficiency increase the risk of severe disease.31 Infection is initiated following the ingestion of food or water contaminated with G. lamblia cysts. Exposure to stomach acids induces the excystation process that releases two trophozoites into the lumen of the proximal small intestine. Colonization occurs when the parasite attaches to the intestinal epithelium and begins to reproduce by binary fission. Trophozoites remain within the lumen and do not invade the epithelial barrier. Parasite migration into the lower intestine triggers encystation, allowing the organism to survive when excreted into the environment. The G. lamblia trophozoite initiates adherence to the intestinal epithelium via a surface mannose-binding lectin. Histopathological changes in symptomatic giardiasis range from a normal appearance to increased crypt-villous ratios, epithelial damage, and chronic inflammatory infiltrate in the lamina propria (see

CHAPTER 30  Host Defenses to Protozoa Fig. 30.3). The factors responsible for the structural changes in the small bowel are not well defined but may include injury from adherence, parasite-induced apoptosis of epithelial cells, and the release of cytotoxins, including proteases. Additional epithelial damage may be mediated by the host cellular immune response. Diarrhea arises from epithelial barrier dysfunction, reduction in microvillous surface area, chloride hypersecretion, and glucose and sodium malabsorption.31

Innate Immunity As G. lamblia does not invade the intestinal epithelium, host defense and immune factors present within the lumen are essential for preventing and controlling infection. Increased intestinal motility contributes to G. lamblia clearance, by impairing the ability of the parasite to attach to the epithelium and resist the luminal bulk flow. Antimicrobial peptides derived from Paneth cells, including cryptdins, neutrophil defensin, and cathelicidin, effectively kill G. lamblia trophozoites in vitro. NO, produced by both epithelial cells and macrophages, inhibits excystation trophozoite division. Despite the protective effect of NO, the parasite can circumvent this defense by competing with host cells for arginine uptake.30 Mast cells have a significant role in protecting against the parasite. Mice deficient in mast cells fail to clear G. lamblia infection, in part because they are unable to mount parasite-specific IgA. Mast cells also contribute to B-cell survival, activation and differentiation into plasma cells, and together with NO induce peristalsis.

Adaptive Immunity Several lines of evidence suggest the importance of the humoral immune response in the control of giardiasis. Infection with Giardia results in the production of anti-Giardia antibodies in the serum and mucosal secretions. Patients with severe B-cell defects or selective IgA deficiency have an increased risk of developing chronic infections.30 Studies in mice have demonstrated key functions of secretory IgA and the polymeric immunoglobulin receptor, which is responsible for IgA transport into the intestinal lumen in controlling parasite burden and eliminating infection.30 There is also evidence for a role of T cell–dependent immunity in the control of giardiasis. A reduction or absence of CD4 T cells can lead to chronic infection. IFN-γ- and IL-17A-producing CD4 T cells develop following infection32 and are important for mediating parasite clearance. Although the mechanisms are still being defined, IL-17A is likely involved in modulating transport of IgA into the intestinal lumen. Epidemiological studies indicate that partial immunity is acquired from Giardia infection, which leads to reduced risk and severity of subsequent infections.30

Evasion of Host Immunity Giardia evades the host humoral immune response by undergoing surface antigenic variation by altering a group of variant-specific surface proteins (VSPs). Selection occurs by an immune-mediated process because switching occurs when intestinal anti-VSP IgA responses are first detected.30 G. lamblia also produces a protease that cleaves IgA. Although Giardia activates dendritic cells for antigen presentation, it also inhibits IL-12 production, in part by enhancing IL-10 release; the net result is the dampening of a local antiparasitical inflammatory response.30 The trophozoite also releases arginine deiminase, which degrades arginine, making it less available for host NO production.31

433

Cryptosporidium parvum AND Cryptosporidium hominis In humans, there are four intestinal coccidians that are intracellular parasites of enterocytes: Isospora belli, Cyclospora cayetanensis, and two species of Cryptosporidium, C. parvum, and C. hominis. Of the four coccidians, Cryptosporidium has the greatest epidemiological significance: in 1993, a huge outbreak involving 403 000 persons occurred in Milwaukee, Wisconsin, in the United States. Because of their similarity, only the immunology of cryptosporidiosis will be discussed.

Pathogenesis Cryptosporidium typically causes self-limited (but often prolonged) diarrhea in the immunocompetent host. However, in the immunocompromised host Cryptosporidium can cause severe diarrhea, with malabsorption and wasting, and cholangiopathy. Infection begins with the ingestion of food or water contaminated with oocysts. The acidic environment of the stomach induces excystation and the release of four sporozoites into the small intestines. Glycoproteins on the surface of sporozoites facilitate attachment and invasion of epithelial cells. After entry into epithelial cells, the parasite resides within a unique intracellular but extracytoplasmic vacuole, that protects the pathogen from environmental and host insults. Inside a vacuole the sporozoites develop into trophozoites and undergo schizogony, with a resultant merozoite-containing schizont. The merozoites are extruded and invade neighboring epithelial cells. The merozoites may continue an asexual cycle or develop into macro- or microgametes that fuse to form oocysts. Before being excreted in feces, oocysts undergo sporulation to become infectious. Histologically, infection causes villous atrophy and blunting, and crypt hyperplasia with increased infiltration of lymphocytes, macrophages, and plasma cells.33 Intraepithelial lymphocytes are uncommon; neutrophils and occasional eosinophils are present between the epithelium and the lamina propria. Disorganized cells undergoing necrosis replace normal enterocyte architecture (see Fig. 30.3). There is an association between the degree of intestinal injury and malabsorption and the intensity of infection, as measured by oocyst excretion. The neuropeptide substance P, which is produced by endothelial cells, lymphocytes, and monocytes in the lamina propria, contributes to diarrhea by increasing intestinal chloride secretion and glucose malabsorption. Increased expression of substance P is observed in patients with AIDS as well as cryptosporidiosis and severe diarrhea.34

Innate Immunity The type I and type II IFNs play a key role in the innate protective response against cryptosporidium.35 Because of the parasite’s intracellular location near the luminal surface of the enterocyte, the macrophages of the lamina propria are spatially isolated from the parasite. Thus the intestinal epithelium mounts its own assault on the invading microbe through TLR2/TLR4–dependent activation of NF-κB and release of the microbicidal peptide β-defensin-2, TNF-α, and the chemokines IL-8, RANTES, and GRO-α, which act as chemoattractants and activators of neutrophils. In patients with AIDS and cryptosporidiosis, the HIV Tat protein may sabotage host defense against Cryptosporidium by inhibiting cholangiocyte TLR4 expression.33

434

Part THREE  Host Defenses to Infectious Agents

IL-15, produced by activated monocytes, stimulates NK-cell proliferation, cytotoxicity, and cytokine production, including IFN-γ. There is significant IL-15 expression in the jejunal mucosa in immunocompetent patients with cryptosporidiosis, and IL-15 levels inversely correlate with parasite burden. However, in patients with AIDS who have chronic uncontrolled cryptosporidiosis, IL-15 is undetectable.33 Mannose-binding lectin (MBL) is a serum protein that binds to various pathogens, including Cryptosporidium. Upon binding, MBL activates complement, thereby promoting opsonization and phagocytosis. Low serum levels of MBL, which may result from malnutrition or polymorphisms in the MBL2 gene, increase susceptibility to cryptosporidiosis.33 Infected intestinal cells also release TGF-β, which decreases necrosis and stimulates the synthesis of extracellular matrix proteins, thereby limiting epithelial damage. Prostaglandins E2 and F2α, released by infected enterocytes, not only promote secretory diarrhea but also upregulate mucin production, which may hinder parasite attachment. In addition, these prostaglandins stimulate the release of β-defensin-2, which has direct anticryptosporidial activity and is chemotactic for T cells and DCs.33

Adaptive Immunity Cell-mediated immunity plays an important role the resolution of cryptosporidiosis and protection from reinfection. In immunocompetent, adult mice CD4 intraepithelial lymphocytes (IELs) initiate early control of infection, whereas cytotoxic CD8 IELs appear later and function in parasite elimination. Resolution of infection depends on a balance of Th1 cytokines (IFN-γ, IL-18) needed to control the infection and Th2 cytokines (IL-4, IL-10, and IL-13) that limit immunopathological damage. In mice, γδ T cells are rapidly recruited to control cryptosporidial infection, but their role in human infection is unknown. Severe intestinal disease or biliary involvement is usually seen in patients with AIDS whose CD4 count is 200/µL usually experience self-limited disease. The role of humoral immunity is less clear. Secretory antibodies produced in the intestines are thought to impair parasite attachment to epithelial cells. Indeed, immunoglobulin deficiencies are often associated with persistent or recurrent infections. However, specific anti-Cryptosporidium IgA levels have been reported in patients with AIDS who are infected with the parasite, suggesting polyreactive T cell–independent antibodies are not sufficient for parasite eradication.33 In experimentally infected human volunteers, serum IgM and IgG directed toward sporozoite proteins protect against the development of symptoms, but not infection.

Evasion of Host Immunity Cryptosporidium evades host defenses primarily by exerting control over infected enterocyte apoptosis. One of the upregulated genes is osteoprotegerin, which inhibits apoptosis by acting as a decoy receptor for TNF-related apoptosis inducing ligand (TRAIL).34 Control of host apoptosis is complex; early inhibition of apoptosis by NF-κB activation allows the parasite to complete its life cycle, whereas the late promotion of apoptosis facilitates merozoite release. Nevertheless, the infected cells secrete FasL, which promotes apoptosis in uninfected bystander cells. In this way, the host counters the antiapoptotic activity of the parasite by surrounding the parasitized cells by a zone of apoptotic cells.

Trichomonas vaginalis Pathogenesis Trichomonas vaginalis is a flagellated protozoan parasite of the human urogenital tract that exists only as a trophozoite. It causes vaginitis, cervicitis, and urethritis. Its adherence to the vaginal squamous epithelium is facilitated by a number of adhesins. Trichomonas causes tissue damage by contact-dependent cytolysis caused by pore-forming proteins and proteases, and secretion of a glycoprotein cell-detaching factor that causes sloughing of the vaginal epithelium. Levels of the cell-detaching factor correlate with the severity of the disease, and vaginal antibodies directed against this factor modulate its effects. Inflammation in the genital mucosa and submucosa leads to copious secretions, and the surface epithelium may slough, causing focal erosions and hemorrhage. The increased risk of HIV transmission in women with trichomoniasis may result from increased recruitment of inflammatory cells, mucosal erosion, or degradation of secretory leukocyte protease inhibitor (SLPI) by trichomonal proteases. Lower levels of SLPI are found in the vaginal fluid of women with trichomoniasis, which can lead to increased tissue damage and HIV transmission.36 The lipophosphoglycan of Trichomonas induces production of the chemokines IL-8 and CCL20, which, which can also facilitate HIV infection by promoting DC recruitment.

Innate Immunity Although trichomoniasis has recently received increased attention as a risk factor for HIV transmission and obstetrical complications, there is little known about the protective immune response against this organism. Trichomonas secretes a factor that promotes neutrophil chemotaxis, causing profuse leukorrhea, but the oxidative microbicidal mechanisms of the neutrophils have decreased efficacy in the anaerobic vaginal environment. Activated macrophages can destroy trichomonads in a T and B cell– independent manner and release IL-lβ and TNF-α, which are chemotactic for neutrophils.37 Trichomonas induces neutrophil apoptosis, and macrophage clearance of these apoptotic cells causes release of IL-10, which may contribute to resolution of the inflammatory response.38

Adaptive Immunity Repeated infections with T. vaginalis do not induce immunity; however, the infection is self-limited in most cases, so there are effective mechanisms of host defense. T. vaginalis induces the production of antibodies in both the serum and vaginal secretions. The serum antibody response correlates with active infection, and serum, but not vaginal, IgG from infected patients displays complement-mediated lytic activity against trichomonads in culture.38

Evasion of Host Immunity Although T. vaginalis activates the alternative pathway of complement, the cervical mucus and menstrual blood are low in complement. Menstrual blood also supplies iron, which upregulates trichomonal adhesins and cysteine proteases, causing the degradation of complement component C3 bound to the surface of the parasite. Parasite virulence is thus enhanced, and the symptoms are exacerbated during menses. Cysteine proteases secreted by T. vaginalis also degrade immunoglobulins, sabotaging the antibody response. The parasite also secretes soluble antigens

CHAPTER 30  Host Defenses to Protozoa that act as decoys for neutralizing antibodies or cytotoxic T cells and disguises itself by binding to host plasma proteins. Phenotypic variation of surface markers is also a means of antibody evasion.38

ON THE HORIZON Anticipated Approaches to Improved Control of Protozoal Diseases • Gaining insight into the role of host genetics, gender, intestinal microbiota, and nutritional status in the outcome of protozoal infections • Defining host immune and inflammatory mechanisms that promote disease caused by protozoal pathogens • Understanding mechanisms for sustained generation of protective immunity against protozoal pathogens • Developing vaccines for several blood and tissue protozoa • Developing new host-directed treatment strategies to overcome immune evasion by protozoal pathogens

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Kumar R, Engwerda C. Vaccines to prevent leishmaniasis. Clin Translat Immunol 2014;3:e13. 2. Bethony JM, Cole RN, Guo X, et al. Vaccines to combat the neglected tropical diseases. Immunol Rev 2011;239:237–70. 3. Crompton PD, Pierce SK, Miller LH. Advances and challenges in malaria vaccine development. J Clin Investig 2010;120:4168–78. 4. Vazquez-Chagoyan JC, Gupta S, Garg NJ. Vaccine development against Trypanosoma cruzi and Chagas disease. Adv Parasitol 2011;75:121–46. 5. Gazzinelli RT, Kalantari P, Fitzgerald KA, et al. Innate sensing of malaria parasites. Nat Rev Immunol 2014;14:744–57. 6. Crompton PD, Moebius J, Portugal S, et al. Malaria immunity in man and mosquito: insights into unsolved mysteries of a deadly infectious disease. Annu Rev Immunol 2014;32:157–87. 7. Schwenk RJ, Richie TL. Protective immunity to pre-erythrocytic stage malaria. Trend Parasitol 2011;27:306–14. 8. Langhorne J, Ndungu FM, Sponaas AM, et al. Immunity to malaria: more questions than answers. Nat Immunol 2008;9:725–32. 9. Stanisic DI, Barry AE, Good MF. Escaping the immune system: how the malaria parasite makes vaccine development a challenge. Trend Parasitol 2013;29:612–22. PubMed PMID: 24176554. 10. Guizetti J, Scherf A. Silence, activate, poise and switch! Mechanisms of antigenic variation in Plasmodium falciparum. Cell Microbiol 2013;15: 718–26. 11. Gurung P, Kanneganti TD. Innate immunity against Leishmania infections. Cell Microbiol 2015;17:1286–94. 12. Ribeiro-Gomes FL, Sacks D. The influence of early neutrophil-Leishmania interactions on the host immune response to infection. Frontiers Cell Infect Microbiol 2012;2:59. 13. Peters NC, Egen JG, Secundino N, et al. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 2008 15;321:970–4.

435

14. Novais FO, Scott P. CD8+ T cells in cutaneous leishmaniasis: the good, the bad, and the ugly. Semin Immunopathol 2015;37:251–9. 15. Soong L, Henard CA, Melby PC. Immunopathogenesis of non-healing American cutaneous leishmaniasis and progressive visceral leishmaniasis. Semin Immunopathol 2012;34:735–51. 16. Geiger A, Bossard G, Sereno D, et al. Escaping deleterious immune response in their hosts: lessons from trypanosomatids. Frontiers Immunol 2016;7:212. 17. Ives A, Ronet C, Prevel F, et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science 2011;331:775–8. 18. Bonney KM, Engman DM. Autoimmune pathogenesis of Chagas heart disease: looking back, looking ahead. Am J Pathol 2015;185:1537–47. 19. Machado FS, Dutra WO, Esper L, et al. Current understanding of immunity to Trypanosoma cruzi infection and pathogenesis of Chagas disease. Semin Immunopathol 2012;34:753–70. 20. Kayama H, Takeda K. The innate immune response to Trypanosoma cruzi infection. Microbes Infect 2010;12:511–17. 21. Tarleton RL. CD8+ T cells in Trypanosoma cruzi infection. Semin Immunopathol 2015;37:233–8. 22. Fernandes MC, Andrews NW. Host cell invasion by Trypanosoma cruzi: a unique strategy that promotes persistence. FEMS Microbiol Rev 2012;36: 734–47. 23. Butler NS, Harris TH, Blader IJ. Regulation of immunopathogenesis during Plasmodium and Toxoplasma infections: more parallels than distinctions? Trend Parasitol 2013;29:593–602. 24. Yarovinsky F. Innate immunity to Toxoplasma gondii infection. Nat Rev Immunol 2014;14:109–21. 25. Aliberti J. Host persistence: exploitation of anti-inflammatory pathways by Toxoplasma gondii. Nat Rev Immunol 2005;5:162–70. 26. Hunter CA, Sibley LD. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat Rev Microbiol 2012;10:766–78. 27. Moonah SN, Jiang NM, Petri WA Jr. Host immune response to intestinal amebiasis. PLoS Pathog 2013;9:e1003489. 28. Nakada-Tsukui K, Nozaki T. Immune response of amebiasis and immune evasion by Entamoeba histolytica. Frontiers Immunol 2016;7:175. 29. Baxt LA, Singh U. New insights into Entamoeba histolytica pathogenesis. Curr Opin Infect Dis 2008;21:489–94. 30. Lopez-Romero G, Quintero J, Astiazaran-Garcia H, et al. Host defences against Giardia lamblia. Parasite Immunol 2015;37:394–406. 31. Cotton JA, Beatty JK, Buret AG. Host parasite interactions and pathophysiology in Giardia infections. Int J Parasitol 2011;41:925–33. 32. Saghaug CS, Sornes S, Peirasmaki D, et al. Human memory CD4+ T cell immune responses against Giardia lamblia. Clin Vaccine Immunol 2016;23:11–18. 33. Kothavade RJ. Challenges in understanding the immunopathogenesis of Cryptosporidium infections in humans. Eur J Clin Microbiol Infect Dis 2011;30:1461–72. 34. Pantenburg B, Dann SM, Wang HC, et al. Intestinal immune response to human Cryptosporidium sp. infection. Infect Immun 2008;76:23–9. 35. Barakat FM, McDonald V, Foster GR, et al. Cryptosporidium parvum infection rapidly induces a protective innate immune response involving type I interferon. J Infect Dis 2009;200:1548–55. 36. Thurman AR, Doncel GF. Innate immunity and inflammatory response to Trichomonas vaginalis and bacterial vaginosis: relationship to HIV acquisition. Am J Reprod Immunol 2011;65:89–98. 37. Han IH, Goo SY, Park SJ, et al. Proinflammatory cytokine and nitric oxide production by human macrophages stimulated with Trichomonas vaginalis. Korean J Parasitol 2009;47:205–12. 38. Petrin D, Delgaty K, Bhatt R, et al. Clinical and microbiological aspects of Trichomonas vaginalis. Clin Microbiol Rev 1998;11:300–17.

CHAPTER 30  Host Defenses to Protozoa

435.e1

MULTIPLE-CHOICE QUESTIONS 1. Which one of the following is characteristic of the immunopathogenesis of cerebral malaria? A. Absence of Plasmodium-specific antibodies B. Impaired interferon (IFN)-γ mediated production of nitric oxide C. Cytoadhesion of parasites and leukocytes to vascular endothelium D. Immune-mediated destruction of infected erythrocytes E. Failure of leukocyte production of leukotriene/tumor necrosis factor (LT/TNF) 2. Which one of the following is NOT a mechanism through which Leishmania can evade the host immune response? A. Complement resistance mediated by the parasite’s surface lipophosphoglycan B. Blunted interferon (IFN)-γ-mediated macrophage activation through the disruption of signal transduction pathways C. Increased synthesis of the immunosuppressive molecules interleukin (IL)-10, transforming growth factor (TGF)-β, and prostaglandin (PG)E2 D. Depletion of L-arginine through the activity of host arginase E. Suppression of an early neutrophil response at the site of infection

3. Which one of the following is a component of the innate immune response to Cryptosporidium infection? A. Decreased intestinal mucin production B. Increased type I and type II interferon production C. Decreased nuclear factor-kappa B (NF-κB) activation D. Antigen-specific CD8 intraepithelial T-cell responses E. Antigen-specific immunoglobulin A (IgA) responses

31  Immune Responses to Helminth Infection Subash Babu, Thomas B. Nutman

Parasitical helminths are complex eukaryotic organisms, characterized by their ability to maintain long-standing, chronic infections in human hosts, sometimes lasting decades. Hence, parasitical helminths are a major health care problem worldwide, infecting more than 2 billion people, mostly in resource-constrained countries (Fig. 31.1). Common helminth infections include those with intestinal helminths, and filarial and schistosome infections are a major medical, social, and economic burden to the countries in which these infections are endemic. Chemotherapy, although highly successful in some areas, still suffers from the disadvantages of the length of treatment, the logistics involved in the distribution of drugs, and, in some cases, the emergence of drug resistance. Vector control measures are at best an adjunct measure in the control of helminth infections but also suffer from the same social, logistic, and economic obstacles as those for mass chemotherapy. Therefore the study of the immune responses to helminth infections attains great importance both in terms of understanding the parasite strategies involved in establishing chronic infection and in the delineation of a successful host immune response to develop protective vaccines against infection.

SPECTRUM OF HOST–PARASITE INTERACTIONS Helminths have characteristically complex lifecycles with many developmental stages.1 Thus the host is exposed during the course of a single infection to multiple lifecycle stages of the parasites, each stage with a shared as well as a unique antigenic repertoire. Thus Schistosoma mansoni infection begins with penetration of the skin of humans exposed to infested waters by the freeswimming cercariae, which then develop into tissue-dwelling schistosomula. In the liver and mesenteric veins, schistosomula differentiate into sexually dimorphic adult worms, which then mate, and the resultant eggs produced migrate through tissues into the lumen of the intestine or bladder for environmental release. Similarly, in lymphatic filarial infection, the host is exposed to infective-stage larvae in skin, lymph nodes, and lymphatics; to adult worms in lymph nodes and lymphatics; and finally to microfilariae in the peripheral circulation. Hence, the host– helminth interaction is complex not only because of the multiple lifecycle stages of the parasite but also because of the tissue tropism of the different stages. Antigenic differences among the lifecycle stages can lead to distinct immune responses that evolve differentially over the course of a helminth infection. In addition, depending on the location of the parasite, the responses are compartmentalized (intestinal mucosa and draining lymph nodes in intestinal

nematode infection, or skin/subcutaneous tissue and draining lymph nodes in onchocerciasis) or systemic (lymphatic filariasis or schistosomiasis). Moreover, the migration patterns of the parasite might elicit varied cutaneous, pulmonary, and intestinal inflammatory pathologies, as seen, for example, in Ascaris or Strongyloides infection during their migratory phase. This is further complicated by the fact that human hosts are often exposed to multiple lifecycle stages of the parasite at the same time. Thus a patient with chronic infection with lymphatic filariasis harboring adult worms and microfilariae might be exposed to insect bites, thereby transmitting the infective-stage parasite. The immune response that ensues will not only be a reaction to the invading organism but will also bear an imprint of the previous exposures and the concurrent infection.

KEY CONCEPTS Helminth Infection Divided into nematodes, trematodes, and cestodes Produce chronic infections that can persist for decades Characteristically cause morbidity rather than mortality Multicellular parasites that do not multiply in the definitive host but can reproduce sexually to produce larval stages that ensure continued transmission

Helminth infections can elicit a spectrum of clinical manifestations mirroring diversity in host immune responses. For example, in lymphatic filariasis, most infected individuals remain clinically asymptomatic despite harboring significant worm burdens; this is thought to reflect the induction of parasite-specific tolerance in the immune system. Others exhibit acute manifestations, including fever and lymphadenopathy, and this is thought to reflect inflammatory processes induced by incoming larvae, dying worms, or superadded infections. Individuals who mount a strong but inappropriate immune response end up with lymphatic damage and subsequent immune-mediated pathology—hydrocele and elephantiasis. Finally, a group of infected individuals mount exuberant immune responses that often result in unusual pathology, such as tropical pulmonary eosinophilia. Thus the clinical manifestations of lymphatic filariasis exemplify the spectrum of host–parasite interactions that occur during helminth infections (Fig. 31.2). Another hallmark of all helminth infections is their chronic nature, with many helminths surviving in the host for decades. For example, adult schistosomes and filariae may survive in host tissues for as long as 30 years, producing eggs and larval stages throughout this time. Similarly, Strongyloides stercoralis, with its

437

438

Part three  Host Defenses to Infectious Agents

ability to “autoinfect,” can maintain its lifecycle for decades. Chronic infections certainly reflect an adaptation that leads to “parasitism,” as mortality induced in the host would prevent parasite transmission if the host died before larval release or egg production could occur. In addition to the long-lived nature of Schistosoma mansoni > 200 million infected

Tre ma tod es

des sto Ce

Brugia malayi Onchocerca volvulus Wuchereria bancrofti 157 million infected

Echinococcus, Taenia spp >100 million infected

Nematodes

Ascaris lumbricoides >1 billion infected

Ancylostoma duodenale Nector americanus 576 million infected

Trichinella spiralis Trichuris trichiura >600 million infected

FIG 31.1  Common and medically relevant helminth infections and their global prevalence.

A

the infection, helminths appear to reflect a harmonious host– parasite interface such that relatively asymptomatic carriers are available as reservoirs for ongoing transmission. Of course, failure to establish this harmonious coexistence does occur, leading to pathological conditions exemplified by cirrhosis and portal hypertension in schistosomiasis and elephantiasis associated with lymphatic filariasis.

PROTOTYPICAL HOST RESPONSES TO HELMINTHS The canonical host immune response to all helminths is of the T-helper 2 (Th2) type and involves the production of cytokines interleukin (IL)-4, IL-5, IL-9, IL-10, and IL-13; the antibody isotypes immunoglobulin G1 (IgG1), IgG4, and IgE; and expanded populations of eosinophils, basophils, mast cells, type 2 innate lymphoid cells, and alternatively activated macrophages2 (Chapter 16). However, it is also being increasingly recognized that while the predominant response is Th2 in nature, a large regulatory component involving both regulatory cytokines and cells are also part of this repertoire.3 The Th2 response induced by helminth parasites is quite stereotypical, but its initiation, progression, and culmination of this response requires interaction with many different cell types, most notably (i) epithelial/stromal cells, (ii) innate lymphoid cells (ILCs), (iii) dendritic cells (DCs) and macrophages; (iv) T cells; (v) B cells; (vi) eosinophils; (vii) mast cells/basophils; and (viii) neutrophils (Chapter 2). In addition, the host–helminth interactions can lead to a variety of modulated immune responses that are mediated largely by the induction of regulatory T cells (Tregs) and alternatively activated macrophages (AAMs) (Fig. 31.3).

B

C

D

FIG 31.2  The clinical manifestations of lymphatic filariasis, including (A) mild lymphedema, (B) severe lymphedema, (C) elephantiasis, and (D) hydrocele.

CHAPTER 31  Immune Responses to Helminth Infection

Epithelial cells

TSLP

TSLP Basophil

439

Langerhans cells

IL-33/ TSLP

TSLP/other cytokines?

IL-25

Relmα/FIZZ1 Natural helper cell

IL-4 IL-13 IL-4 IL-13

Th0

Eosinophil

IL-5 Eosinophil differentiation IL-4 Promotes Th2 differentiation, IgE production IL-10 Role in immunomodulation IL-9 Role in mastocytosis

Th2

Treg

Alternatively activated macrophages

Ar YM gin -1 as e1

Dendritic cells

IL-10 Bystander suppression (allergy, autoimmunity, etc.)

Tfh

B cell IL-4

IgG1 IgE

Basophil

FIG 31.3  Regulation of the T-cell response in helminth infection. IL, interleukin; Th0, precursor T-helper cell; Th2, T-helper 2 cell; Treg, Regulatory T cell; TfH, T-follicular helper cell, RELM-α, Resistin-like molecule-α; Chi3l, Chitinase 3 like protein; IDO, indoleamine 2,3 dioxygenase; TSLP, thymic stromal lymphopoietin.

Helminths and Epithelial Cells

Helminths and Innate Lymphoid Cells

Epithelial cells are the first barrier layer exposed to or breached by most helminths, and the capacity of these cells to respond by initiating an “alarm” response has recently been recognized.4 These epithelial cells mount a prototypical response comprising chemokines and cytokines, such as IL-1, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as alarmins, such as uric acid, ATB, HMGB1, and S100 proteins. These signals program DCs to mount Th2 cell–mediated immunity and in doing so boost type 2 innate lymphoid cell (ILC2), basophil, and mast cell function. Epithelial cells produce chemokines, including CCL17 and CCL22 (acting on ILC2, basophils, Th2 cells, and Tregs), and eotaxins, such as CCL11, CCL24, and CCL26 (acting on eosinophils and Th2 cells). They also produce prostaglandin D2 (PGD2), which acts on the CRTH2 receptor to recruit ILC2, basophils, mast cells, and Th2 cells. More recently, tuft cells, a specialized secretory cell type of the intestinal epithelium, has been identified as a major player in anthelmintic immunity. In addition, epithelial cells in the intestine, for instance, are in constant contact with both beneficial and pathogenic bacteria and hence ideally located for immunological surveillance of the intestinal lumen. This recognition of signals by intestinal epithelial cells is essential to mucosal homeostasis, implicating these cells as central modulators of inflammatory responses. Finally, the production of mucus and mucus-associated bioactive molecules (Mucin5AC, trefoil factor-2, and resistin-like molecule-β [RELMβ]) are important in promoting protection against intestinal helminth infection.

The ILC family includes ILC1, which predominantly express IFN-γ; ILC2, which predominantly express IL-5 and IL-13; and ILC3; which predominantly express IL-22 and/or IL-17.5 ILC2 are defined by their expression of the IL-33 receptor (IL-33R) and the transcriptional regulators, Id2, RORα, GATA-3, and Bcl11b. Unlike T cells, ILC2 rely on cytokines to drive activation rather than on cognate interactions mediated by antigen-specific receptors. ILC2 are a critical innate source of type 2 cytokines, including moderately large quantities of IL-5 and IL-13, but also of IL-4, IL-9, granulocyte macrophage–colony-stimulating factor (GM-CSF), and amphiregulin. These cytokines potently induce eosinophilia, mucus production from goblet cells, activation of AAM, muscle contractility, mastocytosis, and tissue repair.5 They are dependent on IL-2 and IL-7 for their development and activation. In addition, the transcription factors GATA-3 and RORα have been found to be essential for the development of ILC2. Although the function of ILC2 and Th2 cells appear to be largely overlapping, the kinetic differences in the ability to secrete cytokines rapidly and in profuse amounts allows for a coordinated interaction between the two cell types. Moreover, ILC2 can directly regulate the activation of T cells through their expression of major histocompatibility complex (MHC) Class II molecules and the accessory molecules, CD80 and CD86, albeit less efficiently compared with DCs. Finally, recent reports have linked ILC2 with metabolic homeostasis, obesity, and dietary stress, providing an indirect link by which helminths might modulate host metabolic function.

440

Part three  Host Defenses to Infectious Agents

Helminths and Dendritic Cells DCs are professional antigen-presenting cells (APCs) that play an essential role in presenting antigen to T cells to initiate immune responses.2 Although the role of DCs in inducing Th1, Th17, and Treg responses is well established, their role in inducing Th2 responses has remained relatively unclear. Nevertheless, a series of studies have shown that DCs are required for optimal Th2 responses in vivo. Thus in vivo depletion of DCs has been shown to inhibit the induction of Th2 responses to S. mansoni or Heligmosomoides polygyrus. Helminth products can prime DCs for the induction of Th2 responses by interaction with pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). This interaction, which depends on TLR and CLR signaling, can promote Th2 responses by suppressing antigen presentation, costimulation, and/or expression of Th1-promoting cytokines by directly interfering with these pathways. DCs that drive Th2 responses typically exhibit specialized markers, such as CD301b, PDL2, and CD11b, and several receptors for the Th2-related cytokines IL-4R, IL-13R, IL-25R, TSLP-R, and IL-33R. Additionally, the extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 4 (STAT4) pathway upregulates the costimulatory molecules, CD40, OX40L, and Jagged. Activation of the major transcription factors interferon regulatory factor 4 (IRF4) and KLF4 inhibits IL-12 production and increased IL-10 secretion. In addition, DCs expressing FcεRIII can induce murine IgG1-related Th2 responses. These factors typically act individually or in concert to orchestrate Th2 responses in helminth infections. Although Th2 cell–mediated immunity requires IRF4-dependent CD301b+CD11b+ DCs in the mouse, Langerhans cells are the predominant inducers of Th2 cells ex vivo in humans. The modulation of DC function by helminth antigens appears to be generalizable and has been shown to impair their ability to respond to other infectious stimuli (e.g., Mycobacterium tuberculosis).

Helminths and Macrophages Macrophages are the other important class of APCs that can serve as protective effector cells in bacterial and protozoan infections by their production of nitric oxide and other mediators. Helminth interaction with macrophages induces a population of cells preferentially expressing arginase instead of nitric oxide as a result of increased activation of arginase-1 by IL-4 and IL-13.6 These AAMs are characterized by their ability to upregulate arginase-1, chitinase 3-like proteins 3 and 4 (also known as Ym1 and Ym2, respectively), and RELM-α. These AAMs are known to be important in wound healing and have been postulated to play a potential role in repairing wound damage that occurs during tissue migration of helminth parasites. In fact, there appears to be two distinct populations of AAMs, one derived from blood and functioning in an immune regulatory role and the other derived from tissue-resident macrophages apparently responsible for much of the fibrosis seen in chronic helminth infections. By virtue of the expression of regulatory molecules, such as IL-10, TGF-β, and programmed cell death 1 ligand 2 (PDL2), these AAMs may have a predominantly regulatory role in helminth infections. These antiinflammatory macrophages function through arginase-1, PDL2, triggering receptor expressed on myeloid cells 2 (TREM2) and RELM-α to inhibit classic macrophage inflammation and recruitment and T-cell responses. Similarly, macrophage-derived human resistin is induced by

helminth infection and promotes inflammatory responses and increased susceptibility.

Helminths and T Cells Typically, infections with helminths induce a robust Th2 response manifested by enhanced expression of IL-4, IL-5, IL-9, IL-10, and IL-13 in response to live parasites, parasite antigens, or mitogens.7 The central player in Th2 immunity is certainly the CD4+ Th2 cell. It is clear that IL-4Rα, a component of both the IL-4 and IL-13 receptors, is at the epicenter of Th2 immunity, since IL-4 and IL-13, together or individually, are absolutely critical for resistance to most helminth parasites. Recent work has reported that the Th2 cell population is heterogeneous, containing both IL-5+ and IL-5− Th2 cells that express IL-4 and IL-13. In addition, IL-4 and IL-13 production is spatially separated, with IL-13 expression being marked in tissues and IL-4 expression being pronounced in the lymph nodes within the Th2 cell compartment. Finally, induction of GATA-3 and downregulation of T-box expressed in T cells (T-bet) has been shown to be an important step in T resistin-like molecule cell differentiation to the Th2 phenotype in helminth infections. Interestingly, chronic helminth infections are associated with downmodulation of parasite antigen-specific proliferative responses as well as IFN-γ and IL-2 production but with intact IL-4 responses to parasite antigens and global downregulation of both Th1 and Th2 responses to live parasites. Finally, the receptor NLRP3 has been shown to be a key transcription factor in Th2 differentiation. Although the role of tissue resident memory T (TRM) cells is well established in viral and bacterial infections, very little is known about the role of these cells in helminth infections. However, it has been shown that tissue-resident Th2 cells can exert innate (TCR-independent and IL-33–dependent) functions upon appropriate stimuli and confer protection against helminth infection. In addition, although multifunctionality (ability to produce two or more cytokines) has not been well described in the Th2 cell compartment, helminth infections are known to be associated with an antigen-dependent enhancement of mono- and dual-functional Th2 cells and its reversal after treatment. Of interest, a stable subset of parasite induced T-bet+, GATA-3+, Th1/Th2 hybrid T cells has been described to develop directly from naïve precursors and to play a role in limiting pathological inflammation in animal models of helminth infection. Recently, a new subset of T cells expressing IL-9 and IL-10, but not IL-4 (and therefore different from Th2 cells), has been described in allergic inflammation and in response to intestinal parasites.8 These cells appear to be under the control of TGF-β and IL-4 and are dependent on STAT6, GATA-3, IRF4, and PU.1. Th9 cells have been recently shown to be associated with host protection in Nippostrongylus brasiliensis and Trichuris muris infection. Finally, Th9 cells have also been shown to be predominantly associated with lymphatic pathology in filariasis. T-follicular helper (Tfh) cells are a subset of CD4 T cells that migrate to B-cell follicles after activation and promote germinal center formation and B-cell isotype switching.9 These cells, which form an independent lineage of CD4 T cells, have been recently identified to be the predominant IL-4 producing T cells early in helminth infection. In addition, Tfh are major producers of IL-21, a cytokine that plays a crucial role in supporting polarized Th2 responses in vivo. Th17 cells, another subset of CD4 T cells, express the prototypical cytokine—IL-17. In terms of helminth infections, the role of Th17 cells has been primarily studied in animal models of S.

CHAPTER 31  Immune Responses to Helminth Infection

441

mansoni, where it has been strongly associated with infectioninduced, immune-mediated pathology.10 More recently, it has also been demonstrated in human infections, in which children with S. hematobium–associated pathology have higher Th17 responses compared with those who are pathology-free. Similarly, a strong association of Th17 responses with pathological responses has also been demonstrated in lymphatic filariasis. Finally, Th22 cells are yet another subset of CD4 T cells that typically secrete IL-22. To date, only a few studies have examined the role of Th22 cells in helminth infections.11 IL-22 was shown to be induced in the intestinal mucosa after infection with T. trichiura or Necator americanus in humans, whereas the frequency of Th22 cells was shown to be higher in individuals with filarial infection compared with endemic healthy controls.

influence of ILC2. Apart from the rapid kinetics of recruitment, eosinophils in blood and tissue also exhibit morphological and functional changes attributable to eosinophil activation. Eosinophils possess a range of immunomodulatory factors that are released upon cell activation, including cytokines, growth factors, and chemokines. Unlike T and B cells, eosinophils can rapidly release cytokines within minutes in response to stimulation, since most of the cytokines are stored in a preformed fashion in secretory vesicles. Moreover, eosinophils can participate in the regulation of IgE and goblet cell mucus production; they also serve as effector cells in protective immune responses and as regulatory cells influencing both innate and adaptive immunity in helminth infections.

Helminths and B Cells

Basophils are an important component of the immune response to helminth infections.15 Basophils are capable of secreting a variety of mediators, including histamines, cytokines, chemokines, and lipid mediators that promote Th2 responses. Basophils in humans and mice also readily generate large quantities of IL-4 in IgE-dependent and IgE-independent manners. Basophils appear to play an important role in protective immunity to secondary infection (similar to eosinophils) with N. brasiliensis, H. polygyrus bakeri, and L. sigmodontis; they also play an active role in resistance to primary infection (through secretion of IL-4 and IL-13) with T. muris and T. spiralis. In addition, basophils have been shown to be critical APCs for driving Th2 cell differentiation in different models of helminth infection. Mast cells may contribute to inflammatory reactions directed against invasive helminth parasites. These cells express high affinity Fcε receptors that are sensitized with parasite antigen– specific IgE and can be triggered by parasite antigens. It has been postulated that cytokines and other mediators released by sensitized mast cells contribute to (i) the recruitment and activation of effector eosinophils; (ii) increased local concentrations of antibody and complement; and (iii) enhanced mucus hypersecretion and increased peristalsis of the gastrointestinal (GI) tract that plays an important role in resistance to certain GI nematode infections.16 More recently, a role for mast cells (in an IgE-independent manner) in mediating the secretion of epithelial-derived cytokines (IL-25, IL-33, and TSLP) and optimal migration of DCs was shown in H. polygyrus bakeri infection.

Helminth interactions with B cells occur both at the B-cell cytokine level and at the level of antibody production. Interactions at the cellular level primarily result in B-cell activation and cytokine production, most notably by the induction of IL-10. B cells have been shown to be important for the Th2 responses to certain helminths, with IL-2 producing B cells supporting optimal development of effector and memory Th2 cells and LTα1β2expressing B cells supporting the recruitment of a Th2 promoting DCs.12 Immune regulation by B cells has also been recognized in schistosome infection, where B-cell deficiency leads to enhanced Th2 cell–dependent immunopathology. However, it is at the level of antibody production that B cells play a profound role in helminth infections. Susceptibility to secondary infection is increased in the absence of B cells in infection with Litomosoides sigmodontis, S. mansoni, T. muris, and Heligmosomoides polygyrus bakeri.13 IgG is reported as an antibody isotype that is important for protection against intestinal helminths, and IgM (typically produced in a T cell–independent manner) has been linked to timely elimination of filarial parasites. One of the most consistent findings in helminth infections, both in mice and humans, is the elevated level of IgE that is observed after exposure to helminths. Most of the IgE produced is not antigen specific, perhaps representing nonspecific potentiation of IgE-producing B cells or deregulation of a normally well-controlled immune response. Interestingly, these IgE antibodies persist many years after the infection has been treated, indicating the presence of long-lived memory B cells or plasma cells in helminth infections. IgE production both in mice and humans is absolutely dependent on IL-4 or IL-13. Other isotypes that are commonly elevated in humans with chronic helminth infection are IgG4 and IgG1, the former being most dependent on both IL-4 and IL-10. Recent studies have highlighted the role of regulatory B cells in suppression of immune responses to helminth parasites. This B-cell function involves the secretion of IL-10 and IL-35 and is similar to the regulatory activity of B cells in autoimmune diseases.

Helminths and Eosinophils Blood and tissue eosinophilia is characteristic of helminth infection and is mediated by IL-5 (probably in concert with IL-3 and GM-CSF). Recruitment of eosinophils to the site of infection occurs very early in experimental helminth infection—as early as 24 hours after exposure. Kinetics of blood eosinophilia in humans is harder to determine but is postulated to occur as early as 2–3 weeks after infection, as demonstrated in experimental infections of volunteers.14 Both basal eosinophil levels and tissue accumulation during helminth infection appear to be under the

Helminths and Basophils/Mast Cells

Helminths and Neutrophils Although neutrophils are typically considered more important in bacterial and fungal infections, a number of studies have revealed that neutrophils can act in conjunction with macrophages to contain or kill helminth parasites.17 Thus neutrophils are major components of the granulomas forming around filarial parasites and the cysts containing larvae of intestinal helminths. Neutrophils have been demonstrated to collaborate with macrophages in the immobilization and killing of S. stercoralis larvae in a process that is complement dependent and involving neutrophil extracellular traps (NETs). Similarly, neutrophils contribute in the early antifilarial response through oxidative burst, degranulation, and NETosis and protect against infective larvae in skin. A seminal study reported that neutrophils adopt an “N2” phenotype during experimental infection with N. brasiliensis in the lung and express the genes for IL-13, IL-33, RELM-α, and Ym1. These “N2” neutrophils can train macrophages to acquire a memory phenotype that protects against secondary infection. Finally, it was also shown that even during primary infection, the absence of

442

Part three  Host Defenses to Infectious Agents

neutrophils resulted in greater worm burdens because of lack of immunity in the lungs. Thus neutrophils appear to play an unexpected role in immunity to helminths that certainly merits further investigation.

PROTECTIVE IMMUNITY AGAINST HELMINTHS The mechanism of protective immunity to helminths is dependent on the location of the helminth infection.18 Clearly, T cells are central to resistance against helminths. For example, T cells are essential in mediating the expulsion of GI nematodes. Mice lacking T cells are defective in their ability to expel T. muris, but resistance can be reconstituted by transfer of T cells from normal mice. In addition, CD4 T cells from infected mice can transfer protective immunity to severe combined immunodeficiency (SCID) mice (lacking both B and T cells), indicating that CD4 T cells, not CD8 T cells, were important for protective immunity. Similarly, T cells were shown to be required for expulsion in N. brasiliensis infection. Both nude mice (lacking T cells alone) and SCID mice are susceptible to infection with Brugian parasites, whereas mice that lack either CD4 T cells or CD8 T cells are not. In schistosome-infected mice, T cells are essential in forming host-protective granulomas around the eggs deposited in the liver. The role of cytokines in protective immunity has been extensively studied in murine models of both GI helminths and tissue-invasive helminths.19 In general, type 2 (Th2) cytokines target epithelial cells, goblet cells, smooth muscle cells, and macrophages, which together coordinate parasite expulsion by increasing fluid and mucus production, encapsulation and barrier formation, epithelial cell turnover, smooth muscle cell contraction, and production of anthelmintic effector molecules, such as RELM-β. The cytokines involved in both responses are IL-4, IL-5, IL-9, and IL-13. Most of the studies examining resistance to intestinal helminths involve four parasitical GI nematode infections of rodent models—T. spiralis, H. polygyrus bakeri, N. brasiliensis, and T. muris. These studies show that (i) CD4 T cells are crucial for host protection; (ii) IL-4 is required for host protection and limiting host pathology; (iii) IL-13 can substitute for IL-4 in some but not all infections; (iv) IL-2 and IFN-γ inhibit protective immunity; and (v) IL-4 and IL-13 have multiple effects on the immune system and gut physiology leading generally to protection. Type 2 cytokines mobilize a broad range of downstream effector mechanisms. Epithelial cells in the gut, specifically tuft cells, promote goblet cell differentiation, enhancement of mucus secretion, and the production of RELM-β, an innate effector molecule with direct anthelmintic activity. Goblet cells can also secrete gel-forming mucins, which are major macromolecular components of the mucus barrier. Two of these mucins have been shown to be critical in resistance to intestinal nematode infection—Muc2 and Muc5AC. IL-4Rα activation also leads increased intestinal smooth muscle hypercontractility and accelerated epithelial turnover to promote an effector response akin to an “epithelial escalator,” which, together with epithelial secretions, helps expel intestinal helminths. Mucosal mast cells release proteases that can degrade epithelial tight junctions, thereby increasing fluid flow as part of the “weep and sweep” response. AAMs in the gut can also entrap intestinal worms and cause death by compromising worm vitality. Although the role of Th2 cytokines in immunity to GI helminth infection is well defined, their role in protective immunity to tissue-invasive helminths is not as clear.18 In murine models of schistosomiasis, protective immune responses can be generated

by vaccination with irradiated cercariae. This resistance is dependent on a Th1-mediated immune response consisting of macrophages and endothelial cells activated by IFN-γ and TNF-α, producing nitric oxide and Th1-associated antibodies—IgG2a and IgG2b. In contrast, studies in rats and epidemiological studies in humans suggest Th2-mediated effector mechanisms involving IgA and IgE antibodies as well as eosinophils are thought to be central to protective immunity. Protective immunity to filarial infections in mice is dependent primarily on Th2 responses in mice. Thus mice lacking IL-4, IL-4R, or Stat6 are all susceptible to infection with Brugian parasites. In tissue-invasive helminth infections, effector mechanisms involve multiple innate immune cells, with antibodies acting as initiators of immunity by activating Fc receptor expressing cells. Basophils, by their ability to produce high levels of IL-4, act as effectors to promote helminth killing in secondary or challenge infections. For example, basophils are important in immunity to the skin invasive stages of intestinal helminths and, through IL-4 release, promote the activation of macrophages that trap larvae in an arginase-dependent manner. Although eosinophils are crucial players in producing IL-4 early in infection, they are also amplifiers of immune responses, rather than being critical mediators of primary immunity, since depletion of eosinophils does not alter the course of many helminth infections in murine models. The mechanism of protection mediated by eosinophils is thought to be by antibody-dependent, cell-mediated cytotoxicity, as observed in S. mansoni studies in vitro or through release of eosinophil granule contents. In addition, eosinophils play an important role in the protective immunity against primary infection with B. malayi and/or secondary infection with either T. spiralis or N. brasiliensis. The two most abundant granular proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), are required for protective immunity against S. stercoralis and L. sigmodontis. Similarly, neutrophils can attack helminth larvae in response to IL-4 and IL-5, but their importance in resistance to primary helminth infections is not known. Antibodies play a major role in mediating protection to some but not all helminth infections.18 Antibody-mediated passive immunity has been demonstrated in animal models for A. caninum, Schistosoma species, Taenia species, Ascaris suum, S. ratti, T. muris, N. brasiliensis, and H. polygyrus bakeri. Passive immunity has also been shown by using IgG monoclonal antibodies (mAbs) specific for Fasciola hepatica and S. mansoni; IgM (mAbs) specific for B. malayi; and IgG or IgA mAbs specific for T. spiralis. Again using genetically manipulated mouse models, IgM has been shown to be crucial for host protection against B. malayi and to S. stercoralis. B1B cells, a subset of B cells that secrete IgM, appear to be an important component of this protective axis. Finally, antibodies have the capacity to trap tissue migrating helminth larvae and prevent tissue damage by driving an IL-4Rα–independent alternative differentiation of macrophages, in a process dependent on CD11b and FcγR1. In terms of protection, a major mechanism appears to be the formation of multicellular, immune cell aggregates, called granulomas, around incoming infectious larvae or eggs.18 In murine models of schistosomiasis and filariasis, granulomas are primarily composed of T cells (which help in the recruitment of other cell types and mediate alternative activation of macrophages), B cells (particularly the B1 subset), and macrophages and eosinophils. Although the exact mechanism by which granulomas mediate killing of the parasite remains unknown, it is clear that formation of these structures is an important host defense mechanism. One cell type that can mediate effector functions within granulomas

CHAPTER 31  Immune Responses to Helminth Infection KEY CONCEPTS Helminth-Induced Immune Responses Characterized by immunoglobulin E (IgE) antibody production, tissue and peripheral blood eosinophilia, mast cell involvement, innate lymphoid cell type 2 and Th2 cell expansion, and production of type 2 cytokines. Implicated both in pathogenesis of helminth infections and in mediating immunological protection. In mucosal immunity to helminths, T-helper 2 (Th2) cell responses are initiated and sustained by innate populations (including tuft cells and innate lymphoid cells) through interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). In tissues, helminths are acted upon by the host innate effectors, including macrophages, neutrophils, eosinophils, and basophils. Regulated by T cells and other cells producing IL-4, IL-5, IL-9, IL-10, and/ or IL-13. Characterized by the induction of regulatory T cells (Tregs) that mediate downmodulation of immune responses to helminth infections and impact bystander phenomena, such as allergy and autoimmunity.

443

Granulomatous Reactions Granuloma formation is the mainstay of the protective immune response to certain helminths, but it can also lead to deleterious effects in the form of pathology. Although granulomatous reactions occur in many helminth infections (e.g., toxocariasis, Angiostrongylus infections and lymphatic filariasis), parasitical granulomata have been best studied in S. mansoni infections, where granulomatous and fibrosing reactions against tissuetrapped eggs is orchestrated by CD4 T cells and the fibrosis that results from the cellular response is the principal cause of morbidity in infected individuals. The severity of the inflammatory process markedly varies both in humans and in experimental animal models, with severe pathology associated with Th1 and Th17 responses and milder pathology with Th2dominant responses.21 Studies in murine models of granuloma formation have demonstrated the important roles of IL-13 and TNF-α.

Fibrosis is the AAM, which is exemplified by the targeting of the glycan chitin that is frequently expressed by helminths but not by the host. The chitinase and fizz family proteins (ChaFFs), which include chitinase and chitinase-like secreted proteins, are prime candidates for mediating host resistance. These proteins include acidic mammalian chitinase (AMCase) and the RELM family proteins and are capable of enzymatic activities that potentially damage certain helminths.

PATHOLOGY ASSOCIATED WITH IMMUNE RESPONSES IN PARASITIC HELMINTH INFECTION Typically, pathological findings associated with each parasitic infection are different and relate to the presence of the parasites in host tissues, but there are pathological reactions that stem directly from the host response.

Immune Complexes Immune complexes are potent mediators of localized inflammatory processes that form in many parasitic infections presumably as a result of the chronic low-dose antigen release seen in these infections. Circulating immune complexes have been identified in both experimental and human filarial and schistosomal infections. These have been shown to induce lymphatic inflammation and vasculitis in filarial infections as a result of their deposition. In addition, a common manifestation of immune complex–mediated pathology, immune complex glomerulonephritis (ICGN), has been documented by renal biopsy in patients with schistosomiasis and filarial infections. Other manifestations of immune complex–mediated damage, such as reactive arthritis and dermatitis, have also been described in patients with helminth infections.

Autoantibodies and Molecular Mimicry Autoantibodies have been implicated as causing disease in a variety of helminth infections, including filarial infections, schistosomiasis, and hookworm infection, and are thought to reflect a polyclonal B cell expansion that often accompanies these infections.20 Autoantibodies against nuclear material have been found in a vast majority of patients with chronic schistosomiasis, and antibodies against human calreticulin and defensin have been found in onchocerciasis.

Fibrosis is commonly associated with chronic helminth infections that result in chronic inflammation and dysregulated wound healing.22 These infections activate macrophages and fibroblasts, resulting in the production of TGF-β, platelet-derived growth factor (PDGF), IL-1β, and other factors. Macrophages also promote inflammation by recruiting and activating monocytes and neutrophils, as well as activating CD4 T cells. In addition, fibroblasts are stimulated to synthesize matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to extracellular matrix remodeling and fibrosis. Another consequence of chronic schistosomiasis is pulmonary arterial hypertension, which has been shown to be associated with IL-4– and IL-13–mediated type 2 inflammation resulting in TGF-β−induced pulmonary vascular disease. In the same manner, IL-10 and IL-12 are known to modulate IL-13–mediated fibrosis; in the combined absence of IL-10, IL-12, and IL-13Rα, IL-13– dependent fibrosis in chronic schistosomiasis proceeds rapidly to lethal cirrhosis. Infection with Wuchereria bancrofti (one of the causative agents of lymphatic filariasis) is associated with similar fibrotic reactions.

Toll-Like Receptors Immunopathology in lymphatic filariasis is associated with the presence of an endosymbiotic, Rickettsia-like bacteria called Wolbachia. Wolbachia are known to stimulate immune cells through TLR2 and TLR4 and release proinflammatory cytokines, as well as vascular endothelial growth factors (VEGFs), which might contribute to lymphatic pathology.23 Wolbachia-TLR4 interaction has also been shown to be the major mechanism of corneal inflammation in onchocerciasis, and a TLR signaling molecule, IL-1 receptor associated kinase-2 (IRAK-2), regulates pathogenic Th17-cell development in S. mansoni infection.

Immediate Hypersensitivity Responses Immediate hypersensitivity responses are associated with the early and/or acute phase of infections with invasive helminth parasites, such as Ascaris, hookworm, schistosomes, or filariae. Patients do manifest symptoms suggestive of allergic reactivity, such as wheezing or urticaria. Furthermore, in clinical syndromes associated with Loa loa infection (with its angioedematous Calabar swellings), with tropical pulmonary eosinophilia, and with larva currens in strongyloidiasis, IgE-mediated reactions are thought to underlie these signs and symptoms.24 Anaphylaxis is a severe,

444

Part three  Host Defenses to Infectious Agents

life-threatening, generalized, or systemic hypersensitivity reaction, and is associated with IgE interaction with high-affinity IgE receptors on basophils and mast cells (Chapter 23). The risk of anaphylaxis in individuals with helminth infections can vary, depending on the parasite, tending to occur more frequently with echinococcosis or after Anisakis infection, while being extremely rare in most other helminth infections.

Wound Healing Recent studies have shown a close association of type 2 cytokine responses with many aspects of wound healing and repair.25,26 It has been proposed that the type 2 cytokine response has evolved to not only mediate resistance to helminth infection but also activate the wound healing apparatus to repair and reconstruct tissue, since tissue damage is intricately associated with helminth infections. Thus AAMs are intimately involved in this process as they produce MMPs, Arginase-1, insulin-like growth factor 1 (IGF1), VEGF, and TGF-β, which together promote myofibroblast activation, angiogenesis, epithelial cell turnover, and extracellular matrix deposition.

Lymphangiogenesis The anatomical changes in the architecture of lymphatics, which range from lymphangiectasia and granulomatous responses to the development of collaterals, suggest that active lymphatic remodeling involving endothelial cell growth, migration, and proliferation is an important feature of early lymphatic filarial disease. Live filarial parasites (and their excretory/secretory products) have been shown to induce activation, proliferation, and tube formation in lymphatic endothelial cells (LECs) and their differentiation into tubelike networks.27 This was found to be associated with significantly increased levels of MMPs and TIMPs. Recent studies have also implicated the VEGF family in lymphangiogenesis, with VEGF-C being associated with lymphedema and VEGF-A with hydrocele.23 Finally, TLR-mediated events are considered to be the main drivers of this angiogenic/ lymphangiogenic process in filarial disease.27

Carcinogenesis Infection with Opisthorchis viverrini, Clonorchis sinensis, and Schistosoma hematobium are classified as group 1 biological carcinogens (i.e., definitive causes of cancer). The former (liver fluke) is associated with cancer of the bile duct (cholangiocarcinoma) and cancer of the liver (hepatocarcinoma), and the latter is associated with carcinoma of the urinary bladder. The mechanisms of helminth-induced cancer include chronic inflammation, sustained cellular proliferation, modulation of the host immune system, reprogramming of glucose metabolism and redox signaling, induction of genomic instability and destabilization of tumor proteins, stimulation of angiogenesis, resistance to apoptosis, and activation of invasion and metastasis.28

Epileptogenesis Neurocysticercosis, caused by the larval form of Taenia solium, is the most common preventable risk factor for epilepsy worldwide and accounts for nearly 30% of all epilepsies in some endemic areas. The manifestations are variable, depending on the location, number, and size of the cysts in the central nervous system as well as the degree of accompanying inflammation, provoked by cyst degeneration, calcification, and/or perilesional edema.

MECHANISMS OF EVASION AND IMMUNE REGULATION BY HELMINTH PARASITES Helminths exert profound immunoregulatory effects on the host immune system with parasite antigen–specific immune suppression as well as more generalized levels of immune suppression. It has been shown that patients with schistosomiasis or filariasis have markedly diminished responses to parasite antigens and to some measurable attenuation in responses to bystander antigens and routine vaccinations. Thus host immunosuppression is usually antigen specific, whereas chronic infection can be associated with some spillover effects. Among the mechanisms utilized by parasites to avoid immune-mediated elimination are those of evasion—the use of sequestration, camouflage, and antigenic variation—and suppression, regulation, or blockade of immune effector pathways.

Parasite-Derived Factors Parasite-derived products play a very important role in host immune evasion.29,30 Parasite products, such as the schistosomesecreted proteins, alpha-1 and omega-1, promote Th2 differentiation. Alpha-1 (also known as IL-4–inducing principle of schistosome eggs [IPSE]), released by schistosome eggs, induces IL-4 release and degranulation by human and mouse basophils by cross-linking surface IgE. Omega-1 is a ribonuclease abundantly secreted by eggs, shown to condition DCs to drive Th2 polarization. Omega-1 binds to and is internalized by DCs in a mannose receptor–dependent process and then suppresses protein synthesis through degradation of messenger RNA (mRNA). Phosphorylcholine (PC) is a small hapten-like moiety present in the excretory/secretory products of many helminths, and one particular PC-containing molecule, called ES-62, from filarial worms has been shown to have a wide variety of immunomodulatory properties. Thus ES-62 can inhibit the proliferation of CD4 T cells and conventional B cells, decrease IL-4 and IFN-γ production, promote proliferation and IL-10 production by B1B cells, modulate complement activation, and condition APCs to drive Th2 differentiation with concomitant inhibition of Th1 responses. ES-62 has also been shown to exhibit bystander antiinflammatory activity in collagen-induced arthritis, rheumatoid arthritis, chemical contact sensitivity, lupus-associated atherosclerosis, ear inflammation, chronic asthma, and airway hyperreactivity. Helminths utilize glycans within glycoproteins and glycolipids, which mimic host glycans, to regulate host immune responses. In addition, these host-like helminth glycans can directly interact with host glycan–binding proteins, such as C-type lectin receptors and galectins, to shape innate and adaptive immune responses. Similarly, helminth lipids have also been implicated in immune modulation; schistosome phosphatidylserine induces DCs to polarize IL-4–producing T cells, whereas schistosome lysophosphatidyl serine induces DCs to induce IL-10–secreting Tregs. Helminth parasites utilize mechanisms involving cytokine mimicry and interference to establish chronic infection. Thus parasites produce cytokine- and chemokine-like molecules to interfere with the function of host innate immune products. The first helminth cytokines were found to be homologues of TGF-β expressed by B. malayi, and both schistosomes and filarial parasites express members of the TGF-β receptor family. Similarly, E. granulosus expresses a TGF-β ligand, and thus all helminth groups might have the potential to exploit TGF-β–mediated immune suppression. Various helminths, including B. malayi, produce homologues of macrophage migration inhibitory factors

CHAPTER 31  Immune Responses to Helminth Infection (MIFs), which are known to activate an antiinflammatory pathway through SOCS-1, a molecule involved in cytokine signaling. T. muris is known to express a homologue of IFN-γ, which binds to the IFN-γ receptor in vitro and induces signaling. As T. muris is expelled by IL-4, secretion of an IFN-γ–like protein can prolong its survival. Similarly, helminth parasites utilize chemokine- or chemokinereceptor like proteins to evade protective immunity. Ascaris suum is known to express a neutrophil chemoattractant with chemokine binding properties. S. mansoni eggs secrete a protein (S. mansoni chemokine-binding protein [smCKBP]) that binds the chemokines CXCL8 and CCL3 and inhibits their interaction with host chemokine receptors and their biological activity, resulting in suppression of inflammation.31 Similarly, B. malayi (and all of the other filariae sequenced to date) has been shown to express galectins that can bind host immune cells in a carbohydratedependent manner. Helminths secrete two major classes of protease inhibitors called cystatins and serpins, each with proposed immunomodulatory roles. Cystatins inhibit cysteine proteases (cathepsins and aspartyl endopeptidases) required for antigen processing and presentation and therefore inhibit T-cell activation. They also elicit the regulatory cytokine IL-10, leading to direct impairment of T-cell proliferation. The serpins are serine protease inhibitors, which can cause specific inhibition of the neutrophil proteinases cathepsin G and neutrophil elastase. Aspartic proteases from Ascaris lumbricoides have been shown to block efficient antigen processing that is dependent on proteolytic lysosomal enzymes. Other parasite products mediate their effect by blocking effector functions, including recruitment and activation of inflammatory cells and limiting the destructive potential of activated granulocytes or macrophages in the local extracellular milieu. For example, the host chemoattractant platelet-activating factor (PAF) is inactivated by a complementary enzyme PAF hydrolase secreted by N. brasiliensis. Eotaxin-1, a potent eosinophil chemoattractant, is degraded by metalloproteases from hookworms. A. caninum secretes a protein called neutrophil inhibitory factor, which binds the integrins CD11b/CD18 and blocks adhesion of activated neutrophils to vascular endothelial cells and also the release of hydrogen peroxide (H2O2) from activated neutrophils. N. americanus ES products also bind to host NK cells and augment the secretion of IFN-γ, which might crossregulate deleterious Th2 responses. Other modulators, such as prostaglandins, and other arachidonic acid family members, such as PGE2 and PGD2, are known to inhibit IL-12 production by DCs. Finally, helminths susceptible to oxidant-mediated killing express both secreted and membrane-associated enzymes, such as superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase, molecules that are thought to play a significant role in assisting parasite survival in inflamed tissues. Recently, a family of helminth defense molecules secreted by parasitic helminths has been shown to exhibit biochemical and functional characteristics similar to human antimicrobial peptides. These molecules can modulate innate cell activation by classic TLR ligands, such as lipopolysaccharide. It has been also been reported that parasitical helminths can produce exosomes and other secretory vesicles that facilitate the transfer of intracellular cargo. Exosomes derived from helminths have been shown to possess immunomodulatory capacity with the ability to modulate immune responses by altering the function of ILC2. In addition, secreted vesicles from B. malayi, O. volvulus, and S. mansoni have been shown to contain microRNAs that

445

can then act as messengers of communication between the parasite and the host cell. These exosomes (containing microRNA) can then enter the host cell and modulate host gene expression.32

Host-Related Factors

Regulatory T and B Cells Evidence for the involvement of Tregs in helminth-mediated downmodulation of the immune response has been accumulating in recent years33 (Chapter 18). IL-10 and TGF-β, both factors associated with Tregs, are elicited in response to helminth infections, and in vitro neutralization of IL-10 and TGF-β at least partially restores T-cell proliferation and cytokine production in lymphatic filariasis. Similar reversals of immunosuppression are observed in onchocerciasis and schistosomiasis, with IL-10 producing natural Tregs (nTregs) from egg-induced granulomas (in S. mansoni infection) being important for host survival. More recently, Tregs from patients with filarial infections have also been shown to express high levels of other suppressive molecules, such as CCL4, IL-29, LAG-3, and Foxo3. Moreover, filarial infection is associated with an expansion of T cells expressing the IL-10 superfamily cytokine members (IL-19 and IL-24), and inhibition of these cytokines results in increased Th1 and Th2 responses. Tregs play a vital role in limiting host pathology by downregulating harmful Th1/Th17 responses in filarial infection and schistosomiasis. In addition, low level Treg activity is essential for type 2 effector immunity to expel certain intestinal helminths, and transdifferentiation of Th17 cells into Tregs during helminth infection is important for resolution of inflammation. A number of studies have recently reported that B cells might have an active regulatory role in helminth infections. For example, purified B cells from mice infected with B. malayi produce IL-10 in response to filarial antigens. Similarly, IL-10 producing CD1dhi B cells are induced in both mice and humans and are suppressed after anthelmintic treatment. This induction occurs, in part, through a mechanism involving the ICOS-B7RP-1 pathway. Hyporesponsive T Cells Effector T cell responses can be turned off or modulated through a variety of mechanisms, including through cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1). Interestingly, increased expression of CTLA-4 and PD-1 has been demonstrated in filarial infections, and blocking of CTLA-4 can partially restore a degree of immunological responsiveness in cells from infected individuals. Moreover, T cells have decreased induction of T-bet, the Th1 master regulatory gene indicating a failure at the transcriptional level to differentiate into Th1 cells. Finally, T cells from individuals with filarial infection exhibit classic signs of anergy, including diminished T-cell proliferation to parasite antigens, lack of IL-2 production, and increased expression of E3 ubiquitin ligases. Similarly, anergic T cells are found in both humans and mice with F. hepatica infection and schistosomiasis; in the latter case, these T cells express high levels of the anergy molecule GRAIL (gene related to anergy in lymphocytes). Modulation of Apc Function DCs are the first APCs usually to encounter parasites, and helminth modulation of DC function has been well characterized.34 Filarial parasites induce downregulation of MHC class I and class II molecules, as well as cytokines and other genes involved in antigen presentation, thereby rendering DCs suboptimal in their ability to activate CD4 T cells. Schistosomes

446

Part three  Host Defenses to Infectious Agents

have similar effects on DCs, with subsequent Th2 polarization and inhibited responses to Th1-inducing TLR ligands. In addition, schistosomes modulate the activation of Nlrp3 (NLR family, pyrin domain containing 3) inflammasome and thus IL-β production. Excretory/secretory antigens produced by helminths can inhibit DC synthesis of proinflammatory cytokines, chemokines, and costimulatory molecules and promote DC production of the regulatory cytokines IL-10 and TGF-β. Helminth infection has also been shown to induce in vivo differentiation of a CD103CD11clo population of regulatory DCs, which are inefficient in priming effector T cells and instead favor the generation of Tregs. AAMs are able to markedly suppress target cell proliferation, as well as mediate repair of tissue that has been damaged by parasites. In addition, human filarial infection is associated with the expansion of the nonclassic monocyte subset, as well as an immunoregulatory monocyte subset. Helminth antigens can modulate MHC class II and CD80/86 expression on “antigenpresenting” basophils to induce the development of Th2 cells. Finally, a heterogeneous population of immature myeloid cells that share the common property of suppressing immune responses are termed myeloid-derived suppressor cells (MDSCs). Although these MDSCs have been well characterized in cancer immunology, their role in helminth infections is still being explored. Apoptosis Another mechanism of immune evasion is the ability of some helminths to induce host cell apoptosis (Chapter 13). Apoptosis has been described as a host regulatory mechanism in various helminth infections, including schistosomiasis, lymphatic filariasis, and onchocerciasis.

HELMINTHS AND THE MICROBIOTA Recent work has highlighted the importance of the microbiota in influencing host immunological and metabolic functions (Chapter 14). Helminths secrete a variety of products that can directly influence the composition and function of the microbiota, whereas changes in microbiota can have an impact on susceptibility to helminth infection, indicating that helminth–microbiota cross-talk can regulate a variety of host processes.35 Recently, it was reported that infection with helminths (H. polygyrus or T. muris) results in alteration of the composition of the microbiota, with expansion of Lactobacillacea and Enterobacteriacea in the gut, and that the removal of helminths resulted in restoration of the original composition of the microbiota. The consensus in humans is yet to emerge, with some studies showing an effect of helminth infection on microbial diversity and others failing to reveal any differences. Conversely, it has also been demonstrated that introducing higher level of certain commensal microbes enhances susceptibility, whereas removal of microbiota by antibiotics decreases susceptibility to helminth infections.

REGULATION OF ALLERGY, AUTOIMMUNITY, AND METABOLIC DISEASES IN HELMINTH INFECTION The hygiene hypothesis postulates that the stimulation of the immune system by microbes or microbial products protects from the development of inflammatory and atopic disorders36 (Chapter 1). Human studies have demonstrated that people living in areas endemic for helminth infections have a decreased reactivity to skin tests for allergens and milder forms of asthma.37 Experimental

animal models have revealed the protective effect of helminth infections against atopy and asthma. Several mechanisms have been proposed for the helminth-induced protection, the chief of which are the induction of Treg activity, regulatory B-cell activity, and immunosuppressive cytokines including IL-10 and TGF-β. Similarly, exposure to helminth parasites has been shown to prevent the onset of Th1-mediated diseases, such as multiple sclerosis (MS), diabetes mellitus, and Crohn disease in experimental animal models.37 Finally, recent studies in mice have shown that type 2 immunity, induced by helminth infection, can maintain adipose tissue homeostasis and promote adipose tissue beiging, protecting against obesity and metabolic dysfunction; that the immunomodulatory glycan LNFPIII, which is secreted by helminths, can alleviate hepatosteatosis and insulin resistance; and that there exists an inverse association between the presence of helminth infections and the prevalence of type 2 diabetes.

HELMINTH THERAPY FOR INFLAMMATORY DISEASES To date, two species of helminths have been tested as clinical treatment for therapy of inflammatory diseases: T. suis ova and infection with N. americanus. Currently, 28 clinical trials of helminth therapy in 10 autoimmune diseases and allergic or related conditions have been planned, started, or completed.38 These include Crohn disease, ulcerative colitis, MS, celiac disease, autism, plaque psoriasis, peanut and tree nut allergy, asthma, rhinoconjunctivitis, and rheumatoid arthritis. Although well tolerated, recent results suggested that this therapy did not achieve improvement in disease activity or remission rates in Crohn disease. Studies using N. americanus include fairly small trials with patients with Crohn disease or individuals with celiac disease (gluten allergy). Additionally, a few studies have been performed examining the effect of helminth therapy on asthma/allergy. The one minor success of helminth therapy in humans is in the treatment of MS, wherein small trials have successfully demonstrated lower relapses and lower magnetic resonance imaging (MRI) activity, prompting larger phase I or II trial involving either T. suis or N. americanus.

ON THE HORIZON Identification and synthesis of helminth products that can be useful as immune therapy in a variety of inflammatory disorders Deciphering the three way cross-talk among host immunity, helminths, and the microbiota Elucidation of the detailed mechanisms by which helminths manipulate immune responses to bystander antigens Development of clues to production of novel vaccine candidates to protect against not only helminth infection but also helminth-induced morbidity Combined approaches involving genomics, transcriptomics, proteomics, and metabolomics for assessment of host–helminth interactions

VACCINES AGAINST HELMINTH PARASITES Vaccines against helminth infections are a necessary tool for their elimination and eradication for several different reasons.39 Currently, a total of five human anthelmintic vaccines have advanced from discovery through manufacture and are now in phase I or II clinical testing.40 These include three Schistosome

CHAPTER 31  Immune Responses to Helminth Infection vaccine candidates: (i) S. hematobium Sh28GST (phase II); (ii) S. mansoni Sm-TSP-2 (phase I); (iii) S. mansoni Sm-14 (phase I); and two hookworm vaccine candidates: (i) Na-GST-1 (phase I); (ii) Na-APR-1 (phase I). In addition, the two O. volvulus vaccine candidates—Ov-103 and Ov-RAL2—and the S. mansoni candidate—Sm-p80—are in preclinical testing. With rapid advances in the parasite genomics and proteomics, as well the newer, better vaccine delivery systems offering more efficient and quicker assessment, the prospects for newer anthelmintic vaccines are excellent, although the potential lack of commercial markets imposes a significant impediment to their development. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Keusch GT. Immune responses in parasitic diseases. Part A: general concepts. Rev Infect Dis 1982;4:751–5. 2. Allen JE, Maizels RM. Diversity and dialogue in immunity to helminths. Nat Rev Immunol 2011;11:375–88. 3. Mishra PK, Palma M, Bleich D, et al. Systemic impact of intestinal helminth infections. Mucosal Immunol 2014;7:753–62. 4. Hammad H, Lambrecht BN. Barrier Epithelial cells and the control of type 2 immunity. Immunity 2015;43:29–40. 5. McKenzie AN, Spits H, Eberl G. Innate lymphoid cells in inflammation and immunity. Immunity 2014;41:366–74. 6. Kreider T, Anthony RM, Urban JF Jr, et al. Alternatively activated macrophages in helminth infections. Curr Opin Immunol 2007;19:448–53. 7. Grencis RK. Immunity to helminths: resistance, regulation, and susceptibility to gastrointestinal nematodes. Annu Rev Immunol 2015;33:201–25. 8. Noelle RJ, Nowak EC. Cellular sources and immune functions of interleukin-9. Nat Rev Immunol 2010;10:683–7. 9. Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol 2011;29:621–63. 10. Larkin BM, Smith PM, Ponichtera HE, et al. Induction and regulation of pathogenic Th17 cell responses in schistosomiasis. Semin Immunopathol 2012;34:873–88. 11. Bouchery T, Kyle R, Ronchese F, et al. The differentiation of CD4(+) T-helper Cell subsets in the context of helminth parasite infection. Front Immunol 2014;5:487. 12. Shen P, Fillatreau S. Antibody-independent functions of B cells: a focus on cytokines. Nat Rev Immunol 2015;15:441–51. 13. Harris N, Gause WC. To B or not to B: B cells and the Th2-type immune response to helminths. Trends Immunol 2011;32:80–8. 14. Klion AD, Nutman TB. The role of eosinophils in host defense against helminth parasites. J Allergy Clin Immunol 2004;113:30–7. 15. Karasuyama H, Mukai K, Obata K, et al. Nonredundant roles of basophils in immunity. Annu Rev Immunol 2011;29:45–69. 16. Galli SJ, Tsai M. Mast cells in allergy and infection: versatile effector and regulatory cells in innate and adaptive immunity. Eur J Immunol 2010;40:1843–51. 17. Allen JE, Sutherland TE, Ruckerl D. IL-17 and neutrophils: unexpected players in the type 2 immune response. Curr Opin Immunol 2015;34:99–106.

447

18. Anthony RM, Rutitzky LI, Urban JF Jr, et al. Protective immune mechanisms in helminth infection. Nat Rev Immunol 2007c;7: 975–87. 19. Finkelman FD, Shea-Donohue T, Goldhill J, et al. Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models. Annu Rev Immunol 1997;15:505–33. 20. Zandman-Goddard G, Shoenfeld Y. Parasitic infection and autoimmunity. Lupus 2009;18:1144–8. 21. Wynn TA, Thompson RW, Cheever AW, et al. Immunopathogenesis of schistosomiasis. Immunol Rev 2004;201:156–67. 22. Allen JE, Wynn TA. Evolution of Th2 immunity: a rapid repair response to tissue destructive pathogens. PLoS Pathog 2011;7:e1002003. 23. Pfarr KM, Debrah AY, Specht S, et al. Filariasis and lymphoedema. Parasite Immunol 2009;31:664–72. 24. Pritchard DI. The pro-allergic influences of helminth parasites. Mem Inst Oswaldo Cruz 1997;92:15–18. 25. Gause WC, Wynn TA, Allen JE. Type 2 immunity and wound healing: evolutionary refinement of adaptive immunity by helminths. Nat Rev Immunol 2013;13:607–14. 26. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity 2016;44:450–62. 27. Babu S, Nutman TB. Immunopathogenesis of lymphatic filarial disease. Semin Immunopathol 2012;34:847–61. 28. Fried B, Reddy A, Mayer D. Helminths in human carcinogenesis. Cancer Lett 2011;305:239–49. 29. Harnett W, Harnett MM. Helminth-derived immunomodulators: can understanding the worm produce the pill? Nat Rev Immunol 2010;10:278–84. 30. Hewitson JP, Grainger JR, Maizels RM. Helminth immunoregulation: the role of parasite secreted proteins in modulating host immunity. Mol Biochem Parasitol 2009;167:1–11. 31. Smith P, Fallon RE, Mangan NE, et al. Schistosoma mansoni secretes a chemokine binding protein with antiinflammatory activity. J Exp Med 2005;202:1319–25. 32. Coakley G, Buck AH, Maizels RM. Host parasite communications-Messages from helminths for the immune system. Mol Biochem Parasitol 2016;208:33–40. 33. Belkaid Y, Tarbell K. Regulatory T cells in the control of host-microorganism interactions. Annu Rev Immunol 2009;27:551–89. 34. Everts B, Smits HH, Hokke CH, et al. Helminths and dendritic cells: sensing and regulating via pattern recognition receptors, Th2 and Treg responses. Eur J Immunol 2010;40:1525–37. 35. Gause WC, Maizels RM. Macrobiota—helminths as active participants and partners of the microbiota in host intestinal homeostasis. Curr Opin Microbiol 2016;32:14–18. 36. Yazdanbakhsh M, Kremsner PG, van Ree R. Allergy, parasites, and the hygiene hypothesis. Science 2002;296:490–4. 37. Wilson MS, Maizels RM. Regulation of allergy and autoimmunity in helminth infection. Clin Rev Allergy Immunol 2004;26:35–50. 38. Fleming JO, Weinstock JV. Clinical trials of helminth therapy in autoimmune diseases: rationale and findings. Parasite Immunol 2015;37:277–92. 39. Bethony JM, Cole RN, Guo X, et al. Vaccines to combat the neglected tropical diseases. Immunol Rev 2011;239:237–70. 40. Hotez PJ, Strych U, Lustigman S, et al. Human anthelminthic vaccines: Rationale and challenges. Vaccine 2016;34:3549–55.

CHAPTER 31  Immune Responses to Helminth Infection

447.e1

MULTIPLE-CHOICE QUESTIONS 1. The immunity to helminths typically involves a T-helper 2 (Th2) cell response with what other component of the T-cell response? A. Th1 response B. Th17 response C. Regulatory T-cell response D. Th22 response 2. The classic antibody isotypes induced by helminths typically include: A. Immunoglobulin E (IgE) and IgG4 B. IgE and IgG3 C. IgE and IgD D. IgG1 and IgG2

3. These cells are a novel cell type recently shown to play a pivotal role in the initiation of immunity to helminth parasites: A. Natural killer (NK) cells B. Type 1 innate lymphoid cells C. Type 2 innate lymphoid cells D. Type 3 innate lymphoid cells

32  Approach to the Evaluation of the Patient With Suspected Immunodeficiency Javier Chinen, Mary E. Paul, William T. Shearer

Clinical immunologists are often consulted to evaluate patients for suspected immune defects, usually because such patients have an unusual frequency or severity of infectious illnesses. Indeed, immunodeficiency presents with increased susceptibility to infection but may also manifest with conditions that reflect dysregulation of the immune response, such as allergies, autoimmunity, or lymphoproliferation. Prompt diagnosis is essential to reduce the risk of organ damage caused by preventable severe infections. Primary immunodeficiencies (PIDs) are congenital diseases that might affect any aspect of the immune response and are often diagnosed in childhood. Examples of PIDs include severe combined immune deficiencies (SCIDs), complete DiGeorge syndrome, and chronic granulomatous disease (CGD). In contrast to PIDs, secondary immunodeficiencies (Chapters 38, 39) present at any age, as a result of a wide variety of factors that affect the immune function, such as environmental factors, metabolic disease, anatomical abnormalities, or infectious agents. The most known and significant secondary immunodeficiency is caused by human immunodeficiency virus (HIV). The assessment of a patient for PID should include history and physical examination to direct immunology laboratory testing to confirm a diagnosis.

EPIDEMIOLOGY—PRIMARY IMMUNODEFICIENCIES ARE NOT UNCOMMON Estimates of the incidence of PIDs or congenital immunodeficiencies varies from selective immunoglobulin A (IgA) deficiency, a relatively common condition, (1/223–1/1000 people)1 to the less common SCID. Recent analysis from 11 state programs established for universal newborn screening for T-cell deficiencies in the United States reported an incidence of SCID of 1/58 000 live births, comparable with childhood leukemia.2 A householdbased telephone survey suggested that 1 in 1200 persons in the United States has been diagnosed with a PID.3 Although significant progress has been made to stop the acquired immunodeficiency syndrome (AIDS) epidemic, HIV infection continues to be the most prevalent cause of immunodeficiency worldwide, with an estimated 36.9 million people living with HIV.4

PRIMARY VERSUS SECONDARY IMMUNODEFICIENCY Because of their common occurrence, acquired and nonimmunological causes for recurrent infections should be first considered in the differential diagnosis of the patient with a suspected immune disorder. Acquired conditions that might

KEY CONCEPTS Secondary Immunodeficiencies Immunodeficiency is often secondary or transient, caused by nonimmune factors, including: • Previous use of high-dose steroids, or other immunosuppressive medications • Previous use of monoclonal antibodies (mAbs), such as rituximab (anti-CD20) • Immunoglobulin losses via the gastrointestinal or urinary tract • Severe illness requiring critical care • Malnutrition • Human immunodeficiency virus (HIV) infection

increase the frequency of infections include allergic inflammation, HIV infection, and the use of immunosuppressive drugs. Examples of nonimmunological conditions include those that disrupt the usual mucosal clearance mechanisms, such as posterior urethral valves or urethral stenosis in a patient with recurrent urinary tract infection, or cystic fibrosis in individuals who have recurrent sinusitis or pneumonia and/or diarrhea. Disruption of natural barriers can lead to increased risk of infection, for example, in patients with skin lesions caused by eczema or burns or in individuals with cerebrospinal fluid (CSF) leaks following bony injury to the cranium. Patients presenting with low Ig levels might have loss of antibodies as a result of a protein-losing enteropathy, nephropathy, or massive protein loss through skin, such as in severe eczema or burns.5 Secondary immunodeficiency can also result from other conditions affecting cell metabolism (e.g., malnutrition, diabetes mellitus, and sickle cell anemia) or could be secondary to predictable or idiopathic adverse effects of drugs. Optimal management of these conditions often results in improved immunity.

EVALUATING PATIENTS FOR IMMUNODEFICIENCY The evaluation of patients for immunodeficiency is based on a careful assessment of patient history and physical examination, with very limited initial laboratory testing. With this information, the clinician can often tell patients (or parents) whether their (or their child’s) immune system is significantly compromised. The immunologist must take on a role of counselor and advisor of patients and explain the many factors that may result in increased frequency of infections (Fig. 32.1). The limitations of available clinically validated testing need to be considered, as such tests may not be sensitive or specific to identify uncommon

451

452

Part Four  Immunological Deficiencies

1. 2. 3. 4.

History Number, type, course of infection Family history Age of onset, sex Environmental exposure to infection

Suspicious finding or red flag

General screening of immunity 1. CBC, differential, platelets 2. IgG, IgA, IgM, IgE levels 3. Baseline antibody titers: To immunizations (e.g., tetanus, pneumococcus) Isohemagglutinins

Physical examination 1. Growth and development 2. Associated abnormalities (i.e., DiGeorge facies, rash, telangiectasia) 3. Presence or absence of lymphoid tissue

Suspicious finding or red flag

Suspicious finding or red flag

Disease-specific testing of the immune system

FIG 32.1  Evaluation of Immunity in a Patient for Immunodeficiency Starts With a Careful History and Physical Examination. Clues in the history for further evaluation include an excess of respiratory tract infections, of unusual severity; life-threatening infections; infections with unusual organisms; a family history of immunodeficiency. The outlined laboratory tests provide an adequate screen of immunity in a patient with no specific findings.

immune defects, such as impairments in phagocyte function other than oxidative burst deficiency. The medical history and initial laboratory testing often provide clues that suggest a specific immune disorder, and the examination of specific component of the immune response or a diagnostic test for specific immunodeficiencies may be indicated. For example, an increased frequency of infections affecting only the respiratory tract and caused by encapsulated bacteria direct the exploration to defects in humoral immunity and complement; in contrast, a history of Aspergillus pneumonia would suggest neutropenia and CGD. According to the severity of the illness, clinical immunologists may recommend an initial exploration of the major components of the immune system: lymphocyte subset distribution, antibody responses, T-cell function, phagocyte oxidative burst, and the complement system.

EXPLORING THE MEDICAL HISTORY Age and Environment The differential diagnosis of immunodeficiency varies with the age of onset of symptoms. Pediatric patients are more likely to present a PID than a secondary immunodeficiency. Infants from birth to 3 months of age have maternal Igs acquired through the placenta, unless they were born prematurely. Therefore deficiencies in the immune system at this age presenting with frequent infections most probably result from severe deficiencies in other immune components, such as neutrophils, complement components, or T cells. Older patients might present with increased risk of infections secondary to comorbid conditions, such as allergic inflammation or diabetes mellitus, or to a normal decline of immune responses, a process known as immunosenescence (Chapter 38).6 Environmental conditions can influence the risk of infection. Infants frequently exposed to other infants with infections, such as in the setting of a daycare facility, have more infectious illnesses compared with those who are not exposed. By inducing an inflammatory response of the respiratory mucosa, passive cigarette smoke inhalation also predisposes to infections, including otitis media, pneumonia, and bronchitis.7 The hygiene practices of the patient, caregivers, and family members have an impact on the frequency of infections, such as impetigo and furunculosis. For patients with allergies,

exposure to indoor allergens, such as dust mites and molds, often worsens mucosal congestion and increases the risk of sinusitis and otitis media.

Immunization and Previous Infections The immunization history provides significant clues, since the efficacy of vaccines depends on intact immunity. An incomplete schedule of immunizations easily explains the incidence of preventable diseases. A history of an adverse reaction to a live viral vaccine is suspicious for immunodeficiency, as infants with T-cell defects, B-cell defects, and combined T- and B-cell defects are susceptible to potentially fatal or severe infections from live attenuated vaccines. These infections include measles and chicken pox pneumonitis, rotavirus vaccine-induced diarrhea,8 and lymphadenitis caused by the Bacille Calmette-Guérin (BCG) vaccine.9 Historical information regarding the frequency, type, and severity of illnesses and infections should be sought. Individuals with immunodeficiency may have infections with unusually prolonged courses or unusual severity or that may present as unexpected complications (Fig. 32.2). Recurrent infections that involve multiple sites are more suspicious for immune deficiency than those involving a single site. Also suggestive of immune compromise are severe and invasive infections, such as recurrent pneumonia, meningitis, sepsis, septic arthritis, osteomyelitis, or abscess and infections with organisms of low pathogenicity in normal individuals, such as Candida albicans or Pneumocystis jiroveci (Table 32.1) Patients with antibody deficiency disorders tend to present with infections caused by extracellular pyogenic organisms, such as Haemophilus spp., Pneumococcus spp., and Streptococcus spp. In contrast, patients with defects in cell-mediated immunity are more likely to also present with recurrent infections with viruses, fungi, protozoa, and mycobacteria. Furthermore, infections by catalase-positive bacteria, such as Serratia marcescens, may indicate a possible neutrophil oxidative burst defect. Recurrent neisserial infections may be found in individuals deficient in the terminal complement components. A relatively normal incidence of infections followed by a sudden occurrence of repeated infections in an adult or adolescent suggests a secondary immunodeficiency, including HIV infection.

CHAPTER 32  Approach to the Evaluation of the Patient With Suspected Immunodeficiency

453

TABLE 32.1  Clinical Clues of Significance for the Diagnosis of Immunodeficiencies

Recurrent or severe bacterial infections Systemic mycobacterial infections Recurrent or severe viral infections Invasive fungal infections Opportunistic infections Failure to thrive Autoimmunity Lymphoma

T-Cell Function Defect

Antibody Defect

Granulocyte Defects

Complement Defect

X X X X X X X X

X

X (catalase-positive)

X (encapsulated bacteria)

X

X X X X X

X X X (CVID)

IFN-γ/IL-12 Defect X X X

X

CVID, common variable immunodeficiency; IFN, interferon; IL, interleukin.

TABLE 32.2  Nonimmunological Clinical

Findings Present in Immunodeficiency Syndromes Nonimmunological Clinical Finding Small platelets, thrombocytopenia, eczema Conical teeth, ectodermal dysplasia

FIG 32.2  Computed Tomography (CT) Scan in an Infant With Chronic Granulomatous Disease (CGD). Multiple nodular opacities are seen throughout both lung fields as a result of fungal pneumonia in an infant with CGD. (From Seeborg FO, et al. A 5-week-old HIV-1-exposed girl with failure to thrive and diffuse nodular pulmonary infiltrates. J Allergy Clin Immunol 2004; 113: 629, with permission from Elsevier.)

Comorbid Conditions Apart from frequent infections, other important aspects of the history and physical examination may suggest congenital syndromes associated with immune defects (Table 32.2). Neutrophil adhesion defects lead to delayed (beyond 2 weeks of age) umbilical cord separation because of omphalitis and poor wound healing. Patients with DiGeorge syndrome are usually diagnosed in the neonatal period with hypocalcemic seizures associated with hypoparathyroidism, velopalatal insufficiency, or cardiovascular malformations, rather than with recurrent infections. Infants with Wiskott-Aldrich syndrome (WAS)—immunodeficiency, thrombocytopenia, and eczema—present with petechiae and bruises similarly in the neonatal period. Obtaining a history of atopic disease is helpful. Atopic dermatitis has been associated with high risk of infection, including nondermatological infections.10 For some patients with allergic rhinitis, management of allergies reduces the frequency of upper respiratory tract infection. In addition, it is important to inquire for a history of recurrent wheezing. At times, an initial history of recurrent pneumonia is, in fact, secondary to reactive airways disease or asthma. Other significant conditions include renal disease causing proteinuria

Delayed shedding of primary teeth, frequent fractures, hyperextensibility Cerebellar ataxia, telangiectasia Hypoparathyroidism, conotruncal heart defect, velopalatal insufficiency Short limbs Microcephaly

Silvery hair, albinism Pectum carinatum, skeletal dysostosis, pancreatic insufficiency

Immunodeficiency Wiskott-Aldrich syndrome (WAS) Nuclear factor κB (NF-κB) essential modulator (NEMO) defect Autosomal dominant hyper-IgE syndrome (AD-HIES) Ataxia–telangiectasia (AT) DiGeorge syndrome

Cartilage-hair hypoplasia DNAse IV–deficient severe combined immunodeficiency (SCID), Nijmegen syndrome Pigment dilution disorders Shwachman-Bodian-Diamond syndrome (SBDS)

or enteropathies that result in protein losses and secondary hypogammaglobulinemia.

Use of Medications Use of particular drugs might also cause immunodeficiency, which could be predictable, such as the use of rituximab (anti-CD20 antibody) resulting in B-cell depletion and potential antibody deficiency, or idiopathic, such as hypogammaglobulinemia that might develop with the use of anticonvulsants.11

Family and Social Histories Family history is essential in the evaluation of suspected immunodeficiency. A history of early infant deaths and possible consanguinity should be sought. A clear pattern of inheritance may be found to define an X-linked, autosomal dominant, or autosomal recessive genetic syndrome. Many of the most common PIDs have X-linked inheritance patterns. Family members of patients with immunodeficiencies might also have a history of autoimmune disease or of connective tissue disease. Familial cases of selective IgA deficiency and common variable immunodeficiency (CVID) have been reported, and a susceptibility

454

Part Four  Immunological Deficiencies

trait can sometimes be traced back to many generations. A social history should be obtained for risk factors associated with increased risk of acquiring HIV infection. Socioeconomic factors often determine malnutrition, known to be of a significant impact on immune function.12

KEY CONCEPTS Genetic Immunodeficiencies • Diagnosis of genetic immunodeficiencies require high clinical suspicion and confirmation with molecular studies. • In a minority of cases, genetic immunodeficiencies might not present with all clinical elements of classic descriptions. For example, patients with Bruton agammaglobulinemia might have residual B-cell and immunoglobulin production.

PHYSICAL EXAMINATION FINDINGS The physical examination might provide findings that indirectly address the immune system; for example, scarred bilateral tympani suggest recurrent ear infections. More commonly, patients with immunodeficiency might otherwise look like normal individuals, unless severe infections had produced an organ damage or had delayed growth and development. However, attention to details in the physical examination may supply important clues that suggest immune dysfunction. In a normal child, a paucity of lymphoid tissue, such as tonsils and lymph nodes, might reflect impaired development resulting from immune deficiency. This is especially seen in patients with X-linked agammaglobulinemia. Certain physical findings are suggestive of syndromic immunodeficiencies, such as with telangiectasia over the bulbar conjunctivae and face with or without ataxia in ataxia–telangiectasia (AT); chronic eczema and delayed shedding of primary teeth in hyper-IgE syndrome (HIES); severe eczema in immunodeficiency, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome, and WAS; chronic periodontitis in defects of the neutrophils; or silvery hair, pale skin, and photophobia in Chediak-Higashi syndrome. Investigation for Shwachman-Bodian-Diamond syndrome (SBDS) should be considered in patients with neutropenia, especially if they also present with skeletal dysplasia. Patients with DiGeorge syndrome and nuclear factor-κB (NF-κB) essential modulator (NEMO) deficiency present with characteristic facies. Children with severe immune defects are small for their age, with growth delay secondary to recurrent infections. Hepatosplenomegaly and diffuse lymphadenopathy might suggest HIV infection or a disorder of immune dysregulation. Children with leukocyte adhesion defect (LAD) can present with severe gingivostomatitis and dental erosion as a consequence of abnormal leukocyte function (Fig. 32.3). Multiple scars from skin abscesses might suggest neutrophil defects, and scarred tympani with reduced hearing might indicate a history of recurrent otitis media, which can be associated with antibody deficiency.

LABORATORY TESTING FOR IMMUNE FUNCTION Results from commonly ordered tests might provide with a great deal of information about the immune system. The complete blood count (CBC) with differential and platelet determination is ordered to quantitate the total white blood cell (WBC) count and total numbers of neutrophils, lymphocytes, eosinophils, and platelets. Abnormal counts should be determined by using

FIG 32.3  Severe gingivostomatitis and dental erosion in a 2-yearold child with leukocyte adhesion defect (LAD). (Courtesy of Dr. D.C. Anderson.)

age-specific ranges. Leukocytosis, neutropenia, lymphopenia, and abnormalities in WBC morphology can be detected from this test. Persistent neutrophilia might suggest LAD. Anemia may be present in children with chronic disease. Platelet counts may be abnormally low in children with poor bone marrow function or autoimmune disease, and platelets will be reduced in number and morphologically small in children with WAS. Chemistry panels, including serum liver enzymes levels, might suggest organ compromise as a result of infections or autoimmunity associated with immunodeficiency. Low protein levels suggest malnutrition and conditions associated with protein losses, which may cause hypogammaglobulinemia. Examination of the posteroanterior and lateral chest radiographs to look for a thymic shadow can be helpful because its absence suggests impaired T-cell development. This is especially useful in infants because the thymus mass normally involutes with age. In addition, the thymus may shrink in response to such stresses as surgery, infection, or high-dose steroid treatment. HIV infection can be ruled out by screening with measurement of anti-HIV antibodies, by the enzyme-linked immunosorbent assay (ELISA) or the rapid HIV test. In those individuals with suspected humoral immunity defect and in children younger than 18 months of age, a polymerase chain reaction (PCR)–based test to detect HIV viremia should be performed to avoid falsenegative results and confounding maternal anti-HIV antibodies, respectively.

Immunology Testing Specific immunological testing is guided by clues obtained from the history and physical examination and common screening laboratory tests. Serum Immunoglobulin Levels The levels of IgG, IgA, IgE, and IgM can be measured in serum. The IgA level is especially helpful in that it is low in all permanent types of agammaglobulinemia and in selective IgA deficiency.

CHAPTER 32  Approach to the Evaluation of the Patient With Suspected Immunodeficiency KEY CONCEPTS Diagnosis of Immune Deficiencies Features of Congenital Antibody Deficiencies • Free of infections until 6–9 months of age, when maternal antibodies that passed through the placenta to infant are below protective levels • Severe infections with bacterial organisms, especially sinusitis, otitis media, and pneumonias caused by encapsulated bacteria, such as Streptococcus pneumoniae Features of Congenital T-Cell Immunodeficiency • Onset of thrush, diarrhea, and failure to thrive in the first months of life • Severe infections with opportunistic microorganisms, such as Pneumocystis jiroveci, Candida albicans, adenovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV). “BCG-itis” in areas where Bacille CalmetteGuérin (BCG) immunization is mandatory • Absolute and relative lymphopenia Screening Tests for Suspected Immunodeficiency • Evaluation for neutropenia, lymphopenia, thrombocytopenia, and/or small platelets • Immunoglobulin levels and specific antibodies to childhood immunizations • Lateral chest radiography in infants for thymus shadow • Consider flow cytometry to quantify T cells, T-cell subsets, B cells, and natural killer (NK) cells (especially in infants) • Measurement of CH50 activity • Test for oxidative burst in phagocytes

IgE level measurement is of significance for the diagnosis of HIES. Serum IgG subclasses levels can be determined. However, rather than using IgG subclass levels to screen for immunodeficiency, they are best utilized when patients have clinical conditions associated with specific antibody deficiencies but normal total IgG levels. In some of these patients, IgG subclass deficiency, particularly IgG2 and IgG3 deficiencies, might be present. IgG2 subclass deficiency has been linked with selective IgA deficiency and deficiency of antipolysaccharide antibodies. IgA subclass low levels, IgA1, IgA2, have not been associated with a specific immune defect, and there is no validity for measuring these. The variation of normal ranges of human serum Igs with age is an important consideration in children, since IgA and IgG subclass levels may not reach normal adult reference ranges until 6 years of age.1 B-Cell Function: Specific Antibody Production To properly assess B-cell function, specific antibody production must be measured. Patients with normal Ig and Ig subclass levels might exhibit deficient antigen-dependent antibody responses. An initial screen of antibody production may involve the quantification of isohemagglutinins. Isohemagglutinins occur in all individuals except those with blood type AB; isohemagglutinins are natural IgM antibodies to polysaccharide blood group antigens A and/or B, which are not expressed in the red blood cells (RBCs) of the patient tested. Individuals form isohemagglutinins as a result of environmental exposure to ubiquitous antigens that share epitopes with blood antigens. Children less than 1 year of age do not reliably have measurable serum isohemagglutinins because of the limited exposure to the environment. A patient with blood type A should have anti-B IgM; patients with blood type B should have anti-A IgM; and patients with blood type O should have both anti-A and anti-B IgM. These antibodies are

455

normally present in titers greater than 1 : 10; individuals with poor antibody production may have low or absent titers. Specific IgG antibody production can be measured following immunization with protein antigens, such as toxoids derived from Tetanus and Diphtheria organisms, and polysaccharide antigens, such as those produced by pneumococci and Haemophilus influenzae. For pneumococcal immunization, there are two vaccines that need to be differentiated. The conjugated vaccine containing 13 pneumococcal serotypes (PREVNAR13, Wyeth) is currently included in the universal schedule of immunizations for infants and toddlers and induces a robust, T cell–dependent immune response. The unconjugated 23-valent pneumococcal polysaccharide vaccine (Pneumovax, Merck) is available for immunization to adults and children aged 2 years and older. The immune response for this vaccine is considered less dependent on T cells and also less lasting than the conjugated vaccine. The pneumococcal antigen challenge using the unconjugated vaccine is not recommended for children under 2 years of age because healthy children are not thought to reliably respond to the unconjugated pneumococcal antigen at this age. However, this view has been challenged by data showing that 1-year-old children produce normal antibody responses to this unconjugated vaccine.13,14 Normal antibody responses are usually demonstrated with an over twofold rise in specific antibody levels within 2–3 weeks for protein antigens and within 4–6 weeks for polysaccharide antigens.15 Patients with agammaglobulinemia are expected not to produce antibody responses, whereas others, such as those with IgG2 subclass deficiency and normal levels of total IgG, may only have difficulty with antibody production following immunization with polysaccharide antigens. Patients with selective IgA deficiency, alone or with transient hypogammaglobulinemia of infancy, have normal specific IgG antibody production, by definition. The pneumococcal serotypes included in the current conjugated antipneumococcal vaccine, serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, were estimated to be responsible for approximately 90% of invasive pneumococcal disease in children less than 5 years of age worldwide.16 Previous immunization with the conjugate vaccine does not preclude use of the unconjugated pneumococcal polysaccharide vaccine. The 23-valent polysaccharide vaccine provides the potential for stimulation and measurement of a protective immune response to additional 11 serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, 33F) not included in the conjugated vaccine. Testing for antibodies against serotypes not included in the two vaccines and comparing the antibody titers in the pre- and postimmunization blood samples helps in the assessment of specific increase of particular antiserotype antibody titers as a response to the vaccine administration. Evaluation of Cellular Immunity Cellular immune function had been historically screened with the use of the delayed-type hypersensitivity (DTH) skin tests. DTH reactions occur 48–72 hours after antigen exposure, although antigens used to test DTH can occasionally produce immediate hypersensitivity reactions. The clinical response and time of observation were to discriminate between immediate and delayed reactions. DTH reactions involve the production of local edema and vasodilation as a result of inflammatory cytokines secreted by antigen-specific T cells, followed by lymphocyte infiltration and maximal induration 48 hours after antigen intradermal injection of an antigen. Generally, DTH skin tests are performed using vaccine antigens or microbial agents to which the patient

456

Part Four  Immunological Deficiencies 103

A1

103

A2

CD4 PE

CD4 PE

101

A3

A3

A4

101

100

A4

100 A

A2

102

102

100

A1

101

102

100

103

CD3 FITC

B

101

102

103

CD3 FITC

FIG 32.4  Histograms of Fluorescently Stained Lymphocytes. The quadrant of interest, A2, shows lymphocytes that are positive for labeling with both fluorescein isothiocyanate (FITC)–tagged monoclonal antibodies specific for CD3 and phycoerythrin (PE)–tagged monoclonal antibodies (mAbs) specific for CD4. The histogram on the left shows normal fluorescence as a result of CD4 T lymphocytes present in quadrant A2. The histogram on the right shows absence of CD4 T lymphocytes in an infant with SCID.

has had previous exposure. Commonly used antigens include tetanus toxoid, mumps, and extracts from Candida albicans and Trichophyton spp. The purified protein derivative (PPD), or tuberculin, can serve as a negative control in most patients in developed countries with a low incidence of tuberculosis. Likewise, a positive PPD result indicates sensitivity to mycobacteria as well as robust cellular immunity. Virtually all children and adults with previous exposure should respond to at least one antigen in a panel of tetanus toxoid, mumps, and C. albicans. Anergy, or nonresponse to the antigen following previous exposure, may indicate a cellular defect. A nonresponder should be retested with an in vitro lymphocyte evaluation. Lymphocyte subset enumeration. Quantitation of B- and T-cell subsets narrows the differential diagnosis and provides evidence for the diagnosis of combined, cellular, or antibody immunodeficiency (Chapters 34, 35, 93, 94, 95,-96). Both T and B cells can be identified and labeled by using flow cytometry and fluorescent monoclonal antibodies (mAbs) (Chapter 92). T-cell enumeration involves the use of a pan–T cell mAb specific for CD3. The CD4 marker serves as identification for T-helper (Th) cells. CD8 marker characterizes cytotoxic T cells. B cells can be identified by using mAbs against the cell surface markers CD19 or CD20. Natural killer (NK) cells can be identified by using mAbs against CD16 and CD56. Specialized clinical laboratories are available to measure lymphocyte markers of importance to particular diseases; for instance, the proportion of αβ T-cell receptor (αβTCR) and γδTCR double-negative CD3+ T cells are of relevance in the diagnosis of autoimmune lymphoproliferative syndrome (ALPS). T cell subsets can be characterized as naïve or activated based on the expression of CD45RA and CD45RO antigens. B-cell panels and NK-cell panels.  These panels have been designed to characterize the maturation stage of these cells and

provide support for the diagnosis of an specific immunodeficiency. For example, the proportion of class-switched B cells has a predictive value for autoimmune and granulomatous complications in CVID. In flow cytometry, the fluorescence intensity corresponding to cells labeled with each specific antibody is obtained (Fig. 32.4) and the percentage of the specific lymphocyte subset can be estimated. A reference range is available for each subset defining normal values as those whose values fall between the fifth and 95th percentages for this population. Separate ranges should be used for children because infants and children generally have higher absolute numbers of T-cell subsets and higher percentages of CD4 T cells (Fig. 32.5 and Appendix 2). Nonimmune factors, such as age, gender, and adrenocorticoid levels, influence the expression of blood lymphocyte subset populations. Therefore interpretation of lymphocyte phenotyping should take into consideration the clinical status of the patient. For example, transient moderate lymphopenia with predominance of T cells and NK cells might be seen in patients admitted to intensive care units. HIV infection causes progressive depletion of CD4 T cells. These abnormalities resolve when the patient’s condition improves. Lymphocyte functional analysis.  To test lymphocyte function in the laboratory, mitogen- and antigen- induced lymphocyte proliferation or transformation studies are performed (Chapter 93). For these studies, lymphocytes are stimulated to proliferate involving new DNA synthesis and cell division. Lymphocytes from immunized or previously exposed individuals will normally proliferate in response to antigens to which they are sensitized. This response in vitro correlates with the in vivo DTH response. Mitogens, such as concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), stimulate proliferation of normal T cells, as can allogeneic histocompatibility antigens when leukocytes from two donors are mixed in culture. Proliferation

CHAPTER 32  Approach to the Evaluation of the Patient With Suspected Immunodeficiency P1009: CD4 8000

Counts

6000 4000 2000 0 0

5

10

15

Age (years)

FIG 32.5  Change in Distribution of Peripheral Blood CD4 T-Cell Subsets With Age in Healthy Children. Scatter plot indicates peripheral blood CD4 T-cell counts (cells/µL) by age, with lowest curves in healthy children from birth to 18 years of age. (From Shearer WT, et al. Lymphocyte subsets in healthy children from birth through 18 years of age: The Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003; 112: 973, with permission from Elsevier.)

of lymphocytes can be evaluated by the demonstration of cell division or by increased DNA synthesis reflecting this cell process. Increased DNA synthesis is monitored by the incorporation of radiolabeled nucleotides, usually tritiated thymidine, in culture media. A measure of the amount of radioactivity in the cells correlates with DNA synthesis. Other assays to assess mitogen-induced cell proliferation measure deoxybromouridine incorporation, change in pH, or adenosine triphosphate (ATP) concentration of the culture media. These assays are being increasingly used as surrogate markers of cellular immunity; however, a comparison with the traditional assay based on radiolabeled nucleotide is not available. Of note, a flow cytometry assay that measures cell division with the use of carboxyl fluorescein succinyl ester (CFSE), a fluorescent compound that distributes evenly in cells and specific antibodies, is also increasingly used in clinical immunology.17 CFSE is distributed equally in dividing cells, and each progeny cell has half the fluorescence intensity of CFSE compared with the parent cell, providing the basis to identify these dividing cells. After mitogen or antigen stimulation, mononuclear cells can be stained with specifically labeled antibodies, allowing the identification of cell subsets that proliferate. Phagocytes The laboratory evaluation of a patient with a suspected phagocyte deficiency (Chapters 22, 94) should always begin with a CBC. Neutropenia is the most frequently encountered disorder of the phagocyte system.18 Neutrophilia, at values exceeding those associated with acute infection, is a common finding in LAD type 1 (LAD-1). Abnormalities of WBC function involve difficulty with adherence, locomotion, deformability, recognition, attachment, engulfment, phagosome formation, phagocytosis, degranulation, microbial killing, and elimination of engulfed material. Clinical assays to evaluate neutrophil function are limited in number. CGD is diagnosed by demonstrating absent or markedly reduced oxidase activity in neutrophils in response to stimulation.

457

Oxidase activity can be detected by a flow cytometry assay measuring the oxidation of dihydrorhodamine (DHR) 123 in phagocytes, resulting in fluorescent rhodamine-123.19 The nitroblue tetrazolium (NBT) test measures oxidative burst activity as well, but it is a more subjective test and can miss the diagnosis of CGD. For patients with suspected LAD-1 deficiency, neutrophils are labeled with mAb directed against the adhesion molecule CD11/CD18 heterodimer. Absence of fluorescence intensity indicates lack of expression of the adhesion molecule. In addition, an increase of fluorescence intensity after stimulation can be documented in normal individuals, indicating the normal upregulation of this molecule after cell activation.20 Other laboratory techniques used to identify phagocytic defects include assays for chemotaxis and bactericidal activity. A major pitfall for neutrophil studies is the spontaneous cell activation that might occur in vitro when cells are not tested within a few hours of when the sample was drawn, resulting in artifactual values that might falsely suggest poor function. Complement Laboratory tests for complement components include tests for functional activity of the classical pathway with a CH50 assay and the alternative pathway with an AH50 assay, as well as immunochemical methods to measure complement component levels.21 The CH50 evaluation tests the ability of fresh serum from the patient to lyse antibody-coated sheep erythrocytes. This reflects the activity of all numbered components of the classical complement pathway, C1–C9, and terminal components of the alternative complement pathway. A total deficiency of one of the classical complement pathway components will result in a CH50 assay approaching zero (Chapter 21). Patients with complement deficiency are rare, and complement test abnormalities are often transient because of increased consumption or activation. It is usually recommended that that in case of an abnormal result, the complement test be repeated if the sample was taken when the patient had an acute illness. Quantitative tests for components C3 and C4 are utilized in testing for complement deficiencies and for evaluation of complement activation (Chapter 21). Innate Immunity: Interferon-γ Levels, Toll-Like Receptor Assay The importance of the many components of innate immunity are increasingly recognized, as single gene defects in this immune compartment have been found to cause susceptibility to specific infections.22 For example, patients with defects in the proteins that are part of the interferon-γ (IFN-γ) receptor may have elevated serum IFN-γ levels, even when there is no infection to explain these levels. The IFN-induced response associated kinase 4 (IRAK4) defect, observed with susceptibility to pneumococcal infection, might be accompanied with abnormal Toll-like receptor (TLR) assay responses. It should be noted that the clinical value of most of these innate immunity tests as screening or diagnostic tools for immune defects has not been clearly established. The testing of lymphocyte apoptosis in a patient who may have ALPS and the evaluation of NK-cell function for suspected familial hemophagocytic lymphohistiocytosis are examples of specific functional assays that suggest immunodeficiency syndromes. Many patients with increased frequency of infections may not have abnormal results in clinically available immunological testing, which may not give clear evidence of a secondary etiology in the medical evaluation. In these difficult cases, referral

458

Part Four  Immunological Deficiencies

to tertiary care and research centers for investigation of rare diseases is recommended.

MOLECULAR TESTING FOR PRIMARY IMMUNE DEFECTS Molecular testing for specific PIDs is available through commercial and research laboratories.23 Biochemical and genetic testing should be considered. If autosomal recessive SCID is suspected, the adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) enzyme activities in the RBCs should be determined. White blood cells must be used to measure the activity of these enzymes in recently transfused individuals, since donor RBCs will elevate the enzyme activity in deficient patients. AT has the consistent laboratory finding of elevated alpha-fetoprotein (AFP) levels along with variable abnormalities in B- and T-cell function. Nearly 300 defective genes and gene products have been identified to result in congenital immunodeficiency syndromes.24 In these cases, the diagnosis can be confirmed with molecular genetic analysis (Chapter 33). For example, gene mutations in BTK leading to the absence of Bruton tyrosine kinase (BTK) results in arrest of B-cell development at the pre–B cell stage in congenital X-linked agammaglobulinemia. Similarly, abnormal T-cell development leading to SCID results from mutations in at least 15 genes, including IL2RG and JAK3. Patients with gene mutations that may result in disease but have not been investigated need to be carefully evaluated to demonstrate the pathogenic nature of the genetic change. Some genetic changes do not have clinical significance and are known as single nucleotide polymorphisms (SNPs). Standard protocols using Southern, Northern, and Western blot analyses, PCR analysis, and DNA sequence analysis are helpful to identify affected patients, affected fetuses prenatally, and carriers of genetic mutations (Chapter 96). Most recently, the use of whole exome sequencing for immunodeficiency syndromes has facilitated the identification of new genes causing immunodeficiencies by examining all known gene exons without bias, and simultaneously. This methodology for diagnosis is particularly helpful when the clinical presentation does not match any of the already described immunodeficiency syndromes.25

CONCLUSIONS CLINICAL PEARLS Management of Immunodeficiency Patients • Lymphopenia is a hallmark of T-cell immunodeficiency in infancy. Unexplained lymphopenia should be recognized and evaluated. • Normal range for immunoglobulin levels and lymphocyte counts varies with age; age-matched controls should be used for interpretation. • Survival and morbidity outcomes of hematopoietic stem cell transplantation (HSCT) for severe combined immunodeficiency (SCID) are best when performed in the first 4 months of age. • Delays in treatment of immunodeficiency leads to infections causing permanent damage to organ systems, such as the lungs, where bronchiectasis or bronchiolitis obliterans may develop secondary to recurrent pneumonias. • Implementation of universal screening of newborn infants by DNA analysis of dried blood spots on Guthrie cards (T-cell receptor excision circle [TREC]) detect all T-cell deficiencies.

The approach to the patient with suspected immune deficiency requires knowledge of developmental pathways and function of the different compartments of the immune system, as well as

the clinical presentation of these disorders. The medical history, particularly the frequency, severity, and etiology of infections, is most helpful to orient the diagnostic workup. Commonly ordered tests in primary care, such as a CBC and serum Ig levels, are helpful to support possible diagnosis and referral to the clinical immunologist. Immunological testing according to clues obtained from the medical history helps narrow the differential diagnoses to specific immunodeficiencies, which are confirmed by molecular methods. Description of new T-cell subsets (e.g., Th17 and regulatory T cells [Tregs]) has helped explain the immunopathogenesis of certain clinical manifestations, such as the occurrence of autoimmunity in patients with combined immunodeficiency, and “cold abscesses” in the autosomal dominant HIES. Testing for these lymphocyte phenotypes is being integrated in the clinical evaluation. Identification of genetic defects can now be accomplished by increased availability of whole exome sequencing, as an alternative to genetic analysis of candidate genes. Technological advances are making molecular diagnosis available for most patients with immunodeficiency conditions.

ON THE HORIZON • Early diagnosis of severe combined immunodeficiency (SCID) and non-SCID is the key to successful hematopoietic stem cell transplantation (HSCT). • Development of DNA sequence analysis for >300 types of primary immunodeficiency (PID). • Wide availability of whole-genome sequencing methods to determine molecular defects for difficult to diagnose immunodeficiency syndromes

ILLUSTRATIVE CASES Case 1 A 6-month-old Caucasian female infant was brought with a history of rash and otitis media that had been recurrent since 2 weeks of age. The infant had poor weight gain, frequent spitting up, coughing spells, and persistent diarrhea. Maternal HIV testing showed negative results. Physical examination revealed an emaciated infant without palpable lymphoid tissue and severe oral thrush. Cystic fibrosis was ruled out by using genetic studies. Stool viral cultures were persistently positive for rotavirus. Immunodeficiency was suspected because of the infant’s failure to thrive, persistent chronic diarrhea, and recurrent infection. An evaluation of the immune system was performed (Table 32.3). Humoral immunity testing showed Ig levels below or just above the lower limit of normal. Isohemagglutinins were not tested as the patient was less than 1 year old. As she had not been immunized, specific antibody titers to vaccines were not tested. Stool α1-antitrypsin levels were normal, suggesting that protein was not being lost in stool, and urinalysis did not show protein loss. CBC revealed profound lymphopenia and absolute neutropenia. The neutropenia had resolved upon subsequent evaluations, but the lymphopenia persisted. A thymic shadow was not present on the chest radiograph. Evaluation of lymphocyte subsets by flow cytometry revealed that the CD4 and CD8 T-cell and B-cell numbers were all markedly low. The patient’s lymphocytes had no proliferative response when stimulated with phytohemagglutinin. The history and the physical examination, along with laboratory values, including low Ig levels and markedly low lymphocyte numbers with poor proliferative response to mitogen stimulation, strongly suggested the diagnosis of SCID.

459

CHAPTER 32  Approach to the Evaluation of the Patient With Suspected Immunodeficiency TABLE 32.3 Results of Screening Tests and Special Evaluations of Illustrative Cases* CASE NUMBER

Test

1

2

3

4

235 (208–686) 90% of genes associated with human disease. They rarely, but famously, can expand to become hundreds or thousands of nucleotides long, thereby causing such human disorders as fragile X syndrome or Huntington disease. Even without expansion, STRs within exons, some of which may be as small as 9–25 bp, have an outsized impact on the frequency of human disease, since they confer a five- to sixfold increase in the frequency of rare disease-causing indel mutations compared with neighboring exon sequences that do not contain an STR.5 One subclass of indel variants arises from mobile elements. Nearly half of the human genome sequence consists of families of repetitive elements dispersed throughout the genome, of which the two most common are the Alu (a short interspersed nuclear element [or SINE]-usually about 300 bp) and LINE (long interspersed nuclear element) families of repeats. Although most of the copies of these repeats are stationary, some of them contribute to human genetic diversity through retrotransposition, a process that involves insertion of a DNA segment generated through transcription of an Alu or LINE element into an RNA that is then reverse-transcribed into a DNA sequence that is inserted into the genomic DNA. Each mobile element indel consists of two alleles, one with and one without the inserted mobile element. Mobile element polymorphisms are found on all human chromosomes; although most are found in nongenic regions of the genome, a small proportion exist within genes. At least 5000 of them are known to be frequent enough to be polymorphisms, and insertion frequencies of >10% occur in various populations; other mobile elements are rare and have been implicated in causing insertional mutations in human disease genes. As with other indels, difficulties with sequencing, in particular the challenges posed to NGS by repetitive DNA, result in underestimation of Alu and LINE indels throughout the genome.3 Another important type of human variation includes CNVs. These consist of variations in the number of copies of segments of the genome that are larger than those involved in indels and range in size from 1000 bp to many hundreds of kilobase (kb) pairs. Variants larger than 500 kb are found in 5–10% of

CHAPTER 33  Human Genomics in Immunology individuals in the general population, whereas variants encompassing more than a million bases (1 Mb) are found in 1–2%.6 Smaller CNVs, in particular, may have only two alleles (i.e., the presence or absence of a segment), similar to indels. Larger CNVs tend to have multiple alleles because of the presence of different numbers of copies of a segment of DNA in tandem. The content of any two human genomes can differ by as much as 50–100 Mb as a result of copy number differences at CNV loci, compared with some 3–4 Mb of differences because of SNVs and indels. Thus simply on the basis of the number of nucleotides involved in the variation, CNVs represent vastly more human variation than do SNVs or indels. Not only are CNVs a very significant contributor to human variation and disease,7 the areas of the genome where they are found are often the sites of segmental duplications,8 some of the most difficult regions in which to develop an accurate reference sequence. Segmental duplications are ubiquitous throughout the genome. It is difficult to construct an accurate contiguous genome sequence in regions of segmental duplications because the most widely used massively parallel sequencing technologies generate only short reads of a few hundred base pairs and therefore may not be able to differentiate between the sequences of nearly identical DNA segments using unique DNA that flanks them. The assembly algorithms, which always try to create the simplest assembly possible, may consider two nearly identical segments of DNA that have been duplicated in the genome, but differ at a few locations in their sequence, to be the same sequence and collapse them into a single segment of DNA. The fixed sequence differences between the duplicated segments are incorrectly interpreted as polymorphic differences between two alleles of a nonduplicated locus instead of being real sequence differences between different copies of duplicated DNA. The fact that there is a segmental duplication is therefore overlooked. Conversely, the sequence of a unique DNA segment with many SNVs compared with the reference may be incorrectly interpreted as having come from distinct but highly similar duplicated segments, leading to the supposition that there are paralogues or duplicated segments. As a result, segmental duplications are liable to both false-negative and false-positive representations in the human genome assembly. CNVs are particularly difficult to detect and interpret if the number of segments in the reference is incorrect or ambiguous. Many CNVs include genes, from one up to several dozens; thus CNVs are frequently implicated in diseases that result from altered gene dosage.7 One well-known human immunological multisystem disorder, DiGeorge syndrome, is caused by a deletion CNV that occurs de novo in about 1 in 5000 individuals. When a CNV is more frequent, it represents a background of common polymorphism that must be understood if to properly interpret alterations in copy number observed in patients. As with all DNA variations, the significance of different CNV alleles in health and in disease susceptibility is the subject of intensive investigation. The most common SVs in the human genome sequence are inversions, which differ in size from a few base pairs to large regions of the genome (up to several Mb) that can be present in either of two orientations in the genomes of different individuals.8 As with CNVs, inversions are usually flanked by regions of sequence homology, which indicates that recombination of these homologous regions is likely involved in the origin of the inversions.7,8 Most inversions do not involve gain or loss of DNA; in this case, the two alleles corresponding to the two orientations can achieve substantial frequencies in the general population. Such inversions can, however, cause significant gains or losses

465

of DNA in the offspring of inversion carriers because of aberrant recombination during meiosis, leading to serious syndromes brought about by chromosomal imbalance. Furthermore, if inversions interfere with normal gene expression by disrupting a gene or altering the physical relationship between a gene and its regulatory elements, it may be detrimental to health.9,10

CLINICAL IMPACT OF HUMAN VARIATION One of the greatest challenges facing human geneticists is linking variation to phenotype.11 The significance for the health of the vast majority of variants of any type is unknown, and yet this knowledge is essential if we are to apply genomics to clinical care. The impact of variants ranges from completely benign to highly pathogenic, the latter causing devastating disorders of the immune system that may occur as new mutation dominants or as autosomal recessively inherited syndromes. Even common polymorphic variants may affect health or longevity, although their being common means that they are likely to produce a relatively subtle alteration of disease susceptibility rather than directly cause a serious illness.12 Working out the functional impact of human variation will occupy genomics researchers for many years to come. An essential component of this work is to make databases of genetic variants and their impact on human health available to the research and clinical communities, as is being done with the ClinVar database hosted by the National Library of Medicine in the United States and the Leiden Opensource Variation Database (LOVD).13-15

COMPARATIVE GENOMICS Evolution at work is nowhere better illustrated than in the field of comparative genomics, which deals with similarities in the sequence, structure, and chromosomal location of genes between different species whose evolutionary paths diverged from a few years ago to many hundreds of millions of years ago. Direct sequence comparison has revealed that an enormous number of human proteins have orthologues (genes derived from a common ancestor) in other organisms, ranging from 87% in chimps to 79% in mice, 63% in zebrafish, 39% in the fruit fly (Drosophila melanogaster), and 31% in the nematode (Caenorhabditis elegans) (Fig. 33.1). The study of the human genome and the genomic basis for human disease has benefited from studies done in other organisms, particularly mice, in which many decades of inbreeding and gene manipulation have permitted study of the genes and underlying mechanisms by which mutations responsible for immune defects lead to phenotypes. Mouse models of immunity have been highly relevant and informative for humans. However, orthologous genes may serve different functions in different species; therefore one cannot assume that a diseasecausing mutation in humans will cause a similar defect when its orthologue is similarly mutated in mice, and vice versa. For example, the V(D)J recombination activating genes RAG1 and RAG2 in humans and Rag1 and Rag2 in mice appear to have identical functions, with knockout mice showing the same inability to recombine T- and B-lymphocyte antigen receptor genes as humans with RAG1 or RAG2 deficient severe combined immunodeficiency (SCID; Ch. 35).16 As a result, the lymphocyte profile in both species is absent T and B cells with normal natural killer (NK) cells, referred to as T−B−NK+ SCID. A contrasting situation occurs in other SCID genotypes. For example, humans lacking the common γ chain (γc) of receptors for interleukin-2 (IL-2)

466

Part FOUR  Immunological Deficiencies

and other cytokines, caused by mutations in the X-linked gene IL2RG have SCID in which T and NK cells are absent but nonfunctional B cells are present in normal to high numbers, T−B+NK− SCID. Mice with the orthologous gene Il2rg mutated or removed can make T cells but have no B cells, which gives them a T+B−NK− phenotype.17 Genes other than IL2RG alone must be responsible for this difference between species. Such genes, known as modifiers, have not yet been identified. Notably, different strains of mice can also have important phenotypic differences in the presence of a single gene mutation under study; some strains, for example, nonobese diabetic (NOD) and Murphy Roths Large (MRL) mice

Ch

im p Do Mo g us e Ra Ch t ic Pu ken ffe Ze rfish br afi s Fr h u Mo itfly sq uit o C. Be Mu ele e sta gan rd s we ed R A ic C. moe e ce ba re e vis iae E. co li

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

FIG 33.1  Homology Between Human Genes and Genes in Multiple Other Organisms. Comparisons were made by calculating gene homology or similarity between reference protein sequences from human and another species, with homology considered present when the probability of an interspecies match by chance alone was 1 : 1 000) typically have potent antibodies directed against all IgA. These patients are at risk for severe anaphylaxis. Patients with low anti-IgA antibody titers ( IgG1 > IgG3. Most patients are also deficient in IgM and IgE. Patients with uncomplicated conditions demonstrate normal cell-mediated immunity, although a minority of patients may have evidence of T-cell dysfunction as well as other hematopoietic cell types. In some cases, B-cell numbers are reduced, although not to the extent exhibited by disorders of pre-BCR formation or BTK signaling. IgAD and CVID have been associated with congenital infection with rubella virus, CMV, and T. gondii. The administration of certain drugs has also been linked to a depression in serum Ig levels (see Table 34.1). Several medications used to treat epilepsy have been associated with the development of antibody deficiencies. For example, up to 20% of patients treated with phenytoin develop a mild decrease in serum IgA levels, and a minority may progress to a CVID-like phenotype. Medications used for the treatment of rheumatoid arthritis, inflammatory bowel disease, and chronic myelogenous leukemia can also decrease production of antibody. Persistence of antibody deficiency usually requires choosing between discontinuation of the drug and persisting infection with the virus or parasite. Recovery of Ig production may take months to years.

Clinical Manifestations Although some patients with CVID have reduced numbers of circulating B cells, the majority has normal quantities of IgA, IgG, and IgM-bearing B-cell precursors in the blood. Defects in B-cell survival, number of circulating CD27+ memory B cells (including IgM+CD27+ B cells), B-cell activation after antigen receptor cross-linking, T-cell signaling, and cytokine expression have been observed. Both increases and decreases in the relative numbers of CD4 to CD8 T cells are common, and cutaneous anergy is a frequent finding. The clinical manifestations of CVID are similar but more severe than the ones seen in IgAD. Respiratory symptoms often begin with recurrent sinusitis, otitis media, and mild bronchitis. The frequency and severity of the upper respiratory infections worsen in the young adult, and lower respiratory infections, such as pneumonia, become common. Apparently asymptomatic, untreated patients may suffer recurrent subclinical pulmonary infections that can lead to irreversible chronic lung damage with bronchiectasis, unilateral hyperlucent lung, emphysema, and cor pulmonale. Recurrent cellulitis, boils, folliculitis, impetigo, or erythroderma can be presenting complaints. Intermittent or chronic diarrhea due to G. lamblia is a common complaint. Patients can develop a malabsorption syndrome that resembles celiac sprue but is unresponsive to gluten avoidance (Fig. 34.4). Untreated patients often complain of asymmetrical, oligoarticular arthralgias or frank arthritis, which in some cases reflect infections with encapsulated organisms or with Mycoplasma

481

FIG 34.4  Hypogammaglobulinemic Sprue in a 41-Year-Old White Male With Common Variable Immune Deficiency (CVID) and Insulin-Dependent Diabetes Mellitus. The patient suffered from intractable diarrhea. Shown is a hematoxylin and eosin (H&E) stain of a duodenal biopsy obtained by endoscopy. The villi are blunted, and there is a marked increase in intraepithelial lymphocytes. However, unlike typical celiac disease, the villi are not completely blunted, and few plasma cells are seen. The patient is homozygous for the HLA-DQ2, -DR17(3), -B8 haplotype. Although the patient failed to respond to a gluten-free diet, the diarrhea resolved with corticosteroid therapy.

species and thus require antibiotic therapy. Paradoxically, antigenspecific IgE can be produced in sufficient quantities to enable anaphylactic reactions. Patients with CVID are often anergic, but only a minority suffers infections characteristic of cell-mediated immune dysfunction, including mycobacteria, P. jiroveci, and fungi. CD8 T-cell numbers may be depressed in such patients. Most viral infections are cleared normally. Exceptions include enteroviral infections, including meningoencephalitis, as well as hepatitis B and C, which can progress to chronic active hepatitis. Lack of humoral immunity enhances susceptibility to viral reactivation. Untreated patients often complain of recurrent herpes zoster (shingles). Autoimmune diseases are common in CVID. Coombs-positive hemolytic anemia with idiopathic thrombocytopenic purpura, a combination known as Evans syndrome, may predate the diagnosis of CVID. Noncaseating granulomas in the lungs, lymph nodes, skin, bone marrow, and liver reminiscent of or indistinguishable from sarcoid-like syndrome is more common in African Americans but can be seen in up to one-fifth of all patients. Occasionally the granulomas result from mycobacterial and fungal infections. In the majority of cases, the cause remains unclear, and the granulomas resolve spontaneously. There is an increased risk for the development of GI and lymphoid malignancies, especially non-Hodgkin lymphomas. Confounding the diagnosis of malignancy is the patients’ propensity to develop benign lymphoproliferative disorders. Lymphadenopathy, splenomegaly, or a combination of both is common in untreated patients.

Origin and Pathogenesis The typical presenting manifestation of CVID is hypogammaglobulinemia, not agammaglobulinemia, suggesting a partial or

482

Part four  Immunological Deficiencies

varying block in B-cell maturation. Careful analysis of B cells in patients has also revealed a spectrum of immune deficiency ranging from the nearly complete absence of memory B cells to a less severe disorder. All of these findings serve to underline the complex etiology for the disorder, and many details remain to be elucidated. The MHC represents the most common genetic susceptibility locus for CVID.22 Because of linkage disequilibrium, the gene, or genes, within this locus that create susceptibility have yet to be identified with certainty. Non–MHC-associated single-gene defects have been identified, although they represent only a minority of patients with CVID. These include function-loss mutations in genes involved in late-stage B cell–T cell communication, late-stage B-cell growth factors, and B-cell and T-cell signaling and activation pathways (see Table 34.1). These include the genes for ICOS (CVID1), an immune costimulator molecule used by T cells to activate B cells in germinal centers; BAFFR (CVID4) and TACI (CVID2), the receptors for BAFF, CD19 (CVID3), CD21 (CVID7), and CD81 (CVID6), components of the B-cell costimulatory receptor; CD20 (CVID5), an important marker of B-cell differentiation; and LRBA, Cytotoxic T lymphocyte antigen-4 (CTLA-4), PKCδ, Tweak, PIK3CD, PIK3R1, NF-κB2, and KMT2D, which are involved in B-cell and T-cell signaling pathways. TLR7 and TLR9 activation can be deficient in these patients, although the genes are intact.27 The Major Histocompatibility Complex A large array of genes that play important roles in the control of the immune response are located in the MHC on chromosome 6 (Chapter 5). Studies at the University of Alabama at Birmingham have shown that in the Southeastern United States the majority of patients with IgAD and CVID share parts or all of one of three extended MHC haplotypes marked by either HLA-DQ2, -DR17(3), and -B8 or HLA-DQ2, -DR7, and -B44. The combined results of three studies of patients from Alabama, New England, and Australia indicated a 13% prevalence of immunodeficiency in individuals homozygous for HLA-DQ2, -DR17(3), and -B8. These MHC alleles are also more common in patients who suffer from diabetes mellitus, pernicious anemia, celiac disease, autoimmune thyroid disease, and myasthenia gravis. Some individuals with TACI mutations inherited MHC haplotypes associated with the disease,28 and the combination of specific MHC and KIR alleles also increases susceptibility.29 This suggests that epistatic interactions between different susceptibility alleles may influence the penetrance of the disorder.

which is unique for BAFF, is expressed on B cells and on resting T cells. The BAFF/APRIL system plays a key role in mature B-cell homeostasis and development. BAFF and APRIL can also induce class switching in naïve human B cells. Loss-of-function (autosomal recessive) or altered-function (autosomal dominant) TACI alleles have been found in approximately 10% of patients with CVID in many series.31 Two polymorphic alleles, A181E and C104R, are prominent in patients with CVID who have TACI alterations. These alleles are also present in approximately 2% of the normal population, suggesting that the presence of these altered alleles functions as a susceptibility factor for the development of the disease. Family members may have IgA deficiency or may have no evidence of immune dysfunction. However, patients with CVID who have these altered alleles have a higher prevalence of complications from CVID, including lymphoproliferation, splenomegaly, and autoimmune phenomena.32 BAFF-R deficiency has been reported in four patients with CVID to date.30 CD20 (CVID5) CD20 encodes a B-cell membrane–spanning molecule important in B-cell proliferation and differentiation. It is expressed from early pre-B until mature B-cell stage but is lost on differentiation to plasma cell. One case of a female consanguineous patient with CD20 deficiency with low IgG and normal IgA and IgM levels and impaired antibody responses to pneumococcal polysaccharides, has been reported.30,33 ICOS (CVID1) ICOS is a T-cell surface receptor that is important for germinal center formation, terminal B-cell differentiation, effector T-cell responses, and immune tolerance. Patients with ICOS deficiency have low to absent B cells, and some have varying degrees of defective T-cell signaling. They present with recurrent respiratory tract infections and autoimmune complications.30,33

The CD19 (CVID3), CD81 (CVID6), CD21 (CVID7) B-Cell Coreceptor Complex CD21 (complement component C3d/Epstein-Barr virus receptor 2) binds to membrane- IgM-bound antigen when complement C3d is also attached to that antigen (Chapter 21). In association with CD81 and CD19, this coreceptor complex enhances the antigen-binding signal, promoting B-cell activation. Patients with mutations in CD19, CD21, and CD81 have been reported.30

The LRBA (Cvid8)–CTLA-4 Axis Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a cytosolic protein that functions in vesicle trafficking, autophagy, and cell survival. It is expressed by almost all cell types with higher expression in immune effector cells. CTLA-4 is an inhibitory T-cell receptor (TCR) that competes with the costimulatory protein CD28 for binding CD80/86, thereby preventing excessive T-cell activation and maintaining immune tolerance. LRBA plays a role in CTLA-4 surface expression. Patients with LRBA deficiency display early-onset hypogammaglobulinemia with autoimmunity and inflammatory bowel disease. They show reduced levels of at least two Ig isotypes (IgM, IgG, or IgA) and suffer from recurrent infections, autoimmunity, and chronic pulmonary and GI disorders. Patients with haploinsufficiency of CTLA-4 presented with autoimmunity, recurrent infections, benign lymphoproliferation, and varying levels of Igs and B-cells, and T-cell defects.30,33

The BAFF–BAFFR (CVID4)–TACI (CVID2) Axis The TNF family members B-cell activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) bind to two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), both members of the TNF-R family. BCMA is expressed exclusively on B cells, whereas TACI is expressed on activated T cells as well. A third receptor, BAFF-R,

PKCδ Deficiency PKCδ plays a key role in BCR-mediated signaling downstream of BTK and is important in B-cell proliferation, apoptosis, and tolerance. PKCδ deficiency presents with a variable phenotype, with one affected patient having a CVID-like characteristics (hypogammaglobulinemia and severe infections) and others having lupus or autoimmune lymphoproliferative syndrome (ALPS)–like disease.30

CHAPTER 34  Primary Antibody Deficiencies TWEAK Deficiency A patient with TNF-like weak inducer of apoptosis (TWEAK) deficiency displayed low to normal IgG, low IgM, and low IgA and had a history of pneumococcal meningitis, osteomyelitis, thrombocytopenia, and neutropenia. The patient had an autosomal dominant mutation in TNF superfamily member 12 (TNFSF12), which encodes TWEAK.30 NF-κB1 (CVID12) and NF-κB2 (CVID10) Deficiency The NF-κB1 and NF-κB2 (noncanonical) pathways are important in B cell signaling, with NF-κB2 having a more limited set of involved receptors, such as ICOS, TACI, BAFR-R, and BCMA, whereas NF-κB1 affects T-cell and TLR signaling as well. Heterozygous mutations in NF-κB2 were identified in at least 10 patients who presented with early-onset CVID with autoimmunity and recurrent sinopulmonary infections. Patients with NF-κB2-deficiency display panhypogammaglobulinemia, aberrant B-cell subsets, and some degree of T-cell and NK-cell dysfunction. Half of these also suffer from pituitary hormone deficiencies. Patients with NK-KB1 autosomal dominant mutations that create an unstable and rapidly degrading protein have recurrent infections, autoimmunity, benign lymphoproliferative disease, and lymphoma.30 PI3K Mutations Heterozygous mutations in P1K3CD, which encodes the P13K catalytic subunit p110δ, have been reported in over 50 patients with CVID-like disease. The mutation leads to P13K signaling pathway overactivity. Such patients suffer from respiratory tract infections, skin infections, autoimmunity, and lymphoma. The phenotype associated with dominant gain of function P1K3CD mutations is currently referred to as activated phosphatidylinositol 3-kinase δ syndrome (APDS). PIK3R1 encodes the P13K regulatory subunit p85α. Splice-site mutation of p85α results in a loss of an exon of p85α, which is important in inhibiting the catalytic activity of p110d. This also results in autosomal dominant overactive P13K signaling. A single patient with a homozygous P1K3R1 mutation causing complete loss of p85α expression has been found with agammaglobulinemia and absence of B cells, suggesting complete absence of p85α reduces inhibiting P13K signaling.30 Other Genes: BLK, IRF2BP2, IKAROS A heterozygous loss of function mutation in BLK has been noted in related patients with CVID. These patients have respiratory tract infections and bacterial skin infection with panhypogammaglobulinemia. A gain of function mutation in IRF2BP2 has been found in members of a family with CVID. These patients have autoimmune disease and respiratory tract infections. Heterozygous mutation in the gene IKZF1, encoding the transcription factor IKAROS, was recently found in patients with CVID and low B-cell numbers. These patients have progressive loss of B cells and serum Igs.30 Kabuki Syndrome Kabuki syndrome (KS) is a rare but recognizable condition that consists of a characteristic face, short stature, cardiac anomalies, a variable degree of intellectual disability, and immunological defects. Mutations in KMT2D and KDM6A have been identified

483

as the main cause for KS. Similar to CVID, patients with KS have recurrent infections, reduced Ig levels, and autoimmunity.34

Treatment and Prognosis Therapy in CVID begins with the aggressive treatment of ongoing infections and the institution of prophylactic measures to prevent or ameliorate future infections. Patients suffering from moderate upper respiratory tract infections and bronchitis will likely benefit from empiric therapy with agents effective against encapsulated organisms. The concomitant inheritance of MBL protein deficiency appears to further predispose to the development of bronchopulmonary complications, such as bronchiectasis, lung fibrosis, and respiratory insufficiency.3 The course of treatment for patients with immunodeficiency is often prolonged, and intravenous administration of antibiotics may be required. The most effective therapy for hypogammaglobulinemia is Ig replacement. A number of studies have demonstrated a steadily decreasing incidence of infection with increasing frequency of Ig administration. At higher doses, even patients with bronchiectasis may experience improvement in pulmonary function. Each patient may demonstrate his or her own individual response to therapy, exhibiting dramatic differences in the frequency and severity of infections, with moderate changes in the replacement dose. Patients suffering from a serious acute infection often benefit from one-time booster doses of Ig. Ultimately, replacement dosage must be individualized based on the response of the patient. Adverse reactions occur most frequently at the time of the first administration of Ig, likely because of concurrent infection increasing the potential for generation of immune complexes. Some patients with CVID can sustain severe anaphylaxis when given IVIG or other blood products that contain serum or plasma. These patients may possess anti-IgA antibodies, including IgE anti-A antibodies.7 For patients with a history of severe adverse reactions, it is advisable to try batches of IVIG with the lowest IgA possible and to test the patient with the different batches in an intensive care unit. Once the clinician has identified a batch that can be tolerated, the patient may receive therapy under more relaxed conditions. Serum Ig concentrations in patients with CVID may change over time,2 with rare patients regaining normal serum IgG levels and no longer requiring Ig therapy. Careful review of the clinical history of these patients may reveal evidence of exposure to pharmacological agents associated with the development of hypogammaglobulinemia (e.g., phenytoin). However, the overwhelming majority of patients require replacement therapy for life. Although IgG can be replaced, at present IgM and IgA cannot be provided to patients. The absence of these multimeric proteins may help explain why even patients on high-dose replacement therapy may continue to suffer from recurrent sinusitis or GI discomfort.35 Recurrent sinusitis can be ameliorated with continued prophylactic therapy with antibiotics. Patients with CVID also are at risk of infection by G. lamblia as well as other enteric pathogens. Some patients develop lactose intolerance or glutensensitive enteropathy. Gluten avoidance ameliorates symptoms in only a minority of cases. A majority responds to corticosteroids or anti-TNF agents. The use of these agents can be a double-edged sword, however, since resistance to infection will decrease in a patient who already has immune deficiency. Other patients develop a malabsorption syndrome that can lead to hypoalbuminemia and hypocalcemia (as a result of malabsorption of vitamin D),

484

Part four  Immunological Deficiencies

and decreased levels of vitamin A and carotene.36 The cause of diarrhea and malabsorption in this latter patient subset remains unclear, and treatment is limited to supportive measures, with vitamin and mineral replacement as indicated. Patients with bronchiectasis should be treated aggressively with replacement therapy. In severe cases, aggressive pulmonary toilet will benefit the patient, including bronchodilator therapy, position and postural drainage, or other physical therapies. The use of corticosteroids should be avoided. Mothers with IgA deficiency may fail to secrete IgA in their colostrum. Although colostral IgM levels may be elevated in an attempt to compensate for the lack of maternal IgA, the newborn remains relatively unprotected against intestinal pathogens. Of greater concern is the fact that the babies of mothers with untreated CVID are born in a state of humoral immunodeficiency and thus are at risk for life-threatening sinopulmonary infection. To compensate for the loss of IgG across the placenta and to provide the infant with the passive immunity that will be required, the dose of replacement gammaglobulin therapy should be increased by 50% during the third trimester of pregnancy. Splenomegaly is common in untreated patients. Hypersplenism in most patients responds to aggressive therapy with antibiotics and IVIG. The presumption is that the hypersplenism is secondary to reactive hyperplasia of lymphoid follicles within the spleen attempting to respond to infection. Development of esophageal varices or other hematological manifestations of hypersplenism (refractory thrombocytopenia, anemia, neutropenia, and lymphopenia) may require splenectomy as the therapy of last resort. The outcome for most such patients has been good, with resolution of symptoms, although patients with altered TACI alleles tend to do less well. The development of a constellation of pulmonary abnormalities that include granulomatous and lymphoproliferative histopathologic patterns (lymphocytic interstitial pneumonia [LIP], follicular bronchiolitis, and lymphoid hyperplasia), termed granulomatous–lymphocytic interstitial lung disease (GLILD), can be an ominous sign. These patients appear more likely to develop granulomatous liver disease, autoimmune hemolytic anemia, lymphoproliferative disease, and progressive pulmonary disease.37

THERAPEUTIC PRINCIPLES • The primary goal of treatment is to keep the patient infection free. • In patients whose respiratory mucosa is intact, intravenous or subcutaneous replacement immunoglobulin G (IgG) therapy is generally effective in protecting the patient from pulmonary infections. • For those patients who have developed bronchiectasis or who continue to subject themselves to environmental toxins (e.g., smoking), replacement IgG will ameliorate but may not prevent all such infections. • Because mucosal Ig cannot be replaced, even patients on adequate IgG replacement therapy remain at risk for sinus or gastrointestinal infections. • Prophylactic antibiotics that are effective against encapsulated organisms can significantly reduce the frequency of upper respiratory tract infections in patients who continue to suffer in spite of replacement therapy with intravenous immunoglobulin (IVIG). • Prolonged diarrhea in patients with hypogammaglobulinemia is often caused by Giardia lamblia and responds well to metronidazole therapy. • Patients with primary antibody deficiencies should not receive live vaccines.

SELECTIVE IGG SUBCLASS DEFICIENCIES Diagnosis A diagnosis of clinical immunodeficiency should be supported by clear evidence of functional impairment. Most individuals with modest reductions in serum IgG subclass levels are functionally normal. Indeed, individuals with deletions of the heavy chain Ig gene locus, some of whom completely lack IgG1, IgG2, IgG4, and IgA38 have been reported to be asymptomatic. The diagnosis of a functional IgG subclass deficiency can thus be made with confidence only when there is both a significant decrease in the serum concentration of a specific isotype and there is clear evidence of abnormal specific antibody production. Up to 10% of normal males and 1% of normal females have IgG4 deficiency, which makes a diagnosis of immunodeficiency as a result of an isolated IgG4 subclass deficiency problematic. Among patients with a deficiency of IgG1 or IgG3, documentation of the ability to produce protective titers of antitetanus toxin and antidiphtheria toxin antibodies following standard tetanus toxoid and diphtheria immunizations is a strong indication that replacement gammaglobulin therapy is likely unwarranted. Similarly, documentation of a strong antipneumococcal polysaccharide response in patients with an apparent IgG2 deficiency would suggest gammaglobulin replacement is likely not required. IgG2 levels normally begin to rise in childhood later than other subclasses. Conversely, the lack of a response to vaccination calls for appropriate prophylactic antibiotic therapy before a trial of IVIG is attempted.

Clinical Manifestations The clinical spectrum of isolated IgG subclass deficiency is quite variable, and deficiencies of each of the four IgG subclasses have been described. Some individuals are referred to the clinical immunologist with only a mild reduction of total IgG, but most symptomatic patients have marked deficiencies of one or more IgG subclass despite normal total IgG concentrations. Since IgG1 makes up the majority of serum IgG in most patients, a deficiency of IgG1 tends to correlate with depressed serum levels of total IgG. Determination of IgG subclasses is rarely performed on asymptomatic individuals; thus most patients with an isolated IgG2 deficiency come to medical attention as a result of recurrent sinusitis, otitis media, or pulmonary infections. Individuals may have few residual symptoms between infections, but some have severe chronic inflammation with refractory sinusitis, pulmonary fibrosis, or bronchiectasis. Because protective antibodies directed against carbohydrate antigens are usually of the IgG2 subclass, many affected patients exhibit an impairment of their ability to mount specific protective responses to encapsulated pathogens. However, normal responses have also been described.39 Many clinicians would agree, however, that patients with IgG2 deficiency, who suffer from recurrent sinopulmonary infections and who respond to less than half of the polysaccharide antigens with which they have been challenged, meet the standard for functional immune deficiency and thus warrant aggressive prophylactic therapy up to and including Ig replacement should the infections be severe. IgG3 deficiency can occur alone or in association with IgG1 deficiency. Recurrent infection of the respiratory tract with chronic lung disease has been reported. With a serum half-life only 2 weeks, IgG3 levels may be consumed rapidly during the course of an active infection in an otherwise normal individual.40

CHAPTER 34  Primary Antibody Deficiencies

485

Before making the diagnosis of IgG3 deficiency, serum levels of IgG3 should be rechecked when the individual is asymptomatic. Compared with the serum, IgG4 is overrepresented in secretions, and IgG4-committed B cells are present at mucosal sites, suggesting a role in mucosal immunity. Since IgG4 is normally present in the serum in very low concentrations, the significance of a low serum level in a patient with recurrent infection remains unclear.

Analysis of a group of well-characterized patients, mostly female, with a history of RESPIs and normal serum Ig levels revealed a high prevalence of the same MHC haplotypes observed in IgAD, selective IgG subclass deficits, and CVID.23 These patients tend to respond to aggressive antibiotic therapy, including prophylaxis.

Origin and Pathogenesis

Selective deficiencies of either κ or λ light chains have been reported.44-46 In one such case, the patient was the offspring of a consanguineous (uncle–niece) union; and in the second, a molecular analysis demonstrated different loss of function mutations in the patient’s Cκ alleles. The parents of these children had no health difficulties, but each of the patients required medical attention for recurrent sinopulmonary infections and diarrhea. Two of the patients with κ deficiency exhibited IgA deficiency, and the remaining patients with κκ deficiency and λ deficiency had panhypogammaglobulinemia.

The origin of IgG subclass deficiency is unknown. Homozygous deletions of portions of the Ig heavy chain constant locus associated with total absence of IgG2, IgG3, and IgG4 or combinations of these isotypes have been described in healthy individuals. IgG2 deficiency is often found in association with selective IgA deficiency with or without IgG4 deficiency, and patients with selective IgG subclass deficiencies have been shown to have inherited the same MHC haplotypes as those who suffer from IgAD and CVID. These observations suggest that patients with recurrent infections have a more complex defect than the mere elimination of one or more IgG isotype. In some instances, subclass deficiency is associated with a T-cell defect, as in chronic mucocutaneous candidiasis and ataxia–telangiectasia. IgG subclass deficiency can be acquired. Acute infections, medications, chemotherapy, irradiation, surgery, and human immunodeficiency virus (HIV) infection have all been temporally linked to the development of a deficiency in one or more IgG subclass.41

Treatment and Prognosis The natural history of IgG subclass deficiency plus or minus IgA deficiency, especially in children, is not constant.42 Associated allergic rhinosinusitis and asthma must be aggressively treated with conventional therapy for these disorders, as these conditions increase the risk of purulent sinusitis and pneumonia. Causes of anatomical obstruction should be sought when persistent infection of a sinus or pulmonary segment is the presenting complaint; the role of surgical therapy for anatomical obstruction should not be overlooked. Many patients with IgG subclass deficiency do well on prophylactic antibiotics and will never need Ig supplementation. However, Ig replacement therapy can be beneficial in patients with severe, recurrent infections. Patients who begin therapy should improve within the first 2 months, but to avoid the placebo effect, a full 6-month trial is recommended.

ANTIBODY DEFICIENCY WITH NORMAL SERUM IMMUNOGLOBULIN LEVELS Occasional patients may present with normal serum Ig concentrations and a selective inability to respond to infections with pyogenic organisms. Diagnosis requires documentation of an inability to respond to antigenic challenge. These patients may respond to replacement Ig therapy. The antibody response to specific polysaccharide antigens can be very selective. In human, most protective anti-H. influenzae type b (anti-Hib) antibodies utilize the rare VκA2 gene. The Navajo population in the Southwestern United States suffers a 5- to 10-fold increased incidence of Hib disease. This population also exhibits a high prevalence of an A2 allele with a defective recombination signal sequence, preventing use of germline-encoded antibodies that can generate protective antigen-binding sites.43

SELECTIVE LIGHT-CHAIN DEFICIENCY

TRANSIENT HYPOGAMMAGLOBULINEMIA OF INFANCY Diagnosis As infants make the transition from dependence on maternal Ig to reliance on endogenously produced antibodies, they suffer a physiological nadir of serum Ig at 4–6 months of age, a period associated with susceptibility to mild upper respiratory infections and otitis media (see Fig. 34.2). Children who (i) exhibit serum concentrations of one or more of the three major Ig classes that fall below the 95% confidence interval (CI) for age on ≥2 occasions during infancy, (ii) demonstrate a rise in these values to or toward normal over time, and (iii) lack features consistent with other forms of primary immunodeficiency fall within the catch-all diagnosis of transient hypogammaglobulinemia of infancy (THI).47,48 By definition, the diagnosis of THI can be made with certainty only in retrospect.

Clinical Manifestations Ig concentrations are rarely measured in infants unless there is some reason to suspect an immunodeficiency. Most patients with this diagnosis come to medical attention because of either recurrent infections or as a result of routine screening studies of relatives of other patients with immunodeficiency. Yet bearing in mind that 2.5% of normal infants will fall below the 95% CI range at any one time, the diagnosis of THI is remarkably rare. Among two major centers, one in the United States and one in Germany, the diagnosis was given to only 16 of 18 000 children in whom the index of suspicion warranted Ig determinations.49,50 Patients with THI typically are able to synthesize specific antibodies in response to immunization with T-dependent antigens (e.g., tetanus and diphtheria toxoids).51 They may have difficulty, however, responding to polysaccharide antigens (e.g., isohemagglutinins and vaccination with Pneumovax23). Some will fail to sustain protective antibody responses to antigens. Most patients with THI, especially those ascertained as a result of family studies or mild upper respiratory infections alone, exhibit fewer infections over time. The great majority of infants with THI will normalize their serum Ig levels within 24 months of age. However, a minority fails to normalize IgG, continues to suffer with recurrent infections, and may develop evidence of autoimmune disease. These patients often become part of the

486

Part four  Immunological Deficiencies

hypogammaglobulinemia syndrome complex that includes CVID and may ultimately require long-term therapy with gammaglobulin, prophylactic antibiotics, or both.

Treatment and Prognosis Children with suspected THI should be monitored with serial determination of serum Igs and isohemagglutinin titers to confirm acquisition of normal immune function. Some children will not achieve normal levels of IgG for several years, and some will remain IgG subclass or IgA deficient. Treatment of THI with IVIG is generally not warranted unless the child suffers from persistent, recurrent, invasive infections, including pneumonia.

ON THE HORIZON • Elucidation of the molecular basis of selective defects in humoral responses to pathogens, in part through the use of high-throughput sequencing to characterize the precise molecular composition of antibody responses in immune deficiencies • Further elucidation of the molecular basis of common variable immune deficiency, hypogammaglobulinemia, and immunoglobulin A (IgA) deficiency

FRONTIERS IN RESEARCH Bruton reported the first case of agammaglobulinemia in 1952, as well as the first successful therapy for this classic primary antibody deficiency. There has been remarkable progress in the identification of single-gene disorders since that time. However, for the majority of patients the underlying pathogenesis of the most common manifestation of primary antibody deficiency in our population, that is, hypogammaglobulinemia in the adult, still remains unclear. It seems increasingly likely that this disorder is multifactorial in nature, dependent on the inheritance of one or more susceptibility loci in association with either environmental influences or random chance. In rare cases, patients with hypogammaglobulinemia have shown resolution of their symptoms, suggesting that a better understanding of pathogenesis might yield therapies of remission. The molecular basis of selective deficiencies in the response to pathogens in the presence of normal serum Ig levels also remains unclear. The availability of whole exome sequencing and whole-genome sequencing has helped identify the genetic cause of an increasing number of patients with presumed CVID. More genes and likely presumptive alleles will be found in the next decade. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Durandy A, Kracker S, Fischer A. Primary antibody deficiencies. Nat Rev Immunol 2013;13:519–33. 2. Johnson ML, Keeton LG, Zhu ZB, et al. Age-related changes in serum immunoglobulins in patients with familial IgA deficiency and common variable immunodeficiency (CVID). Clin Exper Immunol 1997;108:477–83. 3. Litzman J, Freiberger T, Grimbacher B, et al. Mannose-binding lectin gene polymorphic variants predispose to the development of bronchopulmonary complications but have no influence on other clinical and laboratory symptoms or signs of common variable immunodeficiency. Clin Exper Immunol 2008;153:324–30.

4. De Greef GE, van Tol MJ, Van Den Berg JW, et al. Serum immunoglobulin class and IgG subclass levels and the occurrence of homogeneous immunoglobulins during the course of ageing in humans. Mech Ageing Dev 1992;66:29–44. 5. Stiehm ER, Fudenberg HH. Serum levels of immune globulins in health and disease: a survey. Pediatrics 1966;37:715–27. 6. Soothill JF, Hayward AR, Wood CB. Pediatric immunology. Oxford, UK: Blackwell Scientific Publications; 1983:463–4. 7. Gharib A, Caperton C, Gupta S. Anaphylaxis to IGIV in immunoglobulin-naive common variable immunodeficiency patient in the absence of IgG anti-IgA antibodies: successful administration of low IgA-containing immunoglobulin. Allergy Asthma Clin Immunol 2016;12:23. 8. Carr TF, Koterba AP, Chandra R, et al. Characterization of specific antibody deficiency in adults with medically refractory chronic rhinosinusitis. Am J Rhinol Allergy 2011;25:241–4. 9. Lopez-Herrera G, Vargas-Hernandez A, Gonzalez-Serrano ME, et al. Bruton’s tyrosine kinase—an integral protein of B cell development that also has an essential role in the innate immune system. J Leukoc Biol 2014;95:243–50. 10. Nelson KS, Lewis DB. Adult-onset presentations of genetic immunodeficiencies: genes can throw slow curves. Curr Opin Infect Dis 2010;23:359–64. 11. Sigmon JR, Kasasbeh E, Krishnaswamy G. X-linked agammaglobulinemia diagnosed late in life: case report and review of the literature. Clin Mol Allergy 2008;6:5. 12. Stewart DM, Tian L, Notarangelo LD, et al. X-linked hypogammaglobulinemia and isolated growth hormone deficiency: an update. Immunol Res 2008;40:262–70. 13. Winkelstein JA, Conley ME, James C, et al. Adults with X-linked agammaglobulinemia: impact of disease on daily lives, quality of life, educational and socioeconomic status, knowledge of inheritance, and reproductive attitudes. Medicine (Baltimore) 2008;87:253–8. 14. Conley ME, Dobbs AK, Farmer DM, et al. Primary B cell immunodeficiencies: comparisons and contrasts. Annu Rev Immunol 2009;27:199–227. 15. Boisson B, Wang YD, Bosompem A, et al. A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR(−) B cells. J Clin Invest 2013;123:4781–5. 16. Sawada A, Takihara Y, Kim JY, et al. A congenital mutation of the novel gene LRRC8 causes agammaglobulinemia in humans. J Clin Invest 2003;112:1707–13. 17. Conley ME, Dobbs AK, Quintana AM, et al. Agammaglobulinemia and absent B lineage cells in a patient lacking the p85alpha subunit of PI3K. J Exp Med 2012;209:463–70. 18. Durandy A, Kracker S. Immunoglobulin class-switch recombination deficiencies. Arthritis Res Ther 2012;14:218. 19. Karaca NE, Durandy A, Gulez N, et al. Study of patients with Hyper-IgM type IV phenotype who recovered spontaneously during late childhood and review of the literature. Eur J Pediatr 2011;170:1039–47. 20. Kawai T, Nishikomori R, Heike T. Diagnosis and treatment in anhidrotic ectodermal dysplasia with immunodeficiency. Allergol Int 2012;61:207–17. 21. Hennig C, Baumann U, Ilginus C, et al. Successful treatment of autoimmune and lymphoproliferative complications of patients with intrinsic B-cell immunodeficiencies with Rituximab. Br J Haematol 2010;148:445–8. 22. Schroeder HW Jr, Schroeder HW III, Sheikh SM. The complex genetics of common variable immunodeficiency. J Investig Med 2004;52:90–103. 23. Johnston DT, Mehaffey G, Thomas J, et al. Increased frequency of HLA -B44 in Recurrent Sino-Pulmonary Infections (RESPI). Clin Immunol 2006;119:346–50. 24. Nielsen LK, Dziegiel MH. Recombinant human immunoglobulin (Ig)A1 and IgA2 anti-D used for detection of IgA deficiency and anti-IgA. Transfusion 2008;48:1892–7. 25. Borrelli M, Maglio M, Agnese M, et al. High density of intraepithelial gammadelta lymphocytes and deposits of immunoglobulin (Ig)M anti-tissue transglutaminase antibodies in the jejunum of coeliac patients with IgA deficiency. Clin Exper Immunol 2010;160:199–206.

CHAPTER 34  Primary Antibody Deficiencies 26. Bonilla FA, Barlan I, Chapel H, et al. International Consensus Document (ICON): common variable immunodeficiency disorders. J Allergy Clin Immunol Pract 2016;4:38–59. 27. Yu JE, Knight AK, Radigan L, et al. Toll-like receptor 7 and 9 defects in common variable immunodeficiency. J Allergy Clin Immunol 2009;124:349–56, 56 e1–3. 28. Salzer U, Chapel HM, Webster ADB, et al. Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. Nat Genet 2005;37:820–8. 29. Wang Y, Hwangpo TA, Martin MP, et al. Killer cell immunoglobulin-like receptors are associated with common variable immune deficiency pathogenesis. J Allergy Clin Immunol 2016;138:1495–8. 30. Bogaert DJ, Dullaers M, Lambrecht BN, et al. Genes associated with common variable immunodeficiency: one diagnosis to rule them all? J Med Genet 2016;53:575–90. 31. Sathkumara HD, De Silva NR, Handunnetti S, et al. Genetics of common variable immunodeficiency: role of transmembrane activator and calcium modulator and cyclophilin ligand interactor. Int J Immunogenet 2015;42:239–53. 32. Zhang L, Radigan L, Salzer U, et al. Transmembrane activator and calcium-modulating cyclophilin ligand interactor mutations in common variable immunodeficiency: clinical and immunologic outcomes in heterozygotes. J Allergy Clin Immunol 2007;120:1178–85. 33. Yong PF, Thaventhiran JE, Grimbacher B. “A rose is a rose is a rose,” but CVID is Not CVID common variable immune deficiency (CVID), what do we know in 2011? Adv Immunol 2011;111:47–107. 34. Cheon CK, Ko JM. Kabuki syndrome: clinical and molecular characteristics. Korean J Pediatr 2015;58:317–24. 35. Malamut G, Verkarre V, Suarez F, et al. The enteropathy associated with common variable immunodeficiency: the delineated frontiers with celiac disease. Am J Gastroenterol 2010;105:2262–75. 36. Sneller MC, Strober W, Eisenstein E, et al. NIH conference. New insights into common variable immunodeficiency. Ann Intern Med 1993;118:720–30. 37. Chase NM, Verbsky JW, Hintermeyer MK, et al. Use of combination chemotherapy for treatment of granulomatous and lymphocytic interstitial lung disease (GLILD) in patients with common variable immunodeficiency (CVID). J Clin Immunol 2013;33:30–9.

487

38. Lefranc MP, Hammarstrom L, Smith CI, et al. Gene deletions in the human immunoglobulin heavy chain constant region locus: molecular and immunological analysis. Immunodefic Rev 1991;2:265–81. 39. Shackelford PG, Granoff DM, Polmar SH, et al. Subnormal serum concentrations of IgG2 in children with frequent infections associated with varied patterns of immunologic dysfunction. J Pediatr 1990;116:529–38. 40. Tabata N, Azuma E, Masuda S, et al. Transient low level of IgG3 induced by sepsis. Acta Paediatr Jpn 1995;37:201–2. 41. Morell A. IgG subclass deficiency: a personal viewpoint. Pediatr Infect Dis J 1990;9:S4–8. 42. Kutukculer N, Karaca NE, Demircioglu O, et al. Increases in serum immunoglobulins to age-related normal levels in children with IgA and/ or IgG subclass deficiency. Pediatr Allergy Immunol 2007;18:167–73. 43. Feeney AJ, Atkinson MJ, Cowan MJ, et al. A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease. J Clin Invest 1996;97:2277–82. 44. Bernier GM, Gunderman JR, Ruymann FB. Kappa-chain deficiency. Blood 1972;40:795–805. 45. Barandun S, Morell A, Skvaril F, et al. Deficiency of kappa- or lambda-type immunoglobulins. Blood 1976;47:79–89. 46. Stavnezer-Nordgren J, Kekish O, Zegers BJ. Molecular defects in a human immunoglobulin kappa chain deficiency. Science 1985;230:458–61. 47. Stiehm ER. The four most common pediatric immunodeficiencies. J Immunotoxicol 2008;5:227–34. 48. Moschese V, Graziani S, Avanzini MA, et al. A prospective study on children with initial diagnosis of transient hypogammaglobulinemia of infancy: results from the Italian Primary Immunodeficiency Network. Int J Immunopathol Pharmacol 2008;21:343–52. 49. Tiller TL Jr, Buckley RH. Transient hypogammaglobulinemia of infancy: review of the literature, clinical and immunologic features of 11 new cases, and long-term follow-up. J Pediatr 1978;92:347–53. 50. Dressler F, Peter HH, Muller W, et al. Transient hypogammaglobulinemia of infancy: five new cases, review of the literature and redefinition. Acta Paediatr Scand 1989;78:767–74. 51. Tiller TL Jr, Buckley RH. Transient hypogammaglobulinemia of infancy: review of the literature, clinical and immunologic features of 11 new cases, and long-term follow-up. J Pediatr 1978;92:347–53.

CHAPTER 34  Primary Antibody Deficiencies

487.e1

MULTIPLE-CHOICE QUESTIONS 1. A 50-year-old man presents with a history of two pneumonias in the last 2 years. He has suffered occasionally with sinusitis and bronchitis since childhood, but these infections have readily responded to antibiotic therapy, and he denies dyspnea on exertion. A grandson is receiving therapy for an unknown immune deficiency. Serum immunoglobulin (Ig) levels are drawn. IgM is 2 weeks). Invasive yeast (usually Candida spp.) infections are more commonly seen in patients with severe mucositis and neutropenia. Detailed guidelines for the use of antimicrobial agents in the setting of chemotherapy-induced neutropenic fever have been published.34 Although bacterial infections are the most common infections, when used as immunomodulators, a higher frequency of OIs, such as PJP, and infections with Listeria spp., NTM, Cryptococcus, and VZV, has been described.

Glucocorticoids Depending on the dose and duration, glucocorticoids can induce a vast range of immune defects through decreasing cytokine production (IL-1, IL-6, and tumor necrosis factor-α [TNF-α), and impairing neutrophil and lymphocyte trafficking and function. Infections are a frequent complication of corticosteroid use.32 Bacterial infections are the most common infectious complication, but opportunistic fungal, viral, and mycobacterial infections are also seen, particularly with high doses and long duration of systemic therapy. Some associated infections include GPB and GNB, superficial and invasive candidiasis, invasive aspergillosis and nocardiosis, cryptococcosis, PJP, listeriosis, endemic mycoses, tuberculosis (TB), and infections with NTM, and VZV, among others. Outside of the setting of SOT and HSCT, routine use of antimicrobials for patients receiving corticosteroids is not recommended, except for anti-TB treatment (e.g., isoniazid) for those with a positive tuberculin skin test (purified protein derivative [PPD]) or a positive IFN-γ release assay, and TMP-SMX in those receiving high-dose steroid for extended periods.35 Alternate-day use of corticosteroids has been associated with a lower risk of severe infection; therefore whenever possible, this dosing schedule should be used.31

Calcineurin Inhibitors and Mammalian Target of Rapamycin Inhibitors The calcineurin inhibitors, cyclosporine and tacrolimus, have been cornerstones in improving the outcome of transplant recipients by reducing T-cell function and thus preventing allograft rejection in SOT and GvHD in HSCT. Infectious complications, typically viral, seem to be dose dependent and seen more often when these drugs are used in combination with other immunosuppressants.32,33 The mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus have been associated with a lower incidence of CMV infection, compared with other immunosuppressive agents used in transplantation.32 Although impaired wound healing has been seen, this has not correlated with an increased incidence of infections.

528

Part four  Immunological Deficiencies

Mycophenolate Mofetil

Tumor Necrosis Factor-α Inhibitors

Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid, which inhibits inosine monophosphate dehydrogenase, a key enzyme in purine synthesis. It is a cytotoxic drug with antiproliferative effect on T and B lymphocytes.33 In addition to its potential myelosuppressive effect, it has been associated in SOT to a higher risk of CMV infection.32

TNF-α is a cytokine that plays a central role in macrophage and phagosome activation, differentiation of monocytes into macrophages, recruitment of neutrophils and macrophages, and granuloma formation and maintenance. Blockade results in an increased risk of infection, particularly in the first few months after treatment.37,38 Infections characterized by granulomas have been described most frequently, in particular TB and NTM infections, but also disseminated histoplasmosis and coccidioidomycosis.39 Other notable infections are listeriosis, legionellosis, salmonellosis, recurrent or relapsing leishmaniasis, overwhelming Plasmodium falciparum infection, and invasive mold infections (i.e., aspergillosis and mucormycosis). Risk of certain viral infections also appears to be increased, particularly VZV infection. Several cases of severe hepatitis B virus (HBV) reactivation in patients with positive surface antigen at the start of treatment have been reported, many of whom developed hepatic failure. Patients receiving anti–TNF-α therapy should be screened for TB (latent or active), HBV, and HCV before starting treatment, and treated accordingly.38 Patients with latent TB should receive isoniazid for 6–9 months. Ideally, patients would have received at least 3 weeks of isoniazid before therapy with biologicals; however, in truly urgent cases, this can be done simultaneously. In patients that are hepatitis B surface antigen (HbsAg) positive, baseline HBV DNA levels should be obtained and antiviral prophylaxis or treatment offered. All HBV-seronegative patients should be vaccinated against hepatitis B and seroconversion documented.

Antithymocyte Globulin Antithymocyte globulin (ATG) is a polyclonal Ig prepared by immunizing horses or rabbits with human thymocytes and harvesting the IgG. These antibodies produce a profound lymphopenia that can last well beyond 1 year. Used for induction or rejection therapy in transplantations, their use has been associated consistently with a greater risk of CMV and posttransplantation lymphoproliferative disease (PTLD).36 CMV prophylaxis or preemptive therapy is recommended in transplant recipients that receive ATG. Because these are foreign proteins, serum sickness can also develop between 1 and 2 weeks after treatment.

Monoclonal Antibodies and Small Molecules Monoclonal antibodies (mAbs) and small molecules target specific cellular steps, making it possible to manipulate disease pathways for previously untreatable illnesses. Many of these biological agents interfere with specific aspects of the immune system. Highlighted below are those that have a key infection susceptibility pattern. Others with more subtle increases in infections are also included in Table 37.2.

TABLE 37.2  Selected Monoclonal Antibodies and Small Molecules and Their Risk of Infection Drug Name

Target

Serious Infections

Adalimumab Certolizumab pegol Etanercept Golimumab Infliximab Rituximab

TNF-α*

TB, NTM infection, endemic mycoses, listeriosis, candidiasis, invasive mold infections, nocardiosis, bacterial infections, reactivation of VZV and HBV infections, severe malaria, and leishmaniasis

CD20

Anakinra Rilonacept Alemtuzumab

IL-1

HBV infection reactivation, PML, PCP, enteroviral meningoencephalitis, CMV disease, VZV infection, babesiosis, parvovirus infection, nocardiosis Bacterial infections (cellulitis and pneumonia)

Muromonab

CD3

Basiliximab Daclizumab

CD25

Natalizumab

α-4 Integrin

Abatacept Belatacept Alefacept

T-cell costimulation blockade

Brodalumab Bortezomib Gemtuzumab

IL 17 receptor A Proteasome inhibitor CD33

CD52

Inhibits T-cell activation

Herpes viruses reactivation (EBV, VZV, CMV, HHV-6), severe respiratory viral infections (with adenovirus, RSV, parainfluenza virus, influenza virus), PCP, invasive mold infections, histoplasmosis, cryptococcosis, PTLD, bacterial meningitis, toxoplasmosis, PML, parvovirus infection, nocardiosis, TB, NTM infection, acanthamebiasis, and Balamuthia mandrillaris infection Bacterial infections; listeriosis; nocardiosis; aspergillosis; candidiasis; cryptococcosis; toxoplasmosis; infections with CMV, HSV, VZV, adenovirus, RSV, and enteroviruses; and PCP Bacterial infections; infections with CMV and HSV; EBV-associated PTLD; infections with RSV, adenovirus, influenza, and BK virus; invasive mold infections; candidiasis; nocardiosis; TB; NTM infection; toxoplasmosis PML, respiratory viral and bacterial infections, UTI, viral meningitis and encephalitis, CMV infection, IA, PCP, VZV infection, NTM infection, cryptosporidiosis Bacterial infections, EBV-associated PTLD 1 case of Mycobacterium avium intracellulare (MAI) bursitis, 1 case of nocardiosis when coadministered with infliximab Mucocutaneous candidiasis Herpes zoster Bacteremia, bacterial pneumonia, and HSV reactivation.

*TNF-α, tumor necrosis factor-α; IL-1, interleukin-1; TB, tuberculosis; NTM, nontuberculous mycobacteria; VZV, varicella-zoster virus; HBV, hepatitis B virus; PML, progressive multifocal leukoencephalopathy; PCP, Pneumocystis jiroveci pneumonia; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HHV-6, human herpes virus 6; RSV, respiratory syncytial virus; PTLD, posttransplantation lymphoproliferative disease; HSV, herpes simplex virus; UTI, urinary tract infection; IA, invasive aspergillosis.

CHAPTER 37  Infections in the Immunocompromised Host Rituximab Rituximab is a chimeric murine–human mAb that targets CD20 on mature B lymphocytes. It results in rapid and profound depletion of B cells that can last up to several months.40 In most adults, serum Ig levels remain largely stable, since plasma cells do not express CD20. Initial trials suggested that rituximab had minimal effect on the occurrence of infections; however, more recent meta-analyses have reported a higher risk of infections, especially with repeated administration and in patients with underlying immune defects or concomitant significant immunosuppression.40 HBV reactivation occurs, and there have been reports of fulminant hepatitis and death after rituximab treatment, particularly when used in combination therapy (e.g., rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]). Also a reverse seroconversion phenomenon has been described, with loss of protective HBV surface antibodies and reactivation.38 Assessment of HBV status before starting rituximab is recommended. To reduce the risk of reactivation, HBV suppression with antiviral medication should be considered. Since the initial approval of rituximab, there have been over 280 cases of PML associated with its use. Most cases have been reported in patients with hematological malignancies; however, some have been reported in patients with autoimmune or inflammatory diseases, who take other immunosuppressants as well. Rituximab now carries a black box warning about the risk of PML and death.41 There have been many reports of PJP following use of rituximab, although most patients also received other immunosuppressive therapies. The need for PJP prophylaxis with its use remains to be determined. The greatest risk of infection appears to be related to viral reactivation, particularly in children; as usual, this is confounded by prior or concomitant immunosuppressive medications. In children with immune-mediated neurological diseases, infections after rituximab use are common; even lethal CMV disease has been reported.42 Other rare infections that have been described are enteroviral meningoencephalitis, CMV disease, disseminated VZV infection, refractory babesiosis, parvovirus B19 infection, and nocardiosis.37

Alemtuzumab Alemtuzumab is a humanized IgG1 that targets CD52 on B and T lymphocytes, monocytes, macrophages, and NK cells. It lyses these cell populations and results in profound and sustained deficits in cellular and humoral immunity that lasts for several months; CD4 and CD8 cell count reach their nadir approximately 4 weeks after treatment, but median counts remain at 24 months), and had prior use of immunosuppressants (11.1 cases/1000 patients [95% confidence interval (CI) 8.3–14.5]). The incidence in patients with no detectable anti-JC virus antibodies is calculated at 0.09 cases/1000 patients (95% CI 0–0.48).43 Increases in bacterial and viral infections and OIs have been reported as well, but most patients with these infections were receiving concurrent immunosuppression.

Bortezomib Bortezomib is a dipeptide boronate proteasome inhibitor that causes G2–M cell cycle arrest and apoptosis, which ultimately impacts T-cell immunity. It is now approved for the treatment of multiple myeloma and mantle cell non-Hodgkin lymphoma. An increased incidence of herpes virus infections, in particular VZV infection, has been reported. The incidence in patients

530

Part four  Immunological Deficiencies

with myeloma treated with bortezomib is around 13–22% and in relapsed/refractory mantle cell lymphoma around 10.3%, with age ≥65 years as the only predictive factor.44

INFECTIONS IN SOLID ORGAN TRANSPLANTATION KEY CONCEPTS Determinants of the Risk for Infections in Transplant Recipients • Epidemiological exposures (e.g., residing in a Histoplasma endemic area) • Immunosuppressive drugs • Antimicrobial prophylaxis • Type of transplantation (e.g., cord transplant vs matched related donor transplantation) • Time after transplantation

Despite significant improvement in the survival of SOT recipients, infectious complications remain a major cause of morbidity and mortality. The risk of infection is determined by the interaction of various factors, such as age of the recipient, type of transplantation, invasive procedures, dose and duration of immunosuppressive drugs, epidemiological exposures of both donor and recipient, use of antimicrobial prophylaxis, donor–recipient serological status to certain infections (e.g., CMV infection, EBV infection, toxoplasmosis), and ongoing viral replication (so-called indirect effects).45 Assessment of a recipient’s risk for infection should help tailor specific prophylactic strategies and infectious workup when an infection is suspected. Age is an important determinant of susceptibility to infections; it impacts the likelihood of prior exposures to microbial pathogens, either by primary infection or vaccination. History of exposure can have either positive or negative effects. Older patients are more likely to have encountered pathogens that can remain latent and reactivate at the time of transplantation (e.g., CMV); younger patients have a higher risk of acquiring primary infections after transplantation, and these infections tend to be more severe than disease secondary to reactivation of a latent infection. In addition, preexisting immunity can have a protective effect against clinical disease (e.g., EBV-associated PTLDs).45 The type of allograft affects the specific infectious risk, usually as a result of technical factors associated with the transplantation procedure, but is also inherent to the transplanted organ. For example, urinary tract infections are most common after kidney transplantation, as a result of either catheter placement or ureteric stenting. BK virus is ubiquitous, but BK virus–associated transplant nephropathy is most common after kidney transplantation, and it is an important cause of allograft loss. Infection after liver transplantation frequently results from leaks from biliary and GI anastomoses. Cardiac assist devices in heart transplant recipients are a frequent source of infection. Tracheal anastomotic infections, particularly caused by fungi, are a significant complication of lung transplantation. In single lung transplantations, recurrent infections of the native lung, such as with GNB or fungi, can extend to the transplanted lung. In small intestine transplantation, opportunistic and nonopportunistic viral infections of the GI tract (e.g., with norovirus) are common; these can be severe and even life threatening.45 Despite all the differences in individual risk of infection, the general patterns of infection in the absence of antimicrobial

intervention are similar among those undergoing SOT. This predictable temporal pattern has enabled the institution of specific prophylactic strategies and in the development of an infection differential diagnosis.45

CLINICAL PEARLS Infections in Solid-Organ Transplant Recipients • Most infections occur in the first 6 months following transplantation. • In the first month, most infections are nosocomial or are related to surgical procedures or preexisting infection in the donor or recipient. • Infections between 1 and 6 months following transplantation are commonly secondary to the use of immunosuppressive drugs, with a predominance of cytomegalovirus infection. • Infections after 6 months are usually due either to chronic viral infection, acquired earlier, or to chronic allograft dysfunction, necessitating repeated courses of high-dose immunosuppression.

Infections in the First Month After Transplantation Infections in this period are generally associated with technical complications of the surgery or have a nosocomial etiology. Donor-transmitted infections (e.g., Chagas disease) or reactivation of prior infections (e.g., colonization and infection with multidrug-resistant [MDR] bacteria) can also occur.45

Infections 1–6 Months After Transplantation Before routine antimicrobial prophylaxis, this period was typically characterized by the presence of OIs, such as PJP and CMV infection; the incidence of these infections has been significantly reduced or delayed with TMP-SMX prophylaxis and CMV prophylactic or preemptive therapy. Reactivation of latent infections, such as TB, Chagas disease, endemic mycoses, and cryptococcosis, can occur. Viral infections, such as those with BK virus, adenovirus, RSV, HBV, and EBV, are common (Fig. 37.4). Invasive fungal infections, specifically Aspergillus and nonAspergillus mold infections, can be problematic during this period of heightened immune suppression.45

Infections 6 Months After Transplantation This period is less well defined; patients with satisfactory allograft function develop more severe manifestations of communityacquired infections but have a lower risk of OIs. Patients with poor allograft function and, therefore, increased immunosuppression or those with ongoing chronic viral reactivation are at high risk of OIs, such as invasive fungal infections, late-onset CMV infection, nocardiosis, PJP, listeriosis, and EBV-associated PTLD. In addition, lung transplant recipients with chronic graft dysfunction are at high risk of recurrent bacterial pneumonia. Similarly, liver transplant recipients with chronic graft dysfunction frequently develop biliary strictures and recurrent cholangitis.45

INFECTIONS IN HEMATOPOIETIC STEM CELL TRANSPLANTATION Survival after HSCT has significantly improved during the last decade. Factors contributing to the increase in survival are the use of less toxic, nonmyeloablative conditioning regimens, use of peripheral blood stem cells (PBSCs) that leads to faster neutrophil recovery, and use of antimicrobial preventive therapies.46 Despite these advances, infections are still a frequent cause of morbidity and mortality.

CHAPTER 37  Infections in the Immunocompromised Host

A

B

531

C

FIG 37.4  Disseminated Histoplasmosis in a Patient from an Endemic Area 3 Months After a Heart Transplantation. (A) Chest X-ray with bilateral reticulonodular infiltrates. (B) Computed tomography (CT) scan of the chest with bilateral patchy and nodular infiltrates. (C) Lung biopsy. Gomori methenamine silver (GMS) stain shows yeast forms compatible with Histoplasma capsulatum.

CLINICAL PEARLS Infections in Bone Marrow Transplant Recipients • Infections 2–4 weeks posttransplantation are usually caused by profound neutropenia and damage to mucosal surfaces. • Between the period of engraftment and posttransplant weeks 15–20, OIs predominate and are commonly associated with the development of acute graft-versus-host disease (GvHD) and its treatment. • Serious infections occurring 4–6 months following transplantation are seen predominantly in patients with chronic GvHD.

The risk for infection is influenced by several factors and varies significantly between type of transplantation (autologous vs allogeneic), stem cell source (bone marrow, PBSC, and cord blood), degree of human leukocyte antigen (HLA) matching, hematopoietic potential of the graft (number of infused CD34+ cells), T-cell depletion, type of conditioning (myeloablative vs reduced intensity vs nonmyeloablative), underlying disease, presence of graft rejection or GvHD, donor–recipient serological status to certain infections (e.g., CMV infection), and use of antimicrobial prophylaxis.47 Autologous HSCT (auto-HSCT) refers to the patient serving as his or her own donor to allow administration of myeloablative chemotherapy. Because there is no risk of rejection or GvHD and because after engraftment there is an early and progressive recovery of cell-mediated immunity, the risk for infection is much lower than in allo-HSCT. The major compromises in host defenses occur before engraftment and are usually related to neutropenia and mucosal injury. In contrast, in allo-HSCT, the risk for infection can be divided into three periods: preengraftment, early postengraftment, and late postengraftment periods.

Preengraftment Period The major impairments in host defenses before engraftment are neutropenia and mucosal injury. This period varies widely between different types of conditioning regimens and stem-cell source. In general, when ablative regimens are used, engraftment occurs any time between 15 and 45 days after transplantation and can be as short as 5–7 days with nonablative regimens.47 PBSC grafts

FIG 37.5  Magnetic resonance imaging (MRI) scan of the brain of a patient with human herpesvirus-6 (HHV-6) encephalitis 36 days after a matched unrelated donor hematopoietic stem cell transplantation (HSCT) for diffuse large B-cell lymphoma. Flair axial image shows bilateral medial temporal lobe edema compatible with limbic encephalitis.

are associated with faster neutrophil engraftment compared with cord blood units and therefore lower rates of infection.46 Bacterial infections are most common. However, when neutropenia is prolonged, the risk of invasive fungal infection increases significantly. Without acyclovir prophylaxis, reactivation of severe HSV infection is frequent.

Early Postengraftment Period The resolution of neutropenia marks the beginning of this period and usually lasts 2–3 months (Fig. 37.5). This period is characterized by a progressive but slow recovery of B- and T-lymphocyte function; thus viral and fungal OIs can occur. The risk of infection is higher in patients who develop acute GvHD and therefore require high dose steroids. Common bacterial infections can still occur and are usually related to indwelling catheters or GvHD

532

Part four  Immunological Deficiencies

of the gut. CMV viremia is common during this period. However, routine use of preemptive or prophylactic therapy has been effective in preventing life-threatening CMV disease. The risk of Pneumocystis pneumonia (PCP) has also decreased since the introduction of PCP prophylaxis.47

Late Postengraftment Period This is the period of late immune recovery and ends when the patient regains normal immunity. In the absence of chronic GvHD, this period can last up to 2 years. Viral and bacterial infections of the respiratory tract are common during this period. The development of chronic GvHD delays immune restoration and can extend this period for many years (as long as immunosuppressive drugs are required). Infections with encapsulated bacteria are common in patients with chronic GvHD, as well as opportunistic viral and fungal infections, including VZV and CMV infections, invasive aspergillosis and non-Aspergillus mold infections, and PJP, among others.47

INFECTIONS OF PARTICULAR IMPORTANCE IN TRANSPLANT RECIPIENTS Cytomegalovirus Infection CMV is one of the most important pathogens affecting transplant recipients. It is widely distributed in the general population; seropositivity for CMV varies in different geographical regions and ranges from 30% to 97%.48 Before widespread use of interventions aimed at reducing the incidence of CMV disease (i.e., pneumonitis, gastroenteritis, hepatitis, etc.), it occurred frequently during the first 3 months after transplantation. In SOT, the highest risk for CMV disease is in patients in whom the donor is seropositive and the recipient is seronegative for CMV (D+/R−). CMV has a predilection to invade the allograft, possibly as a result of an aberrant immune response within the graft. The risk of infection varies also with the type of transplantation; recipients of lung, small intestine, and pancreas transplants have a higher risk, whereas the risk in liver and kidney recipients is lower. In SOT, CMV infection has been found consistently to be an independent risk factor for other infectious complications as well as graft rejection.48 In HSCT, infection is usually the result of reactivation of endogenous virus; hence the highest risk is in seropositive recipients. Without prophylaxis, up to 80% of seropositive recipients will experience CMV infection after HSCT. Seronegative individuals have a 30–40% chance of becoming infected when receiving unfiltered blood products or stem cells from a seropositive donor. Risk factors for CMV disease are acute and chronic GvHD, use of high-dose corticosteroids, use of cord blood, T-cell depletion, and use of mismatched or unrelated donors.49 Typically, CMV viremia precedes CMV disease by 1–2 weeks; therefore close monitoring of CMV reactivation by polymerase chain reaction (PCR) assays or pp65 antigenemia allows the detection of early replication, and therefore institution of appropriate antiviral therapy (ganciclovir, valganciclovir, or foscarnet) before the development of end organ disease. This approach, termed preemptive therapy, has the advantage of effectively decreasing the incidence of early CMV disease and limiting drug-related toxicities. In addition, a limited amount of viral replication may allow for the development of CMV-specific immune reconstitution. Preemptive therapy is the preferred method for preventing CMV disease in HSCT49 and is widely used in SOT in patients with intermediate risk for CMV disease.

In high-risk solid-organ recipients (D+/R−), in particular recipients of lung and small bowel, although preemptive therapy is an accepted alternative, universal prophylaxis is preferred. It has the theoretical advantage of preventing reactivation of other herpes viruses and may be more likely to prevent the indirect effects of CMV.48,50 Recently, the development of commercially available CMV- specific IFN-γ release assays, which detect CMVspecific T cells in whole blood, may allow for more precise targeting of prophylactic strategies, beyond those currently used.55

Other Herpes Viruses HSV infection is common in both SOT and HSCT recipients during the first month after transplantation. Routine use of antiviral prophylaxis has successfully decreased the incidence of severe HSV infection. However, breakthrough mucocutaneous HSV infection and even disseminated disease can still occur. Reactivation of VZV infection is also common; however, acyclovir prophylaxis has decreased the incidence of disseminated VZV infection significantly. Both severe VZV and HSV infections are typically treated with intravenous acyclovir.45,46 Although EBV can cause a wide spectrum of disease, EBVassociated PTLD is the most feared complication. The term EBV-associated PTLD is generally used to describe a heterogeneous group of clinical syndromes associated with uncontrolled lymphoproliferation, which can result in true malignancies containing clonal chromosomal abnormalities. Early diagnosis requires a high index of suspicion. EBV viral load monitoring and radiological evaluation can assist in early diagnosis. Reduction of immunosuppression should be the initial strategy in managing the disease. Timing of additional therapies, such as treatment with antivirals and rituximab and chemotherapy, remains controversial.45,47 HHV-6 has been associated with disease after transplantation, and although a variety of clinical manifestations have been described, the more convincing association is with encephalitis. The initial presentation may be subtle with memory loss or disorientation. However, it may progress to severe mental status abnormalities and seizures. The diagnosis is established with a positive PCR in the CSF, and absence of other infectious agents. Treatment is with ganciclovir or foscarnet.47

Invasive Filamentous Fungal Infections Invasive pulmonary aspergillosis is the most common invasive filamentous fungal infection. Dissemination, including to the brain, can occur and should be ruled out in all transplant recipients. Risk factors are prolonged neutropenia, high-dose corticosteroids, cord blood transplantation, CMV disease, GvHD, graft rejection, and lung transplantation. Manifestations of filamentous fungi that are almost exclusively seen in lung transplant recipients are tracheobronchitis, characterized by ulceration and cartilage invasion, and bronchial anastomotic infections.51 Monitoring serum Aspergillus galactomannan in high-risk patients, as well as computed tomography (CT) imaging, can assist in the early diagnosis of invasive aspergillosis. Despite early diagnosis and treatment, the mortality associated with invasive aspergillosis, in particular in HSCT, is still high.51 Antifungal prophylaxis should be considered in patients in whom prolonged neutropenia is expected, in patients with chronic GvHD receiving high-dose corticosteroids, and in lung transplant recipients. Voriconazole is considered the drug of choice for invasive aspergillosis, but newer-generation azoles, such as posaconazole and isavuconazole, are good alternatives.

CHAPTER 37  Infections in the Immunocompromised Host Fusariosis, mucormycosis, and dematiaceous and other mold infections are increasingly recognized as significant pathogens in transplant recipients. Clinical disease can be similar to the infection caused by Aspergillus spp., with largely sinopulmonary disease. Fusarium spp. frequently disseminate hematogenously and can be isolated in blood cultures. Many of these infections are resistant to most antifungal drugs and remain an important cause of transplantation-related mortality.51

Invasive Candidiasis Before routine use of antifungal prophylaxis, invasive candidiasis was the most common invasive fungal infection in transplant recipients. In SOT, most Candida infections occur during the first month after transplantation, usually related to the surgical procedure. Recipients of liver, pancreas, and small bowel are at high risk for invasive candidiasis; lung transplant recipients can develop bronchial anastomotic infections.51 In HSCT, most Candida infections occur in the preengraftment period and are associated with mucosal injury, which results from the conditioning regimen and with the widespread use of antibiotics for the treatment of fever and neutropenia. Fluconazole-resistant Candida spp. are now increasingly isolated, and echinocandins (caspofungin, micafungin, and anidulafungin) are now considered the drugs of choice in invasive candidiasis.52 Antifungal prophylaxis reduces the incidence of invasive fungal infections after HSCT, as well as infection-related and overall mortality. In SOT, antifungal prophylaxis is recommended for high-risk liver transplant recipients and in small bowel and lung transplant recipients.

TRANSLATIONAL RESEARCH ON THE HORIZON • Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus infections remain problematic infections in both hematopoietic-stem-cell and solid-organ transplant recipients, and medications currently available to treat these infections have significant associated toxicities or lack efficacy. • Adoptive immunotherapy, in which virus specific donor-derived T cells are expanded and infused into the transplant recipient, will likely become more widespread in treating these infections.

Viral reactivation and disease are still a major cause of morbidity and mortality among transplant recipients. CMV, EBV, and adenovirus are particularly problematic once disease develops. Preemptive strategies have substantially decreased the incidence of invasive CMV disease; nevertheless, a significant number of patients still go on to develop invasive disease. Preemptive therapy and treatment are limited by the toxicity associated with the available antivirals and their lack of efficacy against EBV and adenovirus. In addition, development of resistance can complicate therapy. Development of new effective antivirals has been slow. Brincidofovir (CMX001) is an ether lipid ester analogue of cidofovir that has been reported to be 1000-fold more potent than cidofovir against DNA viruses, such as CMV and adenovirus. It is available in an oral formulation, but it lacks the nephrotoxic effects of cidofovir.53 However, a recent trial in high-risk recipients in HSCT, in which brincidofovir was used to prevent CMV infection, did not achieve its primary endpoint for the prevention of clinically significant CMV infection after transplantation.53 There are ongoing clinical trials to determine the efficacy and safety of brincidofovir for the treatment of other DNA viruses,

533

such adenovirus and smallpox virus. Letermovir is a new, highly potent anti-CMV agent with a novel mechanism of action targeting the viral terminase subunit pUL56, a component of the terminase complex involved in viral DNA cleavage and packaging. Initial trials have shown that letermovir is effective in reducing the incidence of CMV infection in allo-HSCT and has been used successfully in MDR infections.54 Phase III clinical trials are currently underway. There are other antivirals in the pipeline, but they are at much earlier stages of development. Because of the scarcity of effective, nontoxic antivirals and because uncontrolled viral infections correlate with a lack of cellular immunity against viruses, several groups are working on the development of adoptive immunotherapy, which is the artificial reconstitution of virus-specific T cells with in vitro expanded T cells. A number of small studies have now shown that virus-specific T cells are effective in controlling CMV, EBV, and adenovirus infections in transplant recipients. The translation of adoptive immunotherapy into the clinic has been limited by technical difficulties associated with the generation of viral-specific cells that are not alloreactive, that can be produced from naïve T cells, and that can be developed rapidly and in a cost-effective way. Cells obtained from the original donor can yield long-lasting immunity, but the process of generating these cells can be cumbersome and often not available in a timely manner. Some groups have explored adoptive transfer of HLA partially matched virus-specific T cells derived from third-party donors, that is, healthy individuals other than the donor of the patient’s transplant. Such cells yield high number of virus-specific T-cells, but these cells tend to be short lived.55 Currently, a few international centers have established cryopreserved banks with virus-specific T-cell lines from normal donors, but these are still mostly for investigational use. Significant advances continue to be seen in the field of cellular therapy, and these could become part of the armamentarium against viruses in the next decade.

CONCLUSIONS Immunocompromised hosts are surviving longer as antimicrobials improve and as immunosuppressant agents become less toxic. Both monogenetic primary immunodeficiencies and the increasingly specific immunosuppressant agents allow insight into host control of specific microbes. With this knowledge, new types of immunodeficiencies are being recognized, such as the autoantibody cytokine disorders described above. In addition, the knowledge of specific host immunity will help in developing therapies to boost host responses in the increasing challenges of treatment-resistant microbes. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Holland SM. Chronic granulomatous disease. Clin Rev Allergy Immunol 2010;38:3–10. 2. Rosenzweig SD, Holland SM. Phagocyte immunodeficiencies and their infections. J Allergy Clin Immunol 2004;113:620–6. 3. Greenberg DE, Ding L, Zelazny AM, et al. A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease. PLoS Pathog 2006;2:e28. 4. Siddiqui S, Anderson VL, Hilligoss DM, et al. Fulminant mulch pneumonitis: an emergency presentation of chronic granulomatous disease. Clin Infect Dis 2007;45:673–81.

534

Part four  Immunological Deficiencies

5. Freeman AF, Marciano BE, Anderson VL, et al. Corticosteroids in the treatment of severe Nocardia pneumonia in chronic granulomatous disease. Pediatr Infect Dis J 2011;30:806–8. 6. Leiding JW, Freeman AF, Marciano BE, et al. Corticosteroid therapy for liver abscess in chronic granulomatous disease. Clin Infect Dis 2012;54:694–700. 7. Freeman AF, Holland SM. Antimicrobial prophylaxis for primary immunodeficiencies. Curr Opin Allergy Clin Immunol 2009;9:525–30. 8. Rondeau S, Couderc L, Dominique S, et al. High frequency of voriconazole-related phototoxicity in cystic fibrosis patients. Eur Respir J 2012;39:782–4. 9. Tarlock K, Johnson D, Cornell C, et al. Elevated fluoride levels and periostitits in pediatric hematopoietic stem cell transplant recipients receiving long-term voriconazole. Pediatr Blood Cancer 2015;62:918–20. 10. Boztug K, Klein C. Genetic etiologies of severe congenital neutropenia. Curr Opin Pediatr 2011;23:21–6. 11. Hsu AP, McReynolds LJ, Holland SM. GATA2 deficiency. Curr Opin Allergy Clin Immunol 2015;15:104–9. 12. Hambleton S, Salem S, Bustamante J, et al. IRF8 mutations and human dendritic-cell immunodeficiency. N Engl J Med 2011;365:127–38. 13. Conley ME, Dobbs AK, Farmer DM, et al. Primary B cell immunodeficiencies: comparisons and contrasts. Annu Rev Immunol 2009;27:199–227. 14. Cuccherini B, Chua K, Gill V, et al. Bacteremia and skin/bone infections in two patients with X-linked agammaglobulinemia caused by an unusual organism related to Flexispira/Helicobacter species. Clin Immunol 2000;97:121–9. 15. Murray PR, Jain A, Uzel G, et al. Pyoderma gangrenosum-like ulcer in a patient with X-linked agammaglobulinemia: identification of Helicobacter bilis by mass spectrometry analysis. Arch Dermatol 2010;146:523–6. 16. Van der Burg M, Gennery AR. Educational paper: the expanding clinical and immunological spectrum of severe combined immunodeficiency. Eur J Pediatr 2011;170:561–71. 17. Kobrynski LI, Sullivan KE. Velocardiofacial syndrome, DiGeorge syndrome: the chromosome 22q11.2 deletion syndromes. Lancet 2007;1443–52. 18. Perez EE, Bokszczanin A, McDonald-McGinn D. Safety of the live viral vaccines in patients with chromosome 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome). Pediatrics 2003;112:e325. 19. Milner JD, Brenchley JM, Laurence A, et al. Impaired T(H) 17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome. Nature 2008;452:773–6. 20. Freeman AF, Holland SM. Clinical manifestations, etiology, and pathogenesis of the hyper-IgE syndromes. Pediatr Res 2009;65:32R–37R. 21. Odio CD, Milligan KL, McGowan K, et al. Endemic mycosis in patients with STAT3-mutated hyper IgE (Job) syndrome. J Allergy Clin Immunol 2015;136:1411–13. 22. Zhang Q, Davis J, Lamborn IT, et al. Combined immunodeficiency associated with DOCK8 mutations. N Engl J Med 2009;361:2046–55. 23. Al-Muhsen S, Casanova JL. The genetic heterogeneity of mendelian susceptibility to mycobacterial diseases. J Allergy Clin Immunol 2008;122:1043–51. 24. Holland SM. Interferon gamma, IL-12, IL-12R and STAT1 immunodeficiency diseases: disorders of the interface of innate and adaptive immunity. Immunol Res 2007;38:342–6. 25. Toubiana J, Okada S, Hiller J, et al. Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype: an international survey of 274 patients from 167 kindreds. Blood 2016;127:3154–64. 26. Ram S, Lewis LA, Rice PA. Infections of people with complement deficiencies and patients who have undergone splenectomy. Clin Microbiol Rev 2010;23:740–80. 27. Browne SK, Holland SM. Anticytokine autoantibodies in infectious diseases: pathogenesis and mechanisms. Lancet Infect Dis 2010;10:875–85. 28. Kisand K, Boe Wolff AS, Podkrajsek KT. Chronic mucocutaneous candidiasis in APECED or thymoma patients correlate with autoimmunity to Th17 associated cytokines. J Exp Med 2010;207:299–308. 29. Puel A, Doffinger R, Natividad A, et al. Autoantibodies against IL-17A, IL-17F, and Il-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type 1. J Exp Med 2010;207:291–7.

30. Rosen LB, Freeman AF, Yang LM, et al. Anti-GM-CSF autoantibodies in patients with cryptococcal meningitis. J Immunol 2013;190:3959–66. 31. Allison AC. Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 2000;47:63–83. 32. Mueller NJ. New immunosuppressive strategies and the risk of infection. Transpl Infect Dis 2008;10:379–84. 33. Halloran PF. Immunosuppressive drugs for kidney transplantation. N Engl J Med 2004;351:2715–29. 34. Freifeld AG, Bow EJ, Sepkowitz KA, et al. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2011;52:e56–93. 35. Kang I, Park SH. Infectious complications in SLE after immunosuppressive therapies. Curr Opin Rheumatol 2003;15:528–34. 36. Gaber AO, Knight RJ, Patel S, et al. A review of the evidence for use of thymoglobulin induction in renal transplantation. Transplant Proc 2010;42:1395–400. 37. Salvana EM, Salata RA. Infectious complications associated with monoclonal antibodies and related small molecules. Clin Microbiol Rev 2009;22:274–90. 38. Koo S, Marty FM, Baden LR. Infectious complications associated with immunomodulating biologic agents. Infect Dis Clin North Am 2010;24:285–306. 39. Vergidis P, Avery RK, Wheat LJ, et al. Histoplasmosis complicating tumor necrosis factor–α blocker therapy: a retrospective analysis of 98 cases. Clin Infect Dis 2015;61:409–17. 40. Gea-Banacloche JC. Rituximab-associated infections. Semin Hematol 2010;47:187–98. 41. www.accessdata.fda.gov/drugsatfda_docs/ label/2012/103705s5367s5388lbl.pdf. 42. Dale RC, Brilot F, Duffy LV, et al. Utility and safety of rituximab in pediatric autoimmune and inflammatory CNS disease. Neurology 2014;83:142–50. 43. Bloomgren G, Richman S, Hotermans S, et al. Risk of natalizumab-associated progressive multifocal leukoencephalopathy. N Engl J Med 2012;366:1870–80. 44. Piperdi B, Ling Y, Liebes L, et al. Bortezomib: understanding the mechanism of action. Mol Cancer Ther 2011;10:2029–30. 45. Fishman JA, Issa NC. Infection in organ transplantation: risk factors and evolving patterns of infection. Infect Dis Clin North Am 2010;24:273–83. 46. Gooley TA, Chien JW, Pergam SA, et al. Reduced mortality after allogeneic hematopoietic-cell transplantation. N Engl J Med 2010;363:2091–101. 47. Wingard JR, Hsu J, Hiemenz JW. Hematopoietic stem cell transplantation: an overview of infection risks and epidemiology. Infect Dis Clin North Am 2010;24:257–72. 48. Humar A, Snydman D. Cytomegalovirus in solid organ transplant recipients. Am J Transplant 2009;9:S78–86. 49. Ljungman P, Hakki M, Boeckh M. Cytomegalovirus in hematopoietic stem cell transplant recipients. Infect Dis Clin North Am 2010;24:319–37. 50. Kotton CN, Kumar D, Caliendo AM, et al. Updated International Consensus Guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 2013;96:333–60. 51. Nucci M, Anaissie E. Fungal infections in hematopoietic stem cell transplantation and solid-organ transplantation—focus on aspergillosis. Clin Chest Med 2009;30:295–306, vii. 52. Pappas PG, Kauffman CA, Andes DR, et al. Clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 2016;62:e1–50. 53. Marty FM, Winston DJ, Rowley SD, et al. CMX001 to prevent cytomegalovirus disease in hematopoietic-cell transplantation. N Engl J Med 2013;369:1227–36. 54. Chemaly RF, Ullmann AJ, Stoelben S. Letermovir for cytomegalovirus prophylaxis in hematopoietic-cell transplantation. N Engl J Med 2014;370:1781–9. 55. O’Reilly RJ, Prockop S, Hasan AN, et al. Virus-specific T-cell banks for “off the shelf ” adoptive therapy of refractory infections. Bone Marrow Transplant 2016;51:1163–72.

CHAPTER 37  Infections in the Immunocompromised Host

534.e1

MULTIPLE CHOICE QUESTIONS 1. Mucocutaneous candidiasis is thought to occur due to disruption of interleukin-17 (IL-17) signaling. This is observed in all of the following immunodeficiencies EXCEPT: A. Loss of function mutations in signal transducer and activator of transcription 3 (STAT3) causing hyper IgE syndrome (HIES) B. CD40 ligand deficiency C. Gain-of-function (GOF) STAT1 mutations D. Autoimmune polyendocrinopathy with candidiasis and ectodermal dysplasia (APOCED) 2. Anticytokine autoantibodies are increasingly being recognized as causing immunodeficiency. The following are described associations EXCEPT: A. Anti-interferon (IFN)-γ antibodies and disseminated nontuberculous mycobacteria B. Anti–GM-CSF antibodies and Cryptococcus C. Anti-IL-17 antibodies and mucocutaneous Candida D. Anti-IFNγ antibodies and Staphylococcus aureus E. Anti–granulocyte macrophage–colony-stimulating factor (GM-CSF) antibodies and Nocardia

3. Progressive multifocal leukoencephalopathy has been described in the following settings EXCEPT: A. Rituximab B. GOF STAT1 mutations C. Natalizumab D. Chronic granulomatous disease (CGD) E. Human immunodeficiency virus (HIV) F. Dedicator of cytokinesis 8 (DOCK8) deficiency 4. Photosensitivity is associated with which antifungal? A. Fluconazole B. Caspofungin C. Posaconazole D. Voriconazole E. Isavuconazole

38  Immune Deficiencies at the Extremes of Age Jӧrg J. Goronzy, Claire E. Gustafson, Cornelia M. Weyand

Age is a major factor determining the quality and quantity of an immune response. This age dependency, apparent throughout life, is most obvious at the extremes of age. Both infants and older adults exhibit blunted immune responses to infections and vaccination. However, the underlying mechanisms for immune dysfunction are distinct. Infants are born with limited antigen exposure and an immature immune system, which, however, progressively develops throughout infancy and childhood. The mechanisms underlying the immune dysfunction in older adults are broadly termed immunosenescence. The term is reminiscent of cellular senescence, which is defined as the loss of the ability to proliferate as a result of an irreversible cell cycle block. However, it is misleading to interpret immune aging only as the accumulation of senescent cells, and similar to aging in general, numerous pathways are involved.1 The decline in immune competence is not linear. As early as age 40 years, vaccine responses to selected vaccines (e.g., hepatitis B) start to decline. The incidence rate of herpes zoster, a reactivation of latent varicellazoster virus (VZV), starts to increase at age 50 years, as does morbidity and mortality from influenza infection. A more abrupt transition appears to occur in the eighth decade of life. In terms of public health, infections are major causes of morbidity in the very young and the very old. Although child mortality rates have dropped by almost 50% between 1990 and 2013, pathogenic infections are still one of the largest causes of infant mortality worldwide, accounting for more than 1.6 million deaths a year. Vaccinations have helped change the infectious landscape in children and young adults, but certain vaccines, such as those against Streptococcus pneumoniae, still have limited protective capacity at both extremes of age. Susceptibility to pathogenic infection and ineffectiveness of vaccination is even greater in older adults than in young infants. Moreover, during aging, a functional immune system is important for tissue repair and is of increasing importance in degenerative diseases, and it is vital for cancer surveillance. Immunosenescence is of increasing importance because of the changing population demographics, with increases in the number of individuals older than 65 years in both developed and developing countries.

INFANCY AND THE GENERATION OF AN IMMUNE SYSTEM Infants first start to develop their immune system in utero, maintaining a fine balance between immunological tolerance that helps prevent proinflammatory responses in utero and the ability to respond to foreign antigen exposure upon birth. Immunosuppressive regulatory pathways utilized by the fetus

for protection against possible infections and maternal–fetal rejection in utero are still reflected in the newborn infant immune system, characterized by immune-suppressive, antiinflammatory, and immature cellular responses to foreign antigens. Although newborns and young infants lack the ability to mount effective immune responses against many pathogens, they acquire initial immune protection through the passive transfer of maternal immunoglobulin G (IgG) in utero via the placenta (termed “passive immunity”)2 and from secretory IgA and antimicrobial factors present in maternal breast milk. Alternations in passive immunity (e.g., preterm birth) can lead to increased susceptibility to infections and breakdown of immune tolerance in the infant. Passive immunity can be enhanced by vaccination of pregnant mothers to promote the transfer of vaccine-specific IgG in utero. However, within the first months of life, as maternal IgG wanes and breastfeeding is discontinued, infants must actively develop their own innate and adaptive immune responses to initiate and maintain immune protection. An overview of the development of innate and adaptive cells, as well as the repertoire of diversity during aging, is outlined in Fig. 38.1.

INNATE IMMUNE DEVELOPMENT The innate immune system is typically considered the first line of defense against infection. The development of the innate immune system begins in utero, with all the classic innate cells types present by the end of the first trimester; monocytes and dendritic cells (DCs) are the earliest cells observed at 4 weeks’ gestation (WG) followed by granulocytes and natural killer (NK) cells at 8 WG.3 These innate immune cells expand in number and mature during gestation; however, at birth the functionality of innate immune cells is still diminished significantly compared with later in life.

DYSFUNCTION OF INNATE IMMUNE CELLS During early infancy, all cell types of the innate immune system demonstrate some impairment of function, ranging from reduced mobility to skewed cytokine production in response to innate immune stimulation. The most significant functional limitation of neonatal innate immune cells is their collective inability to kill pathogens. Limited chemotaxis in neutrophils is accompanied by a reduced ability to effectively kill pathogens as a result of poor phagocytosis and decreased secretion of neutrophil extracellular traps under inflammatory conditions, which in combination results in decreased killing of infectious pathogens. Moreover, NK cells and plasmacytoid DCs have reduced capacity to prevent

535

536

Part four  Immunological Deficiencies Development

Homeostasis

Dysfunction

Innate cells Frequency

Monocytes Dendritic cells Natural killer (NK) cells B cells

Frequency

Maturnal Ab (lgG) Naive/Transitional Class switched memory Class-switched Ab T cells

Frequency

RTE Naïve CD4 T cells Naïve CD8 T cells Memory T cells CD4 Tregs

Receptor repertoire Diversity

NK cells (+/- receptors) Naïve B cells (BCR specificity) Memory B cells (BCR specificity) Naïve T cells (TCR specificity) Memory T cells (TCR specificity)

Birth 1

5

30

60

80 years

Age

FIG 38.1  Innate and Adaptive Immune Cell Frequencies and Receptor Repertoire Diversity During Aging. The frequencies of different innate and adaptive B and T cells changes over the course of a lifetime (upper three panels). In addition to compartment size, diversity of antigen receptors is essential for immune function. During infancy, diversity of immune populations expands and is then maintained during adulthood (lower panel). Aging is characterized by the general decline in diversity within naïve B- and T-cell populations. Dotted lines indicate that the progression of these populations is either controversial or unknown.

infection through reduced cytotoxic function and lower secretion of interferon- (IFN-α), respectively. In addition, antigenpresenting cells (APCs) are unable to provide effective help to T cells because of reduced expression of costimulatory molecules and skewed cytokine production toward more antiinflammatory, T-helper 2 (Th2)–inducing cytokines, such as interleukin-6 (IL-6). These combined limitations during early infancy lead to increased susceptibility to viral and bacterial infections and also contribute to the reduced functionality of adaptive immune cells.

KEY CONCEPTS Characteristics of the Developing Innate Immune System • Reduced antibacterial responses (i.e., phagocytosis, secretion of neutrophil extracellular traps [NETs]) by neutrophils and monocytes • Impaired neutrophil chemokinesis (“directed movement”) • Diminished capacity of antigen-presenting cells (APCs; i.e., dendritic cells [DCs]) to provide proper costimulatory help to T cells • Reduced antiviral responses (i.e., interferon (IFN)-α secretion) by plasmacytoid dendritic cells (pDCs)

ADAPTIVE IMMUNE DEVELOPMENT Adaptive immune cells (T and B cells) begin to develop in utero around the start of the second trimester. It is important to note

that although lymphocytes initially develop within the fetal liver (≈7 WG) and then in bone marrow (≈12 WG), T-cell maturation occurs in the thymus, and B cells continue maturation in bone marrow in utero and after birth. Over the course of gestation, both naïve T- cell and B-cell compartments expand, reaching absolute cell concentrations greater than that of the adults by birth. Thus a defect in cellular generation cannot account for the immune limitations observed in infants. However, the composition of the each lymphocyte population within young infants is distinct from that of adults. At birth, both the newborn infant T- and B-cell compartments consist mainly of naïve and immature cells recently migrated out of the thymus or bone marrow (>90% of the population). Both infant T- and B-cell repertoires have less diversity within their receptors’ antigenbinding regions compared with those of adults. T-cell receptors display reduced V-J complexity and fewer amino acid additions. In addition, B cells have decreased B-cell receptor diversity because of less affinity maturation (i.e., somatic hypermutation; Chapter 7) compared with adult populations. The increased levels of recent thymic emigrants and naïve T cells are maintained both in the periphery and in tissues during early infancy. Effector memory T cells can be found selectively in the lungs and gastrointestinal (GI) tract of young infants, likely because these mucosal tissues are the earliest sites of antigen exposure. Class-switch antibodies produced by B cells are required for mucosal protection (primarily IgA) and systemic protection

CHAPTER 38  Immune Deficiencies at the Extremes of Age (primarily IgG) through opsonization, neutralization, or antibodydependent cellular cytotoxicity (ADCC). However, infants demonstrate significantly lower levels of IgG and IgA compared with adults throughout the first year of life, in both the periphery and mucosae.

The reduced antibody titers observed during infancy can be accounted for by the functional inability of infant B cells to undergo class-switching from IgM to IgA or IgG upon antigen stimulation5 and the poor survival of antibody-secreting plasma cells within the infant bone marrow. Moreover, infant B cells have lower rates of somatic hypermutation compared with those of adults, which is important for affinity maturation and the generation of high-affinity antibodies. Although these defects can be partially attributed to extrinsic T-cell defects, such as poor Tfh generation, intrinsic defects in infant B cells (e.g., lack of costimulatory molecules and receptors to T cell–independent factors) also prevent effective antibody responses. Collectively, these neonatal B-cell defects lead to less effective and shorter-lived antibody responses against infectious pathogens and vaccines during early infancy.

INFANCY AND FUNCTIONAL DIFFERENTIATION OF ADAPTIVE IMMUNE CELLS The acquisition of a mature adaptive immune system requires exposure to antigens for cells to form immunological memory. Not only are infants born with limited antigen exposure and thus little memory, infant responses to antigenic stimulation in the T- and B-cell compartments display altered functions compared with that of adults. The most striking alterations are seen within the CD4 T-cell compartment, characterized by significantly increased frequency of immunosuppressive T regulatory cells (Tregs; 30–40% in infants vs 1–10% in adults).4 These changes accompany increased Th2 subsets and defective differentiation to effector Th1 and T-follicular helper (Tfh) cells during early infancy. Moreover, decreased expression of Lck in infant CD4 T cells leads to decreased T-cell activation upon antigen exposure.2 Altered differentiation of CD4 Th subsets and blunted T-cell activation promotes a tolerance-inducing environment good for controlling unnecessary responses to foreign antigen exposure after birth. However, it also leads to inhibition of effective cytotoxic T cells and limits B-cell antibody responses against infectious pathogens and to vaccination (Fig. 38.2).

KEY CONCEPTS Characteristics of the Developing Adaptive Immune System • High frequencies of naïve and immature T and B cells • Skewing of CD4 T-cell population towards immunosuppressive regulatory T cells and T-helper 2 (Th2) cells • Reduced functionality and memory responses of CD8 T cells to viral infections • Limited ability of B cells to produce high-affinity, class-switched antibodies in response to both T cell–dependent and T cell–independent antigens

Infant

Adult

Elderly

Host environment Anti-inflammatory

Pro-inflammatory History of antigen exposure

Monocytes INNATE

TLR

TCR

CD21

BCR

TNFa, IL-1b, IL-6 TCR

PD-1

CD40L

PD-1

CD40L

Treg, Th2 B cells

TLR

IL-12, TNFa,, IL-1b

TLR

CD40L ADAPTIVE

TLR

IL-10, IL-6, IL-23 CD4 T cells

Th1 CD21

BCR

Th17 CD21

BCR CD11c

↓CSR ↓SHM

TACI IgM

537

CSR SHM

TACI IgG, IgA

↓CSR SHM ↓IgG, IgA ↑autoAb

FIG 38.2  Innate and Adaptive Immune Cell Functionality During Aging. The infant immune environment is distinctly antiinflammatory characterized by altered Toll-like receptor (TLR) signaling from monocytes, blunted T-cell and B-cell receptor (TCR/BCR) signaling, and skewing toward T regulatory cells (Tregs). Infant B cells are incapable of class-switch recombination (CSR) or affinity maturation via somatic hypermutation (SHM) upon antigen exposure. Altered monocyte TLR signaling and altered function of adaptive immune cells is also observed in older adults, but with a distinct, more proinflammatory outcome.

538

Part four  Immunological Deficiencies

INFANT IMMUNE DEVELOPMENT AND THE MICROBIOME One of the first environmental exposures that infants experience is with colonizing bacteria. Within hours after birth, the infant intestinal tract is colonized with nonpathogenic bacteria (termed “microbiome”; Chapter 14). The microbiome can reach concentrations of 1010 bacteria per gram of stool by age 7 days, but only mirrors an adult-like composition after age 2 years. Early studies in animal models demonstrated that microbial colonization is essential for the normal development of the immune system during early infancy and throughout life.6 In particular, IgA, the most abundant antibody within the GI tract, requires the presence of colonizing bacterial for its development and long-term maintenance. Moreover, the specific composition of the microbiome influences the generation of innate lymphoid cells, CD4 T-cells subsets (e.g., Treg, Th17, Tfh), and antibody development. Perturbations in the infant microbiome, caused by such factors as mode of delivery, antibiotic treatment, and diet, may significantly alter developing immune responses. It has been described that babies delivered via C-section have a microbiome composition distinct from vaginally delivered babies, the latter dominated by skin bacteria (e.g., Staphylococcus) instead of vaginal bacteria (e.g., Lactobacillus, Prevotella). These differences correlated with an increased incidence of allergy and asthma during childhood suggesting a causal link between microbiome and susceptibility to allergies. These findings are preliminary and more evidence is needed to support the notion that the microbiome influences the development of the infant immune system.

CLINICAL CONSEQUENCES FOR CHILDHOOD VACCINATION Newborn and young infants are highly susceptible to pathogenic infection during the first months to year of life. Indeed, infections are one of the major causes mortality, accounting for more than 24% of global infant mortality. More than 1 million deaths per year are caused by respiratory tract infections (e.g., Streptococcus pneumoniae, Haemophilus influenzae type b, respiratory syncytial virus [RSV]) and almost the same amount by intestinal infections (e.g., rotavirus, Escherichia coli). The development and distribution of vaccines targeting these pathogens have significantly reduced infant infection rates and subsequent mortality; however, vaccine efficacy can vary, depending on the infant’s gestational age at birth, mode of vaccine delivery (oral vs intramuscular), interference by maternal antibodies acquired through passive immunity and via breast milk, and immaturity of infant immune responses, as described above. Additionally, infants require multiple vaccine boosters to elicit and maintain robust protective immunity against infectious pathogens (Chapter 90). Better understanding of the limitations of the human infant immune system that prevent effective immune responses, particularly at mucosal sites where these pathogens initially infect, would facilitate the generation of better vaccines designed to overcome these limitations and induce more rapid and robust immune protection during early infancy.

OLDER AGE AND IMMUNE CELL GENERATION The immune system is in constant demand for cellular replenishment to compensate for peripheral losses and cell death. For

CLINICAL RELEVANCE Consequences of Immaturity of the Developing Immune System • Increased morbidity and mortality from bacterial infections (e.g., pneumococci) • Increased morbidity and mortality from viral infection (e.g., influenza virus, respiratory syncytial virus [RSV]) • Ineffective primary vaccinations; multiple boosters required for most vaccines to elicit effective protection • Susceptibility to allergy and asthma when microbiome colonization disrupted during early infancy

neutrophils, which have a short half-life, the body needs to produce ≈1010 cells/kg per day. Additionally, for lymphocytes, which are more long-lived but are also more numerous because of their wider tissue distribution, the daily need is in the order of several billion cells. Studies in the 1960s showed that the hematopoietic tissue in bone marrow decreases with age. A similar decline is seen for the frequencies of peripheral CD34+CD45+ hematopoietic stem cells (HSCs). In addition to these numerical differences, the functional potential of HSCs changes with age.7 Old HSCs exhibit a reduced capacity to regenerate the hematopoietic system. Telomerase expression in peripheral HSCs is not fully protective, and a shortening in telomeric length is seen with age, similar to differentiated mononuclear cells. Lymphoid differentiation potential diminishes with age in favor of myeloid differentiation as a result of altered lineage specification programs driven by DNA damage and epigenetic changes. This shift may contribute to the clinical observation that HSC-derived leukemia preferentially has a lymphoid phenotype in the young and a myeloid one in older adults.

KEY CONCEPTS Aging Influences Immune Cell Generation and Population Homeostasis Aging influences immune cell generation and population homeostasis. • Hematopoietic stem cells (HSCs) are reduced in frequency and biased toward myeloid lineages and against lymphoid lineages. • Myeloid cell generation is largely intact. • B-cell generation declines and B-cell repertoire selection is disturbed. • As a result of thymic involution, naïve T cells are generated from homeostatic proliferation of peripheral T cells. • Ability to rebuild a T-cell repertoire after lymphocyte-depleting interventions is severely compromised after mid-adulthood. • Virus-specific effector T-cell population accumulates.

As a consequence, lymphocyte replenishment is more affected by age than that of myeloid lineages. Peripheral neutrophil numbers do not decline and are able to recover after therapyinduced depletion (e.g., chemotherapy). Furthermore, there is no loss in the ability to generate robust neutrophilia in response to infection or other stressors. In addition to HSC-intrinsic alterations in lineage commitments, defects in bloodborne factors and bone marrow niches contribute to a decline in B-cell generation.8 As a consequence, recovery after B-cell depletion (e.g., treatment with anti-CD20 antibodies to treat B-cell malignancies or autoimmune diseases) is

CHAPTER 38  Immune Deficiencies at the Extremes of Age incomplete and delayed in older adults. Moreover, the frequency of typical memory B cells expressing CD27 declines, whereas other B-cell subpopulations increase. In particular, a functionally distinct B-cell population that responds to Toll-like receptor (TLR) stimulation has been described.9 These age-associated differences in B-cell selection and expansion may contribute to monoclonal gammopathies (in >5% of individuals older than 70 years; Chapter 80) or increased autoantibodies with increasing age. High-throughput sequencing of Ig genes has the promise to provide insights into the age-associated decline in B-cell responses at the single-cell level. Data so far have indicated that older individuals have prevaccination-expanded B-cell populations that have a high mutation load and are further expanded with vaccination, suggesting that the B-cell response in older individuals relies on the adaptation of a restricted repertoire of memory sequences. T-cell generation is more affected by age than any other myeloid or lymphoid lineage because of the involution of the thymus. The thymus undergoes dramatic structural changes that begin during childhood and puberty.10 Thymopoietic niches disappear, and the numbers of thymic epithelial cells and thymocytes decline. In parallel, the thymic perivascular space increases. One major structural change that does not appear to be a compensatory response but, rather, is an active and regulated developmental step in thymic organ transformation is the accumulation and infiltration of adipocytes in the perivascular space. Thymic resection in children undergoing cardiac surgery reproduces many of the T-cell repertoire changes in 20-year-old individuals that are usually seen in 70- to 80-year-olds, confirming that thymic production during the growth period of childhood and adolescence is important. However, throughout adulthood, homeostatic proliferation of naïve T cells accounts for the bulk of T-cell generation.11 For human CD4 T cells, this process is robust, and frequencies of naïve CD4 T cells only moderately decline with age. In contrast, the naïve CD8 T-cell compartment clearly shrinks. However, repertoire diversity (i.e., the number of different T-cell receptors [TCRs]) remains very high for both naïve CD4 and CD8 T-cell subsets, suggesting that thymic activity is not necessary to prevent holes in the repertoire once a repertoire has formed. Strategies to reactivate thymic T-cell generation will be more important in older patients who cannot restore the naïve T-cell repertoire after a medical intervention, such as chemotherapy or bone marrow transplantation, compared with their healthy counterparts.

T-CELL POPULATION HOMEOSTASIS The adaptive system responds to antigenic challenges with clonal expansion and differentiation into effector cells followed by clonal downsizing and persistence of long-lived memory T cells. Infections therefore leave a permanent imprint on the immune system, a mechanism on which vaccinations capitalize. However, the pathogen-induced clonal expansion also represents a challenge to homeostatic mechanisms that are supposed to maintain a balance among naïve, memory, and effector cells.12 This is particularly evident in persisting infections where the offending pathogen cannot be cleared. Herpes virus infections are highly prevalent in an apparently healthy population without causing active disease, as they establish latency. Classic examples are varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). The effects of these herpes viruses with immune aging differ greatly; VZV tends to relapse with age, with reactivation presenting as shingles. A decrease in the frequency

539

of VZV-specific CD4 memory T cells has been postulated to explain this lack in viral control mechanisms. In contrast, EBV and CMV infections only relapse in severely immunocompromised individuals, but not during normal immune aging. The immune system commits extraordinary resources to controlling CMV, and CMV-specific CD8 T cells can make up a large fraction of the entire T-cell repertoire. Whether this memory inflation has broader implications for immune health remains a matter of controversy. Expansion of the CMV-specific T cells may compromise the size of naïve and central memory T cell repertoires; however, many of the CMV-specific CD8 T cells have the phenotype of end-differentiated effector T cells that lack the expression of CCR7, CD28, and CD27 and therefore do not compete for the same space as naïve cells. CD45RA+CD28− end-differentiated T cells express negative regulatory receptors of the killer lectin-like receptor (KLR), killer immunoglobulin-like receptor (KIR), and immunoglobulin-like transcript (ILT) families that appear to constrain their otherwise unopposed expansion. In spite of these inhibitory receptors, these cells are competent effector T cells capable of producing inflammatory cytokines and therefore possibly contributing to inflammation in older adults. End-differentiated CD45RA+ effector T cells should be distinguished from exhausted CD8 T cells.13 T-cell exhaustion is seen with chronic stimulation by highly replicating viruses or tumor cells and characterized by the expression of inhibitory receptors programmed death 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG3).14 T-cell exhaustion is not a general feature per se of T-cell aging.

INFLAMMATION, AGING, AND THE AGING HOST ENVIRONMENT The aging host environment is characterized by the continuous presence of inflammatory mediators independent of acute or chronic disease (Fig. 38.3).15 Even for the healthy older adult, IL-6 and tumor necrosis factor (TNF) serum levels are twofold to fourfold higher than in young adults. Low-level systemic inflammation plays an important role in the progression of several age-related diseases, including Alzheimer disease, atherosclerosis, and cancer. Moreover, inflammatory markers are associated with several conditions that are characteristic of older adults. IL-6 serum concentrations have been correlated with loss of mobility and advent of disability; increased mortality of older individuals has been shown among those who have higher levels of TNF-α. Increased IL-6 and cross-reactive protein (CRP) serum levels and increased white blood cell (WBC) counts, presumably resulting from increased production of neutrophils, predispose to, and are associated with, frailty. A causative relationship may also exist between the increased production of IL-6 or TNF-α and the age-associated loss in muscle mass, eventually presenting as sarcopenia. Long-lived individuals, such as centenarians, tend to have lower levels of proinflammatory cytokines and increased levels of antiinflammatory mediators, such as cortisone and IL-10, supporting the concept that low-level inflammation is detrimental to healthy aging. Production of inflammatory cytokines is driven by several mechanisms. Failure of the adaptive immune system leads to a less effective control of chronic viral infections as well as incomplete response to exogenous challenges, resulting in increased and prolonged innate immune activation. Defective epithelial barrier function, as well as decline in the mucosa-associated

540

Part four  Immunological Deficiencies Defective GALT

Epithelial dysfunction

Inflammatory mediators TNF-α, IL6

Tissue degeneration

Age-associated adipogenesis Increase in PAMPs and alarmins

Senescence-associated gene activation Accumulation of effector T cells

Defective innate immunity

Frailty

Coronary artery disease

FIG 38.3  Inflammation in Older Adults. The schematic diagram depicts possible mechanisms that account for the increased production of inflammatory mediators with age. These mediators contribute to many of the age-associated diseases.

lymphoreticular tissue (MALT), results in increased leakage and increased systemic levels of lipopolysaccharide (LPS) and innate immune activation. Failure in maintaining T-cell population homeostasis and accumulation of effector T cells also favors an inflammatory response.

KEY CONCEPTS Causes for Increased Constitutive Production of Inflammatory Mediators With Age (Inflammation in Older Adults) • Activation of the innate immune system as a result of defective epithelial barrier function • Activation of the innate immune system as a result of defective adaptive immunity • Accumulation of adipocytes producing inflammatory mediators • Accumulation and activation of T-effector cell populations • DNA damage-induced transcription of inflammatory cytokine genes in senescent cells

The immune system, however, is not the only source of inflammatory cytokines. Adipocytes, in part replacing muscle cells in older adults, produce various inflammatory mediators. Cellular senescence has also been associated with the production of cytokines. Persistent DNA damage response signaling not only induces an irreversible cell cycle block that is characteristic of cellular senescence but also initiates a transcriptional program to secrete numerous growth factors, proteases, and inflammatory cytokines, termed the senescence-associated secretory phenotype (SASP).16

CELLULAR DEFECTS AND SENESCENCE As described so far, immune aging occurs at the system level with organizational restructuring. Equally important are changes at the single-cell level, which are partly cell-intrinsic and partly caused by the host environment. The increased cytokine concentrations in older adults not only activate but also attenuate

signaling pathways. Low responsiveness to cytokine stimuli is frequently seen in those cells that have increased baseline activation of a signaling pathway (e.g., cells that constitutively have increased signal transducer and activator of transcription 3 (STAT3) or STAT1 phosphorylation respond less to IL-6/granulocyte macrophage–colony-stimulating factor (GM-CSF) or type I/II IFNs, cytokines that activate these STATs). Attenuation of signaling pathways by induction of negative feedback loops explains, in part, the reduced responsiveness and functionality of cells of the innate immune system. Although neutrophil and monocyte/macrophage numbers remain normal, many of their functions decline with age.17 Decreased chemotaxis in neutrophils delays tissue infiltration; reduced phagocytosis and respiratory burst compromise the ability to control bacterial infections; and TLR-induced monocyte/ macrophage activation is dampened in older adults. Declines in responsiveness, for example, to TLR stimulation, are partly reversible in vitro, suggesting that they are not intrinsic. Adaptive immune cells are also directly affected by the proinflammatory environment in the aging host. Equally important are cell-intrinsic changes that appear to be a consequence of the replicative history and failures in cellular processes, such as DNA repair and autophagy. The program most obviously influenced by age is cellular senescence. In all hematopoietic cell lineages, including stem cells, telomeric lengths decline with age. This is of particular importance for T cells because much of their response depends on their ability to proliferate and clonally expand.18 Telomeric erosion results not only from cumulative replicative history and DNA damage but also from decline in the ability to express telomerase and repair telomeric ends. Lymphocytes in older adults are more differentiated than those in the young. Although most obvious for CD8 T cells, increasing differentiation can also be noted for B cells and CD4 T cells (Fig. 38.4). Differentiation is generally driven by antigen recognition but could also occur in the absence of exogenous antigen under the influence of cytokines. Proliferation alone may be sufficient to drive initial steps of differentiation. A classic

CHAPTER 38  Immune Deficiencies at the Extremes of Age Infant

Elderly TCR

CD4

TCR CD4

541

Up Down

Lck Zap70

Lck Zap70

pERK

PLC

T cell activation

T cell activation

DUSP6 miR-181a

Internal changes Telomere length DNA damage Epigenetic modifications

+++

+

+

+++

+

+++ Functional outcomes

Proliferation capacity

+++

++*

T cell activation

+

+

Effector function

+

+*

+ = low

++ = normal

+++ = high

*controversial

FIG 38.4  Intrinsic Differences in CD4 T Cells at the Extremes of Age. CD4 T cells from infants and older individuals demonstrate similar dysfunctions, including poor activation and reduced effector functions, although as a result of distinct mechanisms. Signaling cartoons illustrate the best documented defects. Moreover, infant and older adult T cells have very different internal changes to their DNA, including telomere length, the amount of DNA damage, and epigenetic modifications.

KEY CONCEPTS Cellular Dysfunction With Age • Exposure to aging host environment (e.g., inflammatory cytokines) activates negative regulatory signaling loops. • Telomeric erosion impairs proliferative competence and restraints clonal expansion. • End-differentiation reduces functional plasticity. • Activation of specific gene programs modifies cell function: • Gene programs associated with differentiation (e.g., microRNA [miRNA]) • Gene programs associated with T-cell exhaustion (e.g., expression of programmed death-1 [PD-1]) • Loss of CD28 on T cells • Gain in natural killer (NK) cell–associated regulatory receptors on T cells (e.g., killer immunoglobulin-like receptor [(KIR], killer lectin-like receptor [KLR], immunoglobulin-like transcript [ILT]) • Senescence-associated gene activation (e.g., inflammatory mediators)

example is the acquisition of memory-like and effector phenotypes with lymphopenia-induced homeostatic proliferation. So-called virtual memory cells, which presumably have never seen an exogenous antigen, have been identified in mouse models; clonal expansions are also seen within the human naïve T-cell compartment. Some of the changes in gene expression that are seen in naïve T cells with age may represent partial differentiation, such as declines in the microRNA miR181a and changes in the expression of phosphatases and other signaling molecules.18 Increased expression of cytoplasmic phosphatases impairs the TCR-induced

activation of naïve T cells. Changes in cell surface molecules that are seen with terminal differentiation, such as the gain in CD57 and the loss of CD27 and CD28 expression, are the most striking. Of functional importance, predominantly for CD8 T cells, is the gain in expression of cell surface receptors that are usually only found in NK cells. Most of these receptors have inhibitory function, but some of them also stimulate. Since expression of these receptors on individual cells is stochastic, the consequences can range from immunosuppression to autoreactivity.

CLINICAL CONSEQUENCES OF IMMUNE AGING— IMMUNODEFICIENCY, AUTOIMMUNITY, AND ACCELERATED DEGENERATIVE DISEASES The most profound and most noted consequence of human senescence is the increased susceptibility to infections. Upper respiratory bacterial and urinary tract infections are frequent in the older population and less contained by the innate immune system and preexisting adaptive immunity. Not surprisingly, the immune system of an older adult is not able to induce a protective response to new antigens to which the individual has not been exposed to in the past. Clinically important examples are the severe acute respiratory syndrome (SARS) epidemic and West Nile fever virus infection. First-time vaccinations with live viruses, for example, yellow fever virus, are associated with increased morbidity and even mortality in older adults. Despite annual vaccination, influenza infections continue to be associated with high morbidity and mortality. Pneumonia caused by RSV, usually

542

Part four  Immunological Deficiencies

infecting young children, is not uncommon. Immune competence to chronic infections is also compromised with age. The best example here is the reactivation of VZV manifesting as shingles.

CLINICAL RELEVANCE Consequences of Immune Aging • Increased morbidity and mortality from bacterial infections (e.g., pneumococci) • Increased morbidity and mortality from viral infections (e.g., influenza, West Nile fever) • Reactivation of latent virus (e.g., varicella zoster virus) • Ineffective primary and booster vaccinations • Acceleration of degenerative diseases as a result of the production of inflammatory mediators (e.g., atherosclerotic disease, Alzheimer disease, osteoarthritis) • Increased incidence of autoimmune disease (e.g., polymyalgia rheumatica, giant cell arteritis, rheumatoid arthritis)

Vaccinations against pneumococcal antigens, influenza strains, and VZV are recommended in older adults; however, the efficacy of vaccination is reduced. Immune aging also predisposes for autoimmune manifestations and a breakdown in self-tolerance.19 Autoantibodies are a common finding in healthy older adults; many of these autoantibodies are specific for common autoantigens, such as IgG Fc or nuclear components. The risk for several autoimmune diseases, most notably polymyalgia rheumatica and giant cell arteritis, increases with age. Although polymyalgia rheumatica predominantly presents as an activation of innate immunity, giant cell arteritis is clearly a disease of the adaptive immune system with T cell–dependent granulomatous inflammation in the vascular wall of midsized and large arteries. The low-grade inflammation in the aging host has direct clinical consequences in promoting frailty and sarcopenia and accelerating degenerative diseases, including coronary artery disease, osteopenia, and Alzheimer disease. Accelerated immune aging may be one of the reasons that autoimmune diseases, such as rheumatoid arthritis (RA), are associated with a shorter life span and increased risk for cardiovascular morbidity. Inflammation as a manifestation of accelerated aging has been also implicated in the increased morbidity and mortality of patients with HIV infection in spite of highly active antiretroviral therapy (HAART).20

STRATEGIES AND INTERVENTIONS ON THE HORIZON Vaccinations hold the promise for reducing increased susceptibility to infections in young children and older adults, but improving vaccine responses has proven to be a challenge. Current strategies for targeting more elusive pediatric pathogens (e.g., RSV) included maternal vaccination (i.e., the mother is vaccinated during pregnancy and vaccine-specific IgG is transferred in utero to the infant) and the development of infant-specific vaccine adjuvants utilizing newer understanding of the developing infant immune system. Also, further investigations into the role of microbiome and immune development (or dysfunction) may open new strategies to influence immune system development to reduce immune deficiencies and prevent inappropriate hyperactivity. In older adults, a proper T-cell response to infectious organisms or to vaccines depends on the availability of a T-cell repertoire

that includes antigen-receptor specificities to respond to vaccination. Repertoire contraction resulting from thymic involution or memory inflation appears to be less severe than originally thought, and currently explored interventions to restore thymic activity may only be meaningful for selected populations, such as bone marrow recipients. Deficiency in the antigen-presenting system and in costimulatory signals may be overcome by identifying new adjuvants. Increasing the vaccine dose is another promising approach. Live vaccines or self-replicating constructs that also accomplish higher antigen loads may not have a sufficient safety profile in older adults. Direct targeting of signaling defects in older T cells resulting from increased expression of cytoplasmic phosphatases or inhibitory cell surface receptors will be feasible, as shown for checkpoint inhibitors in oncology; however, such approaches need to demonstrate a much better safety profile. Interventions to influence inflammation in older adults at present needs to be nonspecific, given the multitude of underlying mechanisms. Immunomodulatory therapy is obviously standard practice in patients with autoimmune disease who exhibit accelerated aging and increased all-cause mortality. Calorie restriction to slow immune aging to an extent that it is effective is generally not well accepted. Statins and aspirin are being routinely used to prevent cardiovascular disease; their effect may be mostly antiinflammatory. Future interventions will require the development of mild and low-toxicity medications to reduce low-grade inflammation while not impairing the ability to prevent innate immune activation in response to harmful antigens.

ON THE HORIZON • Improved vaccination strategies tailored to the infant or aged immune system (novel adjuvants, novel vaccine delivery systems) • New vaccines for pregnant females to confer passive immunity • Manipulation of the microbiome composition to influence immune system development • Thymic rejuvenation (e.g. with KGF, IL-7 and other mediators) • Prevention of chronic infection that accelerate immune aging (e.g. immunization for CMV) • Pharmacological approaches to improve T and B cell activation, clonal expansion and differentiation • Treatment of inflamm-aging

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Lopez-Otin C, Blasco MA, Partridge L, et al. The hallmarks of aging. Cell 2013;153(6):1194–217. 2. Basha S, Surendran N, Pichichero M. Immune responses in neonates. Expert Rev Clin Immunol 2014;10(9):1171–84. 3. Gollwitzer ES, Marsland BJ. Impact of Early-Life Exposures on Immune Maturation and Susceptibility to Disease. Trends Immunol 2015;36(11):684–96. 4. Simon AK, Hollander GA, McMichael A. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci 2015;282(1821):20143085. 5. Siegrist CA, Aspinall R. B-cell responses to vaccination at the extremes of age. Nat Rev Immunol 2009;9(3):185–94. 6. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009;9(5):313–23. 7. Geiger H, de Haan G, Florian MC. The ageing haematopoietic stem cell compartment. Nat Rev Immunol 2013;13(5):376–89.

CHAPTER 38  Immune Deficiencies at the Extremes of Age 8. Kogut I, Scholz JL, Cancro MP, et al. B cell maintenance and function in aging. Semin Immunol 2012;24(5):342–9. 9. Naradikian MS, Hao Y, Cancro MP. Age-associated B cells: key mediators of both protective and autoreactive humoral responses. Immunol Rev 2016;269(1):118–29. 10. Palmer DB. The effect of age on thymic function. Front Immunol 2013;4:316. 11. Goronzy JJ, Fang F, Cavanagh MM, et al. Naive T cell maintenance and function in human aging. J Immunol 2015;194(9):4073–80. 12. Nikolich-Zugich J, Li G, Uhrlaub JL, et al. Age-related changes in CD8 T cell homeostasis and immunity to infection. Semin Immunol 2012;24(5):356–64. 13. Akbar AN, Henson SM. Are senescence and exhaustion intertwined or unrelated processes that compromise immunity? Nat Rev Immunol 2011;11(4):289–95.

543

14. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15(8):486–99. 15. Kanapuru B, Ershler WB. Inflammation, coagulation, and the pathway to frailty. Am J Med 2009;122(7):605–13. 16. Tchkonia T, Zhu Y, van Deursen J, et al. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J Clin Invest 2013;123(3):966–72. 17. Shaw AC, Goldstein DR, Montgomery RR. Age-dependent dysregulation of innate immunity. Nat Rev Immunol 2013;13(12):875–87. 18. Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to vaccines. Nat Immunol 2013;14(5):428–36. 19. Goronzy JJ, Weyand CM. Immune aging and autoimmunity. Cell Mol Life Sci 2012;69(10):1615–23. 20. Deeks SG. HIV infection, inflammation, immunosenescence, and aging. Annu Rev Med 2011;62:141–55.

CHAPTER 38  Immune Deficiencies at the Extremes of Age

543.e1

MULTIPLE-CHOICE QUESTIONS 1. The developing immune system is characterized by: A. Increased frequencies of regulatory T cells. B. Innate immune system activation. C. Frequent presence of autoantibodies caused by tolerance defects. D. Hematopoietic stem cells preferentially differentiating into myeloid and not lymphoid lineages. 2. An inflammatory environment in older adults is: A. Less pronounced in frail older adults. B. Caused by thymic involution. C. Characterized by the production of cytokines from various cell types. D. Results from failure of naïve T cells to differentiate. 3. The following consideration to vaccinations in older individuals is correct: A. Annual influenza vaccinations are not always protective and are therefore not recommended in older individuals.

B. Vaccinations with live viruses (e.g., yellow fever vaccine) are potentially harmful. C. Only booster, but not primary, vaccine responses are impaired in older individuals. D. Vaccinations even with inactivated or component vaccine carry a higher risk in older adults. 4. T cells are derived from hematopoietic stem cells that differentiate in the thymus. Which of the following statements is correct? A. Thymic involution leads to a rapid loss of naïve T cells. B. Upon thymic involution, peripheral homeostatic proliferation of existing T cells to is effective to at least partially compensate for T cell loss. C. The thymus starts to involute after the age of 50 years. D. Generation of T cells by homeostatic proliferation can increase the T-cell receptor repertoire.

39  Human Immunodeficiency Virus Infection and Acquired Immunodeficiency Syndrome Susan L. Gillespie, Javier Chinen, Mary E. Paul, William T. Shearer

In recent years, there has been a stabilization and possible reversal of the overall growth of the global acquired immunodeficiency syndrome (AIDS) epidemic. The number of new infections has steadily declined since the late 1990s, and there are fewer AIDSrelated deaths as a result of the scale-up of antiretroviral therapy (ART) since 2004. People living with human immunodeficiency virus (HIV) are now living longer and healthier lives. According to the Joint United Nations Programme on HIV/ AIDS (UNAIDS), 36.9 million people worldwide were estimated to be living with HIV or AIDS at the end of 2014 (Fig. 39.1).1 New HIV infections are declining in most countries. The annual number of incident HIV infections peaked at >3 million in the late 1990s and has steadily declined thereafter. In 2014, 2 million people were newly infected, with approximately 1% of these infections (220 000 cases) occurring in children younger than 15 years of age.1 Globally, however, adolescents, particularly adolescent girls, are at particular risk for acquiring HIV infection. In 2012, there were 300 000 new infections reported among adolescents aged 15–19 years, which accounted for about 13% of all new infections. In sub-Saharan Africa, 70% of new infections in adolescents occurred among girls.2 There has been a slow but continued reduction in the number of people dying from AIDSrelated conditions worldwide. AIDS mortality peaked at 2.1 million in 2004, and by the end of 2014, it was down to 1.2 million deaths resulting from AIDS-related illnesses.1 The decline reflects increased access to ART and improvements in the care and support of infected individuals. Mortality among children younger than 15 years of age has also declined as a result of the expansion of services to prevent mother-to-infant transmission of HIV and an increase in access to care and treatment for children. Although access to care and treatment services for HIV-infected children in resource-limited settings is expanding, it is estimated that only 31% of children in need of life-saving ART received it, and an estimated 150 000 children had died of AIDS-related illnesses in 2014. Of concern, HIV/AIDS is the most common cause of death among adolescents in sub-Saharan Africa and the second leading cause of death among adolescents globally.

KEY CONCEPTS Trends in Human Immunodeficiency Virus (HIV) Infection • Global rates of HIV infection have stabilized, but there is great heterogeneity in disease incidence among countries and regions. • Although most countries had a reduction in disease incidence between 2001 and 2009, seven countries had increases in disease incidence by more than 25% over that period. • The age demographic most affected in the developing world, that is, those aged 25–44 years, includes men and women who are economically productive and women of childbearing potential. • Worldwide, most infections are acquired through heterosexual contact. • In the United States, African Americans and men who have sex with men (MSM) are disproportionately infected. • The numbers of infections transmitted from mother to child are declining as access to prophylactic medications to prevent infection improves.

of HIV infection, the number of newly diagnosed infections in the United States has remained unchanged from 2006–14, approximately 40 000–50 000 per year. Within the United States, there is great variability in both the geographical and demographic distribution of the disease, with certain segments of the American population being disproportionately affected, specifically men who have sex with men (MSM) and ethnic and racial minorities, including African Americans and Hispanic Americans (Fig. 39.2).3 In the United States and other developed countries, in contrast to more resource-limited parts of the world, the number of children newly infected with HIV has decreased dramatically as a consequence of successful interventions against perinatal mother-to-child transmission. In 2014, 174 children were diagnosed with HIV infection, 73% of whom had acquired the infection through perinatal transmission.4 At the same time, new pediatric AIDS cases and AIDS deaths also have plummeted, in large part as a result of powerful combinations of antiretroviral (ARV) drugs.

HIV PATHOGENESIS

US Perspective

HIV Lifecycle

The Centers for Disease Control and Prevention (CDC) estimate that almost 1.2 million adults and adolescents were living with HIV in the United States at the end of 2014, including 156 300 (12.8%) whose infection was undiagnosed. Despite ongoing prevention efforts designed to reduce the number of new cases

HIV is a lentivirus that targets CD4 T cells by specifically binding the viral Env protein to two cell surface proteins, the CD4 receptor, and either the CCR5 or the CXCR4 chemokine receptor (Fig. 39.3). Other cell targets for HIV are monocytes and dendritic cells (DCs), although they present less expression

545

546

Part four  Immunological Deficiencies Adults and children estimated to be living with HIV, 2014 By WHO region

Number of people, by WHO region Eastern Mediterranean: 330 000 [200 000–460 000]

Americas: 3 400 000 [2 500 000–4 400 000]

Western Pacific: 1 400 000 [1 200 000–1 800 000]

South-East Asia: 3 500 000 [3 200 000–3 700 000]

Europe: 2 500 000 [2 200 000–2 800 000]

Africa: 25 800 000 [24 000 000–28 700 000]

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal states of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries, Dotted and dashed lines on maps represent approximate border lines for which there may not yet be full agreement.

Total: 36 900 000 [34 300 000–41 400 000] 0

875 1,750

Data Source: World Health Organization Map Production: Information Evidence and Research (IER) World Health Organization © WHO 2016. All rights reserved.

FIG 39.1  Adults and children estimated to be living with human immunodeficiency virus (HIV), 2014: by World Health Organization (WHO) region, 2014.

12,000

11,4000

Estimated new HIV infections in the US 2009, for the most affected subpopulations 10,800

Number of new HIV infections

10,000 8,000 6,000

6,000

5,400

4,000 2,400 1,700

2,000

1,700

1,200

940

Black male IDUs

Black female IDUs

0 White MSM*

Black MSM

Hispanic MSM

Black heterosexual women

Black heterosexual men

3,500 Kilometers

Hispanic heterosexual women

White heterosexual women

FIG 39.2  US trends in human immunodeficiency virus (HIV) infection by race and mode of transmission. (From Centers for Disease Control and Prevention. HIV surveillance—United States, 1981–2008. MMWR Morb Mortal Wkly Rep 2011; 60: 689–93. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention; 2011. (Also available at http://www.cdc/gov/hiv/topics/surveillance/resources/reports.gov.)

CHAPTER 39  HIV and Acquired Immunodeficiency Syndrome

547

The HIV Life Cycle HIV medicines in six drug classes stop HIV at different stages in the HIV life cycle 1 Binding (also called attachment): HIV binds (attaches itself) to receptors on the surface of a CD4 cell. CCR5 antagonist

2 Fusion: The HIV envelope and the CD4 cell membrane fuse (join together), which allows HIV to enter the CD4 cell. Fusion inhibitors CD4 receptors

CD4 cell membrane

3 Reverse transcription: Inside the CD4 cell, HIV releases and uses reverse transcriptase (an HIV enzyme) to convert its genetic material– HIV RNA– into HIV DNA. The conversion of HIV RNA to HIV DNA allows HIV to enter the CD4 cell nucleus and combine with the cell’s genetic material– cell DNA.

HIV RNA Reverse transcriptase HIV DNA

Non-nucleotide reverse transcriptase inhibitors (NNRTIs) Nucleotide reverse transcriptase inhibitors (NRTIs)

Membrane of CD4 cell nucleus

5 Replication: Once integrated into the CD4 cell DNA, HIV begins to use the machinery of the CD4 cell to make long chains of HIV proteins. The protein chains are the building blocks for more HIV.

Integrase 4 Integration: Inside the CD4 cell nucleus, HIV releases integrase (an HIV enzyme). HIV uses integrase to insert (integrate) its viral DNA into the DNA of the CD4 cell.

HIV DNA

Integrase inhibitors CD4 cell DNA 6 Assembly: New HIV proteins and HIV RNA move to the surface of the cell and assemble into immature (noninfectious) HIV.

7 Budding: Newly formed immature (noninfectious) HIV pushes itself out of the host CD4 cell. The new HIV releases protease (an HIV enzyme). Protease acts to break up the long protein chains that form the immature virus. The smaller HIV proteins combine to form mature (infectious) HIV. Protease inhibitors (PIs)

FIG 39.3  The human immunodeficiency virus (HIV) lifecycle and stage susceptibility to antiretroviral therapy. (From https://www.aidsinfo.nih.gov/education-materials/fact-sheets/19/73/the-hiv-lifecycle.)

Protease

548

Part four  Immunological Deficiencies

of these receptors compared with CD4 T cells. After the virus is attached to the cell, the viral membrane fuses with the cell membrane and viral RNA enters the cytoplasm. It is transcribed by the viral reverse transcriptase into double-stranded DNA, which is translocated to the cell nucleus, and the virus integrase enzyme mediates its integration into the cell genome. The viral proteins Tat and Nef, in addition to T-cell activation factors, induce active transcription and expression of viral RNA and proteins to produce new virus particles that exit the host cell to infect other cells. CD4-independent viral entry has been demonstrated in B cells, astrocytes, and kidney epithelial cells; however, efficient viral replication is not likely to occur in these cells (see Treatment).

blood. They might produce very low levels of viral RNA and proteins and therefore might not be detected by immunosurveillance. HIV reservoirs are established during the acute phase of the infection, hence the recommendation for early intensive treatment to reduce the population of latently infected CD4 T cells (nonreplicating monocytes, astrocytes, and glial cells are other latently infected cells). Molecular mechanisms that inhibit HIV replication from viral DNA and maintain latent infection include histone deacetylases and methyl transferases with methylation of viral genes, especially of the long-terminal promoter repeat. These epigenetic modifications can be reversed by using T-cell activation signals.

HIV Entry Through Mucosal Surfaces

ANTI-HIV IMMUNITY

Using dilution techniques that can precisely determine unique virion genome sequences, it was found that about 80% of HIV infections via heterosexual transmission start with a single virion.5 This is remarkable, considering the high rate of mutation and diversity of viral genome sequences found in infected individuals. In contrast, HIV transmission occurring in MSM and in intravenous drug users is caused by several viral variants, indicating that the multilayered epithelia of the vaginal mucosa and the cervical mucus are likely deterrents for lentiviral infection.6 Regardless of the route of infection, the time to develop viremia and immunological changes is similar to that of all forms of transmission. Viral replication can be detected in the germinal center of lymph nodes within a week, and viremia is found within 21 days of infection. The most active replication occurs in the gut-associated lymphoid tissue (GALT), with massive depletion of CD4 T cells. Activation and destruction of CD4 T cells are associated with an increase of serum cytokines, which might explain the flu-like syndrome that patients might experience during acute HIV infection.7 Simultaneously, HIV reservoirs become established with viral infection and integration of viral DNA in resting cells.

Immunity against HIV depends mostly on specific cytotoxic CD8 T cells, which recognize and destroy infected cells.7 These antiviral cells are most efficient when certain combinations of human leukocyte antigen (HLA) and virus strain occur in the host, such as the presence of a cell bearing the HLA-B27 allele and infected with clade B viral strain. However, HIV infection almost always results in global T-cell destruction and exhaustion. Antibody responses are initially directed against the gp41 portion of the Env protein. This response and subsequent anti-HIV antibodies become ineffective because of the rapid production of escape mutants.

T-cell Depletion Progressive T-cell depletion in HIV infection is induced, in part, by a state of chronic immune activation, which also contributes to noninfectious complications, such as cardiovascular disease.7 In individuals with HIV infection, immune activation is caused by increased microbial translocation in the gut, direct Toll-like receptor (TLR) stimulation by HIV, and coinfection with other pathogens. This activated state results in increased levels of inflammatory cytokines, such as interleukin-6 (IL-6). Also detected is an increase of lipopolysaccharide (LPS) in blood, which, in turn, can also activate the coagulation cascade and promote thrombosis. Low or normal levels of T regulatory cells (Tregs) are observed in long-term nonprogressors, suggesting their role in anti-HIV immunity, especially in modulating CD8 T-cell immunity. It has also been suggested that Tregs may play a beneficial role by reducing chronic immune activation.8

HIV Latency and HIV Reservoirs The most difficult problem in the efforts toward achieving a cure for HIV infection is the presence of HIV reservoirs, defined as resting or nonreplicating cells infected with HIV.9 Anti HIV drugs are able to efficiently suppress HIV replication; however, when these drugs are stopped, activation of the resting cells results in the production of new virus. Most viral DNA is detected in memory CD4 T cells within lymphoid tissues and peripheral

HIV Vaccines: Basic Concepts Several efforts to develop an effective anti-HIV vaccine have attempted to address the genetic diversity of HIV that develops in the infected individual, by identifying epitopes that remain relatively conserved and by inducing neutralizing antibodies as well as cell-mediated immunity.10 Broadly neutralizing antibodies, which target conserved Env epitopes from different strains and can prevent HIV from establishing infection, might develop in some individuals with chronic infection, offering hope for disease prevention. Such antibodies are found to develop after approximately 2 years of infection, when many generations of escape virions, inducing new antibody epitopes, allow the immune response to refine and select for best affinity. This concept suggests that several booster immunizations might reproduce the process in shorter time. Current approaches involve viral vectors that drive the expression of HIV proteins in host tissue; an example is a canary pox virus carrying the HIV env gene. No current vaccines have been able to induce high titers of broadly neutralizing antibodies against HIV Env. Of note, an unexpected, although modest, increased rate of HIV infection was observed in individuals immunized with an adenovirus-based HIV vaccine compared with nonimmunized individuals and was highest in persons who had antiadenovirus antibodies before the trial. The mechanisms explaining this increased infection rate are not clear. Results from the RV144 clinical trial conducted in Thailand showing a 31% reduction of HIV transmission demonstrated that vaccine protection may be a reality. New vector approaches, improving T-cell responses and achieving high titers of broadly neutralizing vaccines, are being investigated to increase vaccine efficacy (see HIV preventive vaccines).

ROUTES OF INFECTION HIV is transmitted through three routes of infection: sexual, parenteral, and perinatal. In general, higher viral loads, lower

CHAPTER 39  HIV and Acquired Immunodeficiency Syndrome CD4 T-cell count, and larger viral inoculum size are all associated with a greater risk of transmission. Sexual transmission is the most common mode of infection, responsible for 70–80% of worldwide infections. Besides the factors mentioned above, recipients in penetrative intercourse are more likely to become infected. Anal intercourse carries the greatest risk, followed by vaginal intercourse, and oral intercourse is the least likely to spread the virus. The presence of other sexually transmitted diseases, especially ulcerative lesions, such as those seen in herpes simplex or syphilis infection, increases the risk of transmission. Parenteral transmission is the second most common route of HIV infection, accounting for 8–15% of all HIV infections. Examples include contaminated needles used by people who are addicted to intravenous drug use (IDU), accidental needle sticks occurring in health care workers, improperly sterilized hospital equipment, and contaminated blood products. Use of contaminated needles by people addicted to IDU is the largest risk factor for parenteral transmission. In 2013, 7% of the estimated 47 500 new HIV infections in the United States were attributed to IDU.11 Although infection control efforts have greatly reduced the risk, HIV transmission via transfusion of contaminated blood products remains significant, particularly in resource-limited settings where there is a dependence on replacement of blood by family member donors and paid blood donors and where the infrastructure for routine blood screening is suboptimal. Perinatal transmission accounts for the majority of pediatric HIV cases and for 5–10% of HIV infections in patients of all ages. The virus is capable of infecting the child in utero, during labor, and after delivery through breastfeeding. Without preventive therapy, the risk of a child contracting HIV from the mother during gestation or during labor is about 15–40%. The majority of transmission events occur during the passage of the fetus through the birth canal, by exposure of the baby to infected maternal blood, amniotic fluid, and/or cervical and vaginal secretions.12 Although breastfeeding increases the risk of transmission of the virus from mother to child by another 15–29%, breastfeeding is still recommended to prevent morbidity and mortality related to diarrheal diseases in settings where access to replacement feeding is limited.

IMMUNOPATHOGENESIS The mechanisms of immunodeficiency induced by HIV are not limited to depletion of its main target cell, the CD4 T cell; HIV infection ultimately leads directly or indirectly to the impairment of every arm of the immune system.6 The time of progression from infection to the development of AIDS is not the same for all infected individuals, and several explanations have been proposed, including genetic resistance genes and the presence of low-virulence mutant viral particles. The study of long-term nonprogressor individuals has helped to define T-cell and antibody features associated with anti-HIV immunity in the search for the optimal vaccine design, focusing on minimizing the escape variants and inducing persistent specific viral neutralization or inhibition of replication.10 HIV researchers have also explored the role of chronic immune activation processes that are associated with progression to AIDS, which also helps explain the diverse clinical impact of this viral infection in different individuals.

Mucosal Dendritic Cells: Myeloid Versus Plasmacytoid Following a mucosal inoculation of HIV, CD4 T cells are generally infected by CCR5-tropic virus, also called R5 or M-tropic virus.13

549

After approximately 1 week, the virus becomes detectable in draining regional lymph nodes. Myeloid DCs are not infected with HIV; rather, HIV gp120 binds to DC-specific intercellular adhesion molecule–grabbing nonintegrin (DC-SIGN) in the cell membrane. The resulting complex is internalized as a phagosome and then presented on the cell surface. As there is no fusion of the HIV Env with the DC membrane, infection does not occur. Plasmacytoid DCs, in contrast, express CD4 and the coreceptors CXCR4 and CCR5 and thus become infected by the virus. Infection leads to expression of CCR7, which acts as a homing signal for the lymph nodes; it is in lymph nodes that the virus infects CD4 T cells, and significant viral replication occurs, resulting in detectable viremia and dissemination through lymphoid tissues. Of interest, the target cells for HIV infection, CD4 T cells and monocyte/macrophage cells, are also reservoirs for the virus. HIV-infected cells carry a stable provirus integrated in the cell genome, which is transcriptionally silent until the cell is activated. These cells can reestablish HIV viremia even after prolonged antiviral treatment, since viral reservoirs are unlikely to be soon eradicated.

Gastrointestinal System: Early Target Within days of infection, 20% of CD4 T cells found in the GALT are infected. Of these, up to 80% are killed, by lytic infection and Fas-mediated apoptosis of both infected and noninfected cells, mainly CD4+CCR5+ memory T cells. By the time of peak viremia, 60% of the mucosal memory CD4 T cells are infected. Because their destruction is less drastic, circulating CD4 T-cell counts do not reflect the magnitude of CD4 T-cell death taking place in the GALT. Following acute HIV infection, a massive activation of the immune system occurs. Inadequate memory T-cell responses can lead to infection and inflammation of the entire bowel. The presence of immune activators over the massive surface area of the bowel has been hypothesized to result in the profound immune activation seen in HIV. Mucosal Th17 cells are a preferential target for HIV, further weakening mucosal immunity and favoring microbial infection. The viremia decreases spontaneously from as high as 10 million copies of HIV RNA per milliliter during the acute illness phase to a stable level many orders of magnitude lower called the viral set point. It is known that higher set points of viral load and lower T-cell counts are loosely predictive of shorter periods of clinical latency. Eventually, a prolonged period of homeostasis between the virus and the immune system collapses, and AIDS ensues.

Chronic Immune Activation and Progression to AIDS After the acute stage, HIV infection induces a high degree of proliferation and turnover of CD4 and CD8 T cells. The massive immune activation can be explained by the circulation of many soluble factors, including TLR ligands. HIV infection appears to activate plasmacytoid DCs by stimulation of TLR7 to secrete interferon-α (IFN-) and proinflammatory cytokines, using both TLR8 and DC-SIGN to infect these cells.14 In turn, stimulated DCs activate T cells, which are also being directly activated through TLR ligands. Bacterial products from the mucosa (e.g., LPS) and HIV protein products, such as Nef, Env, Vif, and so on, are mediators of this immune activation. Other microbial infections (viral, bacterial, or fungal) can stimulate the different TLRs and result in CD4 and CD8 T-cell activation and apoptosis. This rampant immune activation is thought to lead to the eventual collapse of the immune system.

550

Part four  Immunological Deficiencies

Anti-HIV Cellular Immunity

INNATE IMMUNITY

In individuals with HIV infection, the initial control of viremia occurs with the initial expansion of HIV-specific CD8 T cells. The importance of anti-HIV cytotoxic T cells is suggested by the development of viral escape mutations that are driven by immune selection pressure and evade the immunological response. In addition to their cytotoxicity, CD8 T cells secrete chemokines, such as RANTES and MIP-1β, which inhibit viral entry via CCR5, and IFN-γ, which activates immune cells and increases HLA expression (Chapter 10). Although severely reduced in number, IFN-γ–secreting HIV-specific CD4 T cells are present in individuals with HIV infection. The role of adaptive cellular immunity in protection against natural infection is currently challenged, since about 40–60% of noninfected but exposed individuals do not present with detectable anti-HIV responses.

Innate immunity mechanisms are characterized by their lack of antigen specificity and include epithelial barriers, the complement system, phagocytes, and antigen-presenting cells (APCs). Their role in HIV pathogenesis is complex, as their function is of most importance during acute infection but their contribution to immune activation may be deleterious in the chronic stage. Studies including individuals who were exposed to HIV but did not develop infection have suggested that an increased innate immune response generated in the mucosal microenvironment may explain the failure to develop a specific anti-HIV response.17

Mechanisms of T-Cell Depletion Three major mechanisms of T-cell depletion have been reported: direct lytic infection, apoptosis, and autophagy. Direct lytic infection is the result of massive activation of the immune system with production of inflammatory cytokines, as in the GALT (see Cytokines in HIV infection). Apoptosis Increased apoptosis of T cells in HIV infection was described early in the history of the epidemic. Several explanations have been offered: (i) HIV-induced apoptosis of infected cells (viral cytopathic effect); (ii) bystander effect from HIV-infected neighbor cells releasing viral proteins; (iii) death of HIV-specific effectors following their migration to infected sites; (iv) perturbation of proapoptotic signaling molecules on immune cells secondary to the chronic immune activation; and (v) destruction of HIVinfected cells by immune effectors. Late HIV infection is associated with the dominance of a syncytia-inducing form of the virus. Syncytia formation (clustering of CD4 T cells and membrane fusion) occurs through the interaction of HIV Env and CD4/CXCR4 on neighboring cells. These cells are more prone to undergoing apoptosis through a Fas-dependent pathway.15 Most of the HIV proteins have been implicated at one time or another in HIV-induced apoptosis. Autophagy Autophagy is a process by which cytoplasm and organelles are sequestered and directed toward lysosomal pathways and histone deacetylase.16 This process has been implicated in both the prevention and induction of apoptosis, reflecting common regulatory factors shared by both (e.g., tumor necrosis factor [TNF]–related apoptosis inducing ligand [TRAIL], FADD, DAPk, ceramide, and Bcl-2). HIV Env has been shown to be a stimulus for autophagy in uninfected CD4 T cells via its interaction with CXCR4. Inhibition of the autophagic pathway prevents Envinduced cell death in uninfected cells in vitro, demonstrating that this mechanism contributes to the loss of uninfected cells.

Anti-HIV Humoral Immunity After establishment of HIV infection, neutralizing antibodies are produced; however, the virus quickly mutates to avoid them, such that the host continues to respond to evolving viral mutants. The fact that antibodies can be effective in protecting hosts from HIV infection has been demonstrated in macaques and in a humanized mouse model of HIV infection.

NK Cells in HIV Infection NK cells are dramatically altered in HIV infection.18 A small subset of NK cells has been found to express both CD4 and CCR5 or CXCR4. These NK cells can be infected by HIV and may serve as one of the sites of latent infection or can be activated and contribute to the state of immune chronic activation. CCR5 and inhibitory receptor expression are higher on NK cells in patients with HIV infection than in HIV-seronegative subjects. In contrast, there is reduced expression of surface receptors on NK cells that induce cytotoxic activity, such as NK-cell protein 30 (NKp30), NKp44, and NKp46. Antibody-dependent cellular cytotoxicity (ADCC) is reduced in patients with HIV infection, as well as the NK-cell responsiveness to IL-2. The expression of HLA-C–specific inhibitory NK-cell receptors has been found to be increased in patients with HIV. Interestingly, studies of Vietnamese patients who were exposed to HIV through IDU but were seronegative showed that the NK cells of these patients not only secreted greater quantities of chemokines compared with those of the controls but they also had greater direct cytotoxicity. Genetic studies have suggested that inheritance of particular killer inhibitor receptor (KIR) alleles with their HLA ligands delays disease progression.19 The two subsets of DCs, myeloid and plasmacytoid, play an additional role in HIV infection by stimulating innate immune responses, such as the type 1 IFNs, against HIV via plasmacytoid DCs. DCs may have opposing roles, with myeloid DCs acting as reservoirs and favoring HIV dissemination and plasmacytoid DCs inhibiting HIV replication by cytokine secretion. In fact, low levels of plasmacytoid DCs correlate with high viral loads, low CD4 T-cell counts, opportunistic infections, and disease progression.

Cytokines in HIV Infection The dysregulation of the immune system produced by HIV infection includes significant perturbation in the balance of Th1 and Th2 cytokine levels. The Th1 cytokines IL-2, TNF-α, IFN-γ, and IL-12 decrease during HIV infection, whereas the Th2 cytokines, IL-10, and IL-4 increase or remain normal; levels of proinflammatory cytokines, such as IL-1, IL-6, and IL-8, also increase. Viral replication in HIV-infected T cells and monocytes is induced by IL-2, IL-7, and IL-15, as well as by the proinflammatory cytokines. These cytokines appear to induce viral replication by activating the host cell, a requirement for HIV productive replication. Some cytokines, such as IL-10, decrease HIV production, likely by inhibiting the synthesis of the activating cytokines and by decreasing the expression of CCR5 and other chemokine receptors. In addition, the HIV long terminal repeat (LTR) promoter contains sequences that bind cellular factors that are activated as a response to cytokine binding, such as nuclear factor (NF)-κB and AP1. IFN-α and IFN-β have activity against HIV, although their role of these cytokines in vivo is not well established and may have only an adjuvant effect.20 Their

551

CHAPTER 39  HIV and Acquired Immunodeficiency Syndrome

If the HIV infection is left untreated, its natural history involves the progression through four clinical phases: acute retroviral syndrome, asymptomatic or latent infection, symptomatic HIV infection, and finally AIDS. Each clinical phase correlates with specific events in the interaction between HIV and the host immune system. A small percentage of patients become long-term nonprogressors, and an even smaller percentage become elite controllers (see Long-term nonprogressors/elite controllers).

Acute HIV Infection Soon after infection, unopposed by effective host immune responses, HIV rapidly replicates and disseminates to lymphoid tissues (see Immunopathogenesis: gastrointestinal system) and to the systemic circulation, with viremia reaching as high as 10 million copies per milliliter. Plasma viremia typically peaks in 3–4 weeks after transmission, and then, as a result of the depletion of susceptible CD4 T cells and HIV-specific immune responses, the virus load precipitously declines, followed by more gradual decline for several weeks before reaching the set point. Clinically, the acute phase of HIV infection is manifested by a flu-like illness, referred to as acute retroviral syndrome.21 Two to 4 weeks after transmission, coinciding with the period of high plasma viremia and dissemination of virus to lymphoid organs, the majority of infected individuals experience a nonspecific infectious mononucleosis–like illness that lasts from a few days to several weeks. As the host develops HIV-specific immunity, the virus load decreases, CD4 and CD8 T cells recover, and the symptoms of the acute infection resolve. Although up to 90% of patients seek medical care for this illness, the nonspecific nature of the symptoms makes diagnosis of acute infection difficult, and most newly infected individuals are not diagnosed until much later. The public health implications of the acute HIV infection are enormous because the risk of transmission from individuals with acute infection appears to be much higher than that from those with established infection, in part because of the high viral load in the former.

Asymptomatic HIV Infection The acute infection is followed by a prolonged asymptomatic or latent period that may last 8–10 years in adults but is much shorter in children. During this time, the HIV viral load fluctuates around a relatively stable set point.22 The viral set point is a major determinant of infectivity and risk of disease progression, with higher viral loads being associated with more likely viral transmission, more rapid disease progression, and greater risk of death. The host immune response is insufficient to eradicate the infection but is enough to contain viral replication for many years. Although commonly thought to represent a stalemate between viral replication and CD4 T-cell production, this period is actually characterized by a steady and inexorable decline of CD4 T cells (50–75 cells per year).

Acute Infection Is an Opportunity for Early Human Immunodeficiency Virus (HIV) Diagnosis • Acute HIV infection is a nonspecific viral syndrome, often described as being similar to infectious mononucleosis. • Many patients present to a clinician at this stage, but most are not recognized as HIV infection. • Irreparable damage to the host immune system occurs during this stage of HIV infection resulting in chronic immune activation and the eventual collapse of the immune system. • HIV viral latency is established during the acute infection as HIV DNA integrates with the host genome resulting. Integrated viral genetic material makes cure of HIV impossible even after prolonged viral suppression with antiretroviral therapy (ART). • ART initiated during the acute infection can halt the destruction of the body’s memory T cells in the gut-associated lymphoid tissue (GALT) and lead to better long-term outcomes for the patient. • The practical importance of early therapy of acute infection remains in question because patients who are able to achieve and maintain undetectable viral loads on ART do well despite having commenced treatment long after their acute infection.

Symptomatic HIV Infection (pre-AIDS) As the infection progresses, most individuals develop clinical symptoms. The ability of the immune system to contain viral replication is overcome, and the viral load begins to increase. There is usually an inflection point in the CD4 T-cell curve marking the start of a period of more rapid decline in CD4 T-cell counts. As these counts fall, immunodeficiency, symptomatic disease, and AIDS eventually occur (Fig. 39.4).

End-Stage HIV Infection: AIDS As the CD4 T-cell count drops to 75% of cocaine users in the United States are exposed to levamisole. Clinical manifestations of AAV induced by levamisolecontaminated cocaine include constitutional features; arthralgia; retiform purpura involving the ears, face, and extremities; and, less commonly, renal and lung diseases. Laboratory abnormalities include leukopenia, neutropenia, and high-titer p-ANCA, directed against MPO-ANCA or against atypical p-ANCA–associated antigens, such as HNE, lactoferrin, and cathepsin G. PR3-ANCA, ANA, and antiphospholipid autoantibodies have also been described in these patients. This multiplicity of antibodies helps distinguish AAV induced by levamisole-contaminated cocaine from primary AAV, which usually targets just one antigen. Levamisole has an estimated mean half-life of 5.6 hours; therefore serum testing is likely to be negative if the most recent exposure occurred >24 hours prior to sample collection. Urinary detection of levamisole is highly suggestive of drug-induced disease and is useful if exposure occurred 90% in some series), and eradication of hepatitis C appears to be effective in reducing the manifestations of disease. True PAN typically presents with neuropathies, systemic inflammation, and ischemic abdominal pain (difficulty eating because of medium-vessel supply to the gut and nerves). If it is associated with hepatitis B, the patient may or may not have obvious signs of liver disease. Isolated cutaneous vasculitis typically presents as purpuric lesions, skin ulceration, or broken livedo, and biopsy of the skin will show typical changes of a necrotizing vasculitis. KD usually presents in childhood as an acute illness, with relapsing high fevers, significant lymphadenopathy, and a

The investigation of patients with suspected small- or mediumvessel vasculitis should follow a careful history and examination to determine the likely diagnosis and underlying illness. The differential diagnosis is very wide. It is important to be vigilant in looking for positive signs of vasculitis, but it is equally important not to forget to look for more common causes of the clinical presentation. Many of the studies performed can result in nonspecific findings, such as an elevated white blood cell count, platelet count, or the erythrocyte sedimentation rate. The C-reactive protein (CRP) level is typically raised. Patients may be anemic. Liver or, more importantly, renal function may be abnormal. The presence of hypereosinophilia can be suggestive of a diagnosis of EGPA, but there are other causes of hypereosinophilia, particularly drug reactions. It is important to test renal function in patients with suspected vasculitis in case there is nephritis, and it is always important to perform urinalysis for microscopic hematuria or proteinuria. Although these might be explained by the presence of a kidney infection or other causes, they raise a strong suspicion of glomerular inflammation. An abnormal urinary sediment in combination with hypertension should alert the physician to the possibility of kidney involvement by small-vessel vasculitis. Since the 1980s, the discovery of ANCA12 has transformed the recognition of renal vasculitis, so that these patients can be managed more effectively. Histology remains a very important diagnostic test, not only to make a positive diagnosis but also to exclude other causes. Although histology from the airways can be nondiagnostic, this may still assist in ensuring that the patient does not have cancer, sarcoidosis, o tuberculosis, or IgG4-related disease, all of which could present in a similar way with inflammation of the upper or lower airway. Renal histology is still the gold standard to diagnose suspected glomerulonephritis and may be useful in predicting the prognosis.76 Four categories of renal lesion have been proposed: focal, crescentic, mixed, and sclerotic. Follow-up of patients with different patterns has shown outcomes: progressively worse renal outcome from focal (the best) through to sclerotic (the worst) over the subsequent 5 years.

ASSESSMENT The outcome of small- and medium-vessel vasculitis has been completely transformed by immunosuppressive therapy. With treatment almost all patients with KD recover from their initial illness. In AAV, >70% survive at least 5 years from starting treatment. The majority of patients with PAN respond to initial therapy. However, the risk of recurrence of disease is high in small-vessel vasculitides. Relapse rates are likely to exceed 50% over time.46 Patients experience comorbidities as a consequence of the disease or its treatment and can suffer from infections or other complications that contribute to their overall poor health. In the long term, small-vessel vasculitides and their treatment can be associated with effects on the cardiovascular system with increased risk of hypertension, coronary heart disease, and stroke.

800

Part SIX  Systemic Immune Diseases

Immunosuppressants given to patients can significantly improve disease control but may increase the risk of malignancy. The continued use of high doses of glucocorticoid therapy contributes to hypertension, diabetes, heart diseases, osteoporosis, and infection. It is therefore obvious that careful evaluation of a patient’s status is required throughout the course of disease. Constant vigilance is required to detect and manage any flare-up of disease because the consequences of untreated inflammation of vessels to vital organs can be severe. Although for most patients, initial therapy is very successful and improvements in disease status is obvious to the patient, the subsequent disease course can be much more complicated because of comorbidities, the evolution of disease-related damage, and morbidity caused by treatment as well as disease flare-ups. There are no suitable biomarkers that can be universally applied in small- and medium-vessel vasculitides to determine the patient’s disease state to provide an evidence-based rationale for adjustment of therapy. ANCA titers fluctuate during the course of disease, and although ANCA remains a very useful diagnostic test, its value in managing variations in disease activity is very limited. Up to 40% of patients have an elevation of ANCA without any new deterioration in clinical state, but a recent study has shown that patients with renal involvement as part of their presentation are likely to have relapses associated with ANCA rises.77 Among 166 patients with AAV, all were positive for ANCA and 104 had renal involvement (a mixture of PR3-ANCA and MPO-ANCA). The hazard ratio for ANCA rises predicting subsequent relapse was 11.09 (CI 5.01–24.55), suggesting that this test may be of value in detection of future relapse in this subset of patients. However, for the majority of patients with systemic vasculitis, careful clinical evaluation remains a cornerstone of effective disease management as recommended by the EULAR guidelines on management of systemic vasculitis.78 In simple terms, at the onset of the condition, disease activity is the dominant problem, and treatment should be directed toward it; but over the course of time, with the development of consequences of vasculitis or its treatment, there is an increasing component of damage or scarring and also side effects of drug therapy. Similarly, different patients function at different levels with the same amount of disease, or damage, and therefore the ability to perform normal tasks is an important component of their overall condition to consider (Table 58.3). The primary tool used is the Birmingham Vasculitis Activity Score (BVAS) in adults and the Paediatric Vasculitis Activity Score (PVAS) in children (reviewed in Ponte et al., 2014).79 Training is recommended for assessment in the Birmingham Vasculitis Activity Score (BVAS). BVAS provides a quantitative score based on individual items, providing an effective means to define the patient’s status with regards to response to therapy. Many recent studies of different immunosuppressive agents have made use of the BVAS either to define improvement in terms of a fall in BVAS score or to define a cut-off representing active disease, or inactive disease, or flare-up, depending on the number of items present. Although it is subject to observer variability, it provides an effective means by which groups of patients can be compared against each other and allows individual patients to be followed up over the course of their condition. The score is weighted according to the organ system and the individual manifestation, to reflect the severity of the disease. The range of scores for BVAS and PVAS are 0–63. For BVAS GPA, items are divided into major (scoring 3 points) and minor (scoring 1

point each); with 15–19 major items and 19–23 minor items, the range of sores is 0–76. The PVAS was developed as a pediatric version of the BVAS, but validated in children with vasculitis and demonstrated to be effective at discriminating different disease states and is increasingly used as a research tool in pediatric vasculitis.

KEY CONCEPTS Assessment of Disease Activity in Vasculitis • For small- and medium-vessel vasculitis in adults, version 3 and a specific version of the BVAS for granulomatosis with polyangiitis (GPA) are used. • BVAS version 3 is the most generic and applicable across different forms of vasculitis; BVAS for GPA has been specifically designed for use in GPA and contains very specific items related to that condition. • During sequential monitoring, a time frame of 3 months is recommended so that disease activity is considered to be of most relevance during this time. The time frame is based on pragmatic clinical experience that this is the usual time taken during which immunosuppressive therapy is likely to have a significant effect on active disease manifestations.

Damage Assessment in Vasculitis The concept of damage in patients with vasculitis is about the permanent consequences of having vasculitis. It is an attempt to measure disease burden regardless of cause. The Vasculitis Damage Index (VDI) is the most widely used and validated measure for assessment of damage in vasculitis (reviewed in Ponte et al., 2014).79 The VDI captures the long-term consequences of a diagnosis of vasculitis and its treatment and associated comorbidities. Damage is defined as lasting at least 3 months or occurring at least 3 months ago for single time point events (e.g., stroke or myocardial infarction) and should be recorded as a permanent change to the patient’s damage status. A VDI of >5 points recorded within 6 months of disease carries a significant increased risk of subsequent mortality compared with a lower damage index at 6 months (reviewed in Ponte et al., 2014).79 VDI is a useful index of future harm. A further development of the damage index has been the combined damage assessment (CDA). In a comparative study with the VDI, CDA was shown to be inferior to it (reviewed in Ponte et al., 2014).79

TREATMENT Once a diagnosis of small- and medium-vessel vasculitis has been established, treatment should be focused on patients and their problems rather than the specific diagnosis. Treatment for different forms of vasculitis may look very different, but many aspects of different forms of vasculitis require the same therapeutic approach. Without a clear understanding of the underlying pathogenesis of disease, we are inevitably led by the need to suppress inflammation and reduce damage to prevent mortality and improve survival. However, as well as modifying immune dysregulation, it is important to consider other aspects of the patient’s condition, such as comorbidities and the prevention of future comorbidities.

CHAPTER 58  Small- and Medium-Vessel Primary Vasculitis

801

TABLE 58.3 Assessing Disease Activity in Vasculitis System Assessed

BVAS

BVAS WG

PVAS

Comments

Myalgia, arthralgia/ arthritis, fever, weight loss Infarct, purpura, skin ulcer, gangrene

Arthritis, fever

Myalgia, arthralgia/ arthritis, fever, weight loss Polymorphous exanthema, livedo, panniculitis, purpura, skin nodules, infarct, ulcer, gangrene, other skin vasculitis

Mouth ulcers, genital ulcers, adnexal inflammation, proptosis, red eye, (epi)scleritis, conjunctivitis, blepharitis, keratitis, blurred vision, visual loss, uveitis, retinal vasculitis Bloody nasal discharge, paranasal sinus involvement, subglottic stenosis, conductive hearing loss, sensorineural hearing loss Wheezing, nodules or cavities, pleural effusion, infiltrates, endobronchial changes, massive hemoptysis, respiratory failure

Mouth ulcer, conjunctivitis/ episcleritis, retro-orbital mass, uveitis, scleritis, retinal exudates

These are typical features in many forms of systemic vasculitis. Skin is a common organ involved in most forms of small and medium vessel vasculitis. Skin manifestations in ANCA-associated vasculitides tend to be less serious compared with manifestations in kidneys, lungs, and upper airways. For skin vasculitis in children the manifestations are more diverse. Eye involvement is most common in GPA and less often seen in MPA or EGPA.

Cardiovascular system

Loss of pulses, ischemic cardiac pain, cardiomyopathy, congestive cardiac failure, valvular heart disease, pericarditis

Pericarditis

Abdominal system

Ischemic abdominal pain, peritonitis, bloody diarrhea

Mesenteric ischemia

Renal system

Hypertension, proteinuria, hematuria, impaired renal function, deterioration in renal function

Hematuria, red cell casts, or glomerulonephritis, deterioration in renal function

Hypertension, proteinuria, hematuria, impaired renal function, deterioration in renal function

Nervous system

Headache, meningitis, organic confusion, seizures, stroke, cord involvement, cranial nerve, lesion, sensory or motor neuropathies

Meningitis, stroke, cord involvement, cranial nerve lesion, sensory or motor neuropathies

Headache, meningitis, organic confusion, seizures, stroke, cord involvement, cranial nerve, lesion, sensory or motor neuropathies

General Skin

Mucous Membrane/ eyes

Ear, nose, and throat

Chest

Purpura, skin ulcer, gangrene

Blood nasal discharge, paranasal sinus involvement, swollen salivary glands, subglottic inflammation, conductive hearing loss, sensorineural hearing loss Nodules or cavities, pleurisy, infiltrates, endobronchial changes, alveolar hemorrhage hemoptysis, respiratory failure

Mouth ulcers, genital ulcers, adnexal inflammation, proptosis, red eye (epi)scleritis, conjunctivitis, blepharitis, keratitis, blurred vision, visual loss, uveitis, retinal vasculitis Blood nasal discharge, paranasal sinus involvement, subglottic stenosis, conductive hearing loss, sensorineural hearing loss Wheezing, nodules or cavities, pleural effusion, infiltrates, endobronchial changes, massive hemoptysis, respiratory failure

Loss of pulses, bruits, blood pressure discrepancy, claudication, ischemic cardiac pain, cardiomyopathy, congestive cardiac failure, valvular heart disease, pericarditis Abdominal pain, peritonitis, bloody diarrhea, bowel ischemia

ENT manifestations are most common in GPA and rarely seen in MPA or EGPA. However, EGPA is often characterized by the presence of nasal polyps, which are inflammatory in nature.

The chest is commonly affected in all three forms of ANCA vasculitis. Wheezing is a common feature of EGPA as are infiltrates. In contrast, infiltrates, nodules, and endobronchial disease dominate in GPA. Massive hemoptysis can occur in patients with GPA or MPA and less frequently in patients with EGPA, but may also be a typical feature with GBM (glomerular basement membrane) disease. Cardiovascular manifestations are most widely recognized in KD, which is not particularly well covered by PVAS. Although CVD manifestations do occur in small- and medium-vessel vasculitis, they are more typically seen in large vessel diseases, such as Takayasu arteritis. Gut involvement is more typical in medium-vessel vasculitis, especially polyarteritis nodosa, but is well recognized in patients with GPA especially with colitis giving rise to bloody diarrhea. Renal involvement in small-vessel vasculitis is one of the major manifestations leading to organ failure and death and should be carefully assessed. Renal involvement in medium-vessel vasculitis is much less common and takes the form of infarction of segments of the kidney leading to hematuria and hypertension with consequent impairment of renal function. Neurological involvement is a common feature in small- and medium-vessel vasculitis; often it does not lead to immediate loss of life. Strokes are less common, whereas peripheral neuropathies are more common and can cause long-term disability.

802

Part SIX  Systemic Immune Diseases

No Treatment/Symptom Relief Small-vessel vasculitis, such as isolated cutaneous vasculitis resulting from infection or use of pharmaceutical agents, may respond to simple withdrawal of the offending agent or resolution of the infection without the need for specific treatment. However, for more recalcitrant disease, symptom relief may be required and occasionally systemic steroids. Symptom relief could be provided in the form of antipruritic agents or topical cream to reduce skin inflammation and/or topical steroids. NSAIDs can be helpful in relieving symptoms of joint pain or swelling. As monotherapy they are unlikely to resolve skin manifestations but could be tried in combination with other therapies. Colchicine has been used for skin vasculitis and occasionally can be effective, although the doses required should remain below 2 mg/day to avoid the predictable side effects of abdominal cramps and diarrhea.

Target-Directed Therapies In diseases where there is a clearly defined provoking agent, as in hepatitis B–related PAN or hepatitis C–related cryoglobulinemic vasculitis, eradication of the virus is a key part of treatment of the disease. Effective antiviral agents play a vital role in the management of virus, associated with the need for immunosuppression. Hepatitis B–related PAN is treated with a combination of antiviral therapy plus plasma exchange to remove immunecomplexes and other inflammatory mediators combined with a course of glucocorticoid therapy. For hepatitis C–related cryoglobulinemic vasculitis, recent reports of virus eradication may also be transforming the outcome of this disease. Unfortunately, the toxicity of these regimens can be considerable, with >40% requiring erythropoietin, red blood cell transfusions, and or G-CSF.

Specific Therapies In KD, although the etiological factors have not yet been defined, it seems likely that this is related to some kind of infectious agent (see earlier section on pathogenesis). The most effective therapy is use of high doses of IVIG (0.4 g/kg/day for 5 days) combined with high doses of aspirin, which is usually curative. Whether or not this will prevent long-term harm to the cardiovascular system, particularly the coronary arteries, remains to be explored. Glucocorticoids Glucocorticoids (Chapter 86) remain a cornerstone in the management of most forms of multisystem vasculitis. They are relatively contraindicated in KD because they may potentially worsen the development of coronary artery aneurysm, but they have been used in combination with IVIG and aspirin with beneficial outcomes. However, they are an integral part of almost all therapeutic regimens for management of vasculitis. In some instances, such as isolated skin vasculitis, they are the only treatment required, but more often they are insufficient on their own without causing significant morbidity from side effects. Typical doses of glucocorticoid therapy are 1 mg/kg/ day over a period of 2–4 weeks, reducing to around 10–15 mg/ day within 6 months, and then slowly withdrawing steroids in the next 6–12 months. The use of high-dose intravenous methylprednisolone is popular but lacks evidence. The only randomized trial of intravenous methylprednisolone compared it against plasma exchange in patients with severe AAV.80 This

study demonstrated the superiority of plasmapheresis over IV prednisolone, both used as adjunct therapies (in combination with cyclophosphamide and high doses of oral prednisolone) for treatment of severe AAV (mainly MPA plus some cases of GPA) with significant renal impairment (creatinine levels above 500 µmol/ L [5.66 mg/dL]). The role of glucocorticoid therapy is been increasingly challenged by more recent trials using smaller doses for shorter periods or even eliminating steroid use completely in some instances. Glucocorticoid side effects are well known: weight loss, increased appetite, mood swings, hypertension, risk of diabetes, risk of infection, risk of cataract, and skin striae. The risk of osteoporosis is largely preventable with concurrent use of bisphosphonate therapy (unless there is significant renal dysfunction) with supplementary calcium and vitamin D replacement. Other Immunosuppressive Therapies Cyclophosphamide (Chapter 87) has been available since the 1950s but was first used for the management of systemic vasculitis in the 1970s and remains the most effective agent we have for managing multiorgan systemic vasculitis. Initially it was used as a daily oral agent at 2–3 mg/day, and it transformed the outcome of patients with AAV from inevitable mortality to a high likelihood of survival. It is a cytotoxic agent and carries with it the risk associated with chemotherapy, including increased risk of malignancy, especially in the bladder because it is predominantly excreted through the kidneys and accumulates in the bladder. Initial protocols were associated with excessive risks of bladder cancer (approximately 33-fold), but despite this, the daily oral cyclophosphamide dosing regimen was not effective in maintaining control of disease. Therefore over the past 20 years or so, there have been a number of trials comparing reduced doses of cyclophosphamide, high-dose intermittent pulse therapy,81 or with combination strategies of induction with short courses of cyclophosphamide followed by a switch to another drug for maintenance,81 or by replacing cyclophosphamide with another agent, such as methotrexate. All these studies81 have demonstrated the equivalence of using shorter courses of daily oral cyclophosphamide and, more recently, of using of high-dose intermittent pulses of cyclophosphamide to reduce the total dose even further. The total cumulative dose from six cycles of cyclophosphamide over a period of 3 months would be 6 g (based on 15 mg/kg/ treatment on six occasions). This compares with 9–12 g of cyclophosphamide given as daily oral therapy over 4–6 months.81 Although the relapse rate for patients given high-dose intermittent cyclophosphamide was higher during the subsequent 5 years compared with that for patients who were not given daily oral cyclophosphamide, relapse was always effectively managed with reintroduction of therapy and never led to mortality.78 The consequences of exposure to cyclophosphamide are likely to be risk of cancer (more recent studies suggest that this risk is relatively modest now that the regimens include much lower total doses), infertility, hair loss, nausea, vomiting, diarrhea, cytopenia, and increased risk of infection. Less common complications of cyclophosphamide include hyponatremia. The introduction of rituximab for AAV has had a significant impact on the use of cyclophosphamide, with increasing numbers of patients being managed with rituximab in place of cyclophosphamide, especially if patients are of child-bearing years, or if there is a potential contraindication to using cyclophosphamide, such as a previous history of bladder cancer.

CHAPTER 58  Small- and Medium-Vessel Primary Vasculitis Because of the nature of AAV, relapse is common. Therefore a single course of therapy rarely achieves long-lasting remission. Repeat cycles of treatment are likely to be required, which accounts for the accumulation of higher doses of cyclophosphamide, especially in the pre-rituximab era. Therefore although each individual course of therapy may only contain 6–9 g, during a patient’s life time, they may require treatment for several relapses, which would then start building up the total exposure to cyclophosphamide. Nevertheless, even though it is being slowly replaced, it remains an important aspect in the therapy of vasculitis even in the present time. Azathioprine is an immunomodulator with cytostatic properties. It inhibits cell division; it has been an effective immunosuppressant agent for decades. It was first used in combination with steroids and found to reduce mortality in systemic vasculitis in an open-label retrospective study of 64 patients.82 The 5-year survival of patients given no therapy was 12%, and those given steroids alone had a 53% survival rate, whereas those treated with steroids plus another agent (mainly azathioprine but a few had cyclophosphamide) had a survival rate of 80%. It is an oral medication given at 2–2.5 mg/kg/day, and it has largely been superseded as an induction agent by cyclophosphamide. Azathioprine is usually now used as maintenance therapy once the disease has been controlled with another agent. It has been shown to be equivalent to methotrexate and superior to mycophenolate76 as maintenance therapy. It is a relatively safe immunosuppressant and can be used safely throughout pregnancy. Methotrexate (MTX) is popular among rheumatologists but less so among renal physicians who regard it with some suspicion because of its potential for nephrotoxicity. The latter is only the case for patients who have well-established renal disease (typically with a creatinine level >300 µmol/L). MTX is an effective immunosuppressant agent used very widely in the management of inflammatory arthritis and has found its place in the management of GPA, where studies have demonstrated its efficacy in comparison to oral cyclophosphamide.78 However, it needs to be given continuously, rather than as induction regimen over the short period of time. Although MTX is as effective as cyclophosphamide in inducing remission in GPA, stopping the drug inevitably leads to relapse. MTX is recommended for non–life-threating AAV, usually in combination with steroid treatment. MTX is available either as oral, intramuscular, or subcutaneous administration. The dose is 20–25 mg/ week in most studies of vasculitis. It is contraindicated in pregnancy. Cotrimoxazole, an antibiotic containing sulfonamide and trimethoprim, was fortuitously discovered to have beneficial effects in patients with GPA who had been treated coincidentally for infections. There has been a significant advance in our understanding of GPA, partly as a result of this historical experiment and partly based on suggestions that S. aureus plays a role in initiating disease by its effect on the nasal mucosa. Eradication of the organism has been suggested as one mechanism by which it works, although the drug is not particularly effective against this organism. It is more likely that the drug has an immunosuppressive effect in itself; it has been demonstrated to be effective in combination with low-dose steroids in a randomized trial of localized forms of GPA.78 It is also commonly used as a prophylactic agent against Pneumocystis jiroveci infection; it is given 3 times per week for patients receiving other more potent immunosuppressant therapy, such as cyclophosphamide or even MTX (despite the potential for drug interactions leading to anemia).

803

Mycophenolate mofetil is a widely used transplantation drug that has been tested in AAV but is less effective than azathioprine as maintenance agent for patients who have achieved remission. It is currently being tested against cyclophosphamide as an induction agent in AAV. Is typically given as 2–3 g/day as an oral dose, along with reducing courses steroids. It is contraindicated in pregnancy. Cyclosporine is a well-established immunosuppressive drug that has been used for transplantation for many decades. Cyclosporine has been given to limited numbers of patients with systemic vasculitis, with one small well-conducted trial82 in 32 patients with GPA. In combination with plasmapheresis, it was as effective as continuous oral cyclophosphamide as a maintenance agent. It is generally limited by its toxicity and is not routinely used. Leflunomide, an antilymphocyte agent used extensively for the management of inflammatory arthritis, has been tested in patients with AAV in limited trials demonstrating its ability to maintain remission. Indeed, in a recent meta-analysis, it was reported to be superior to azathioprine, MTX, and mycophenolate as a maintenance agent for AAV, but more trial data are needed. It is an oral agent that is characterized by a very long half-life, and it is not suitable for use in pregnancy. The role of hydroxychloroquine in mild forms of vasculitis is uncertain. There is anecdotal evidence that it is beneficial in patients with skin manifestations with small-vessel vasculitis. Its use probably stems from its known effects in the treatment of connective tissue diseases with skin and joint manifestations, such as SLE. Other similar agents, such as mepacrine and dapsone, have also been used for skin vasculitis with occasional reported positive outcomes. However, the potential toxicity for each of these agents should be considered alongside the relatively limited evidence for benefit. Specific Immunotherapy Better understanding of the pathogenesis of vasculitis (see section on pathogenesis) has led to the development of targeted immunotherapy for some of these diseases. Rituximab is an mAb against B cells (Chapter 89) and has been in widespread use for treatment of RA. Its role as an effective agent in AAV is well established with two randomized trials demonstrating efficacy comparable with cyclophosphamide.78 Rituximab induction therapy is just as effective as cyclophosphamide for moderate and moderateto-severe AAV. Maintenance therapy with rituximab is a real possibility with the potential to provide long-term control and reduce relapse risk. The long-term consequences of repeat cycles of rituximab, however, are unexplored. The risks are hypogammaglobulinemia, which occurs in most cases, and the potential increase in incidence of infections. The most feared complication is the risk of reactivation of the JC virus leading to the complication of progressive multifocal leukoencephalopathy (PML), which has a very high mortality rate. Other Therapies Belimumab is currently undergoing clinical trials as a maintenance agent for AAV. The drug is a BAFF inhibitor and may be an effective means to control the disease over the long term. Mepolizumab is an mAb directed against IL-5, which controls eosinophil production. Mepolizumab has been successfully used in the treatment of hypereosinophilic syndrome and is undergoing a trial in EGPA. IVIG (Chapter 84) has been available as a replacement therapy for patients with hypogammaglobulinemia

804

Part SIX  Systemic Immune Diseases

for several years, and it is the standard of care for KD, but its use in AAV has been limited. An initial study suggested benefit, but the likelihood is relatively short-term control.82 Plasmapheresis has been available for several decades. It is not entirely clear how it works; there are many theories suggesting the removal of circulating immune mediators is effective in reducing inflammation. It has been successful for rescue therapy in patients with very aggressive AAV or anti-GBM disease. The MEPEX trial demonstrated that it was able to reduce renal dysfunction in patients with severe AAV.78 However, the long-term follow-up of the MEPEX trial demonstrated that the difference between patients treated with plasma exchange and those given methylprednisolone pulses (both as adjunct treatment alongside cyclophosphamide and steroid) did not last.83 It has been suggested this is accounted for by the fact that many patients with severe renal disease had already developed irreversible changes to their kidneys and that renal dysfunction would have been secondary to damage rather than active disease. A new study is underway to test the role of plasma exchange in patients with less severe renal disease, but results are not yet available. Plasmapheresis is effective in conjunction with antiviral therapy and steroids in the management of hepatitis B–related PAN.

OUTCOMES The majority of patients have a successful initial outcome. Either the condition is self-limiting in the case of more isolated forms of skin vasculitis or the initial immunosuppressive therapy is successful. Over 94% of patients with generalized AAV would expect to survive the first 18 months,78 whereas patients with more severe disease, especially with significant renal impairment, have a mortality of around 25% after 2 years.82 This contrasts with >80% likelihood of dying without adequate treatment. However, with current therapy, 5-year survival figures suggest around 25–30% mortality among AAV-affected patients.84 The bigger problem, however, is morbidity. The quality of survival for most patients with multiorgan disease is complicated by episodes of relapse in 50–70% of cases and low-grade grumbling disease, which never quite goes into full remission in up to a third of cases.85 This is added to the comorbidity experienced by older patients, usually a combination of vasculitisrelated damage, steroid-induced side effects, and the long-term consequences of immunosuppressive agents. In the first year of diagnosis, the most likely cause of mortality is active vasculitis or infection,62 the latter being a surrogate measure of the severity in immunosuppressant therapy required to control the disease. Long-term adverse outcomes in vasculitis can be measured using a structured VDI (see Assessment section above). One of the most important outcomes is the development of end-stage renal failure and the requirement for dialysis. It is likely that this is significantly reduced as a result of effective therapy given within the first 4 months of diagnosis. Transplantation is successful in patients with AAV, and these patients should be offered this treatment. Ten-year survival rates (32.5%) are similar to those reported for other patients without diabetes receiving a kidney transplant.86 The immunosuppressive regimens used for maintaining the transplant (Chapter 81) are often sufficient to keep the vasculitis in remission, but there is a need for ongoing review. Infection is a significant concern, especially in the early course of disease when potent treatment being commenced, especially high doses of steroids. The risk of serious infection requiring hospitalization is very high in the first year,62 especially if the

steroid doses remain high after 6 months. Interstitial lung disease (nonspecific interstitial pneumonia) is reported in around 20% of Japanese patients, especially those with MPA. This is a higher prevalence than that seen in other populations and may reflect genetic and environmental differences unique to Japan. Chronic neuropathy occurs in 15% of patients with AAV87 and can be very distressing for patients. Upper airway disease generally continues to cause long-term problems in 65% of patients with GPA because of chronic mucosal damage causing symptoms of chronic nasal congestion, discharge, and discomfort.88 Symptom relief is only partially successful in alleviating these problems. The risk of cardiovascular disease among patients with smallvessel vasculitis is probably around four times greater in patients with AAV compared with the general population. The risk of cardiovascular events is around 14% within 5 years of diagnosis, especially in older patients who have baseline hypertension and MPO antibodies.89 Cancer is associated with the presence of small-vessel vasculitis. It may predate as well as occur at the same time as diagnosis or develop subsequently, but it is recognized as a risk among patients treated with immunosuppressive and cytotoxic agents. Cancer of the bladder particularly has been an established risk arising from treatment with cyclophosphamide for many years; the original data from the 1970s suggested up to 33-fold increased risk of bladder cancer among patients treated with cyclophosphamide for vasculitis compared with background controls. However, this risk has been reduced with the use of more limited courses of cyclophosphamide (typically 3–6 months duration) and particularly with use of intermittent cyclophosphamide delivery. In a recent large series from the European Vasculitis Study Group (EUVAS), the only increased risk of cancer was for nonmelanoma skin cancer, and this may also have reflected the use of azathioprine as well as use of cyclophosphamide.55 Shang et al.90 showed that in a meta-analysis of over 2500 patients, the standardized incidence rate of late-occurring malignancies, particularly nonmelanoma skin cancer, leukemia, and bladder cancer, was 1.74 (95% CI = 1.37–2.21). We advise all of our patients to wear sun protection. The ability to work is significantly affected by AAV; among 410 patients interviewed, 26% of those of working age were classified as work disabled.91 The strongest influences on this outcome were fatigue, depression, high levels of damage (measured using the VDI), and being overweight. Patients’ functional outcome can be variably affected by vasculitis and its treatment. Patients report impairment of function as measured by using generic tools, such as EQ-5D or the short form 36.92 The impairment is similar to that found in other chronic diseases. Physical functions tend to be more affected than mental functions, especially in older patients with evidence of neurological involvement, usually peripheral neuropathy. Functional outcome is not directly correlated to disease activity, although in a Japanese cohort, 18 months after initiation of therapy, many aspects of function had started to improve.93 One of the problems of determining long-term outcomes in patients with vasculitis is the compounding effect of the very intensive immunosuppressive therapy required to control disease. Over the last 3 decades, we have seen dramatic shift away from long course cyclophosphamide toward short courses of intermittent dose therapy, but we are now witnessing an era when targeted biological therapies are able to take the place of cyclophosphamide. Therefore eliminating the use of cyclophosphamide altogether in some patients may reset potential future outcomes. If this is coupled with a reduced use of glucocorticoid therapies and maintaining better disease

CHAPTER 58  Small- and Medium-Vessel Primary Vasculitis control with less frequent relapses, the outcome may well be improved considerably for patients in the future.

ON THE HORIZON • Improved understanding of the underlying pathogenic mechanisms in different forms of vasculitis is starting to lead to more logical, rational therapies, some of which have become established as standard of care or should be available in the near future. • Rituximab is available and is being increasingly used in place of cyclophosphamide in the management of ANCA-associated vasculitis (AAV). Rituximab therapy is also effective in patients with cryoglobulinemic vasculitis, even when associated with the presence of hepatitis C. There is a concern that this might lead to reactivation of the virus and increase the risk of hepatoma or induce the development of the B-cell clone. However, better control of hepatitis C with effective antiviral agent is now possible. • Multiple proinflammatory cytokines are being explored as therapeutic targets, although this will only allow for the suppression of common pathways in the end stage of inflammation. • Mepolizumab has recently been shown to be effective in the treatment of eosinophilic granulomatous polyangiitis (EGPA) in a large randomised controlled trial. • Blocking interleukin (IL)-6 may be of benefit for some patients. • The discovery of the involvement of the complement pathway (C5) in AAV has led to the testing of therapies directed against C5a in humans. • The ultimate goal remains to identify upstream pathologies leading to vasculitis, which promises to fundamentally change the management of these devastating diseases, as it may permit the induction and maintenance of drug-free remission.

ACKNOWLEDGEMENTS The authors thank Jana Vaskova for administrative and secretarial support. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Kawasaki T, et al. A new infantile acute febrile mucocutaneous lymph node syndrome (MLNS) prevailing in Japan. Pediatrics 1974;54(3):271–6. 2. Andrews M, Edmunds M, Campbell A, et al. Systemic vasculitis in the 1980s-is there an increasing incidence of Wegener’s granulomatosis and microscopic polyarteritis? J R Coll Physicians 1990;24(4):284–8. 3. Watts RA, Mooney J, Skinner J, et al. The contrasting epidemiology of granulomatosis with polyangiitis (Wegener’s) and microscopic polyangiitis. Rheumatology (Oxford) 2012;51(5):926–31. 4. Mohammad AJ, et al. Prevalence of Wegener’s granulomatosis, microscopic polyangiitis, polyarteritis nodosa and Churg-Strauss syndrome within a defined population in southern Sweden. Rheumatology 2007;46(8):1329–37. 5. Nesher G, et al. The Incidence of Primary Systemic Vasculitis in Jerusalem: A 20-year Hospital-based Retrospective Study. J Rheumatol 2016;43(6):1072–7. 6. Holman RC, et al. Racial/ethnic differences in the incidence of Kawasaki syndrome among children in Hawaii. Hawaii Med J 2010;69(8):194–7. 7. Arora A, et al. Incidence of Leukocytoclastic Vasculitis, 1996 to 2010: A Population-Based Study in Olmsted County, Minnesota. Mayo Clinic Proceedings 2014;89(11):1515–24. 8. Watts RA, Scott DGI. Epidemiology of the vasculitides. Semin Respir Crit Care Med 2004;25(5):455–64.

805

9. Nakamura Y, et al. Epidemiologic Features of Kawasaki Disease in Japan: Results of the 2009-2010 Nationwide Survey. J Epidemiol 2012;22(3):216–21. 10. Reinhold-Keller E, et al. Stable incidence of primary systemic vasculitides over five years: Results from the German vasculitis register. Arthritis Rheum 2005;53(1):93–9. 11. Herlyn K, et al. Doubled prevalence rates of ANCA-associated vasculitides and giant cell arteritis between 1994 and 2006 in northern Germany. Rheumatology 2014;53(5):882–9. 12. van der Woude FJ, et al. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet 1985;1(8426):425–9. 13. Xiao H, et al. Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest 2002;110(7):955–63. 14. Primo VC, et al. Anti-PR3 immune responses induce segmental and necrotizing glomerulonephritis. Clin Exp Immunol 2010;159(3):327–37. 15. Little MA, et al. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system. PLoS ONE 2012;7(1):e28626. 16. Roth AJ, et al. Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis. J Clin Invest 2013;123(4):1773–83. 17. Lyons PA, et al. Genetically distinct subsets within ANCA-associated vasculitis. N Engl J Med 2012;367(3):214–23. 18. Stassen PM, et al. HLA-DR4, DR13(6) and the ancestral haplotype A1B8DR3 are associated with ANCA-associated vasculitis and Wegener’s granulomatosis. Rheumatology (Oxford) 2009;48(6):622–5. 19. Cao Y, et al. DRB1*15 allele is a risk factor for PR3-ANCA disease in African Americans. J Am Soc Nephrol 2011;22(6):1161–7. 20. Papiha SS, et al. Association of Wegener’s granulomatosis with HLA antigens and other genetic markers. Ann Rheum Dis 1992;51(2):246–8. 21. Spencer SJ, et al. HLA class II specificities in vasculitis with antibodies to neutrophil cytoplasmic antigens. Kidney Int 1992;41(4):1059–63. 22. Schreiber A, et al. Membrane expression of proteinase 3 is genetically determined. J Am Soc Nephrol 2003;14(1):68–75. 23. Alberici F, Martorana D, Vaglio A. Genetic aspects of anti-neutrophil cytoplasmic antibody-associated vasculitis. Nephrol Dial Transplant 2015;30(Suppl. 1):i37–45. 24. Kamesh L, et al. CT60 and +49 polymorphisms of CTLA 4 are associated with ANCA-positive small vessel vasculitis. Rheumatology (Oxford) 2009;48(12):1502–5. 25. Langford CA, et al. An open-label trial of abatacept (CTLA4-IG) in non-severe relapsing granulomatosis with polyangiitis (Wegener’s). Ann Rheum Dis 2014;73(7):1376–9. 26. Wieczorek S, et al. Functionally relevant variations of the interleukin-10 gene associated with antineutrophil cytoplasmic antibody-negative Churg-Strauss syndrome, but not with Wegener’s granulomatosis. Arthritis Rheum 2008;58(6):1839–48. 27. Ciavatta DJ, et al. Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis. J Clin Invest 2010;120(9):3209–19. 28. Chen M, Kallenberg CG. The environment, geoepidemiology and ANCA-associated vasculitides. Autoimmun Rev 2010;9(5):A293–8. 29. Gomez-Puerta JA, Gedmintas L, Costenbader KH. The association between silica exposure and development of ANCA-associated vasculitis: systematic review and meta-analysis. Autoimmun Rev 2013;12(12):1129–35. 30. Tervaert JW, Stegeman CA, Kallenberg CG. Silicon exposure and vasculitis. Curr Opin Rheumatol 1998;10(1):12–17. 31. Rihova Z, et al. Silica and asbestos exposure in ANCA-associated vasculitis with pulmonary involvement. Ren Fail 2005;27(5):605–8. 32. Eden A, et al. Lack of association between B19 or V9 erythrovirus infection and ANCA-positive vasculitides: a case-control study. Rheumatology (Oxford) 2003;42(5):660–4. 33. Wu YY, et al. Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection. Clin Exp Immunol 2002;128(2):347–52.

806

Part SIX  Systemic Immune Diseases

34. Calhan T, et al. Antineutrophil cytoplasmic antibody frequency in chronic hepatitis B patients. Dis Markers 2014;2014:982150. 35. Kain R, et al. Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis. Nat Med 2008;14(10):1088–96. 36. Hurtado PR, et al. CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis. BMC Immunol 2008;9:34. 37. Noh JY, Yasuda S, Sato S, et al. Clinical characteristics of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis caused by antithyroid drugs. J Clin Endocrinol Metab 2009;94(8):2806–11. 38. Waldhauser L, Uetrecht J. Oxidation of propylthiouracil to reactive metabolites by activated neutrophils. Implications for agranulocytosis. Drug Metab Dispos 1991;19(2):354–9. 39. Nakazawa D, et al. Abnormal conformation and impaired degradation of propylthiouracil-induced neutrophil extracellular traps: implications of disordered neutrophil extracellular traps in a rat model of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Rheum 2012;64(11):3779–87. 40. Berman M, Paran D, Elkayam O. Cocaine-Induced Vasculitis. Rambam Maimonides Med J 2016;7(4). 41. Wiesner O, et al. Antineutrophil cytoplasmic antibodies reacting with human neutrophil elastase as a diagnostic marker for cocaine-induced midline destructive lesions but not autoimmune vasculitis. Arthritis Rheum 2004;50(9):2954–65. 42. Tsurikisawa N, et al. Decreases in the numbers of peripheral blood regulatory T cells, and increases in the levels of memory and activated B cells, in patients with active eosinophilic granulomatosis and polyangiitis. J Clin Immunol 2013;33(5):965–76. 43. Abdulahad WH, Boots AMH, Kallenberg CGM. FoxP3(+) CD4(+) T cells in systemic autoimmune diseases: the delicate balance between true regulatory T cells and effector Th-17 cells. Rheumatology 2011;50(4):646–56. 44. Marinaki S, et al. Persistent T-cell activation and clinical correlations in patients with ANCA-associated systemic vasculitis. Nephrol Dial Transplant 2006;21(7):1825–32. 45. Holden NJ, et al. ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process. Ann Rheum Dis 2011;70(12):2229–33. 46. Nakazawa D, et al. Enhanced Formation and Disordered Regulation of NETs in Myeloperoxidase-ANCA-Associated Microscopic Polyangiitis. J Am Soc Nephrol 2014;25(5):990–7. 47. Yuan J, et al. C5a and its receptors in human anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Arthritis Res Ther 2012;14(3). 48. Chen SF, et al. Plasma complement factor H is associated with disease activity of patients with ANCA-associated vasculitis. Arthritis Res Ther 2015;17. 49. Fukui S, et al. Antineutrophilic cytoplasmic antibody-associated vasculitis with hypocomplementemia has a higher incidence of serious organ damage and a poor prognosis. Medicine (Baltimore) 2016;95(37). 50. Choi HK, Merkel PA, Niles JL. ANCA-positive vasculitis associated with allopurinol therapy. Clin Exp Rheumatol 1998;16(6):743–4. 51. Hirohama D, et al. Development of myeloperoxidase-antineutrophil cytoplasmic antibody-associated renal vasculitis in a patient receiving treatment with anti-tumor necrosis factor-alpha. Mod Rheumatol 2010;20(6):602–5. 52. Jarraya F, et al. Myeloperoxidase-antineutrophil cytoplasmic antibody-positive crescentic glomerulonephritis associated with benzylthiouracil therapy: report of the first case. Nephrol Dial Transplant 2003;18(11):2421–3. 53. Bienaime F, et al. D-Penicillamine-induced ANCA-associated crescentic glomerulonephritis in Wilson disease. Am J Kidney Dis 2007;50(5): 821–5. 54. Carmona-Rivera C, Purmalek MM, Moore E, et al. A role for muscarinic receptors in neutrophil extracellular trap formation and levamisoleinduced autoimmunity. JCI Insight 2017;2(3):e89780. 55. Heijl C, Harper L, Flossmann O, et al. Incidence of malignancy in patients treated for antineutrophil cytoplasm antibody-associated

vasculitis: follow-up data from European Vasculitis Study Group clinical trials. Ann Rheum Dis 2011;70(8):1415–21. 56. Guma M, et al. Frequency of antineutrophil cytoplasmic antibody in Graves’ disease patients treated with methimazole. J Clin Endocrinol Metab 2003;88(5):2141–6. 57. Lenert P, Icardi M, Dahmoush L. ANA (+) ANCA (+) systemic vasculitis associated with the use of minocycline: case-based review. Clin Rheumatol 2013;32(7):1099–106. 58. Zhang AH, et al. Inhibition of oxidation activity of myeloperoxidase (MPO) by propylthiouracil (PTU) and anti-MPO antibodies from patients with PTU-induced vasculitis. Clin Immunol 2007;122(2): 187–93. 59. Denissen NH, et al. Can sulfasalazine therapy induce or exacerbate Wegener’s granulomatosis? Scand J Rheumatol 2008;37(1):72–4. 60. Takahashi K, Oharaseki T, Yokouchi Y. Update on etio and immunopathogenesis of Kawasaki disease. Curr Opin Rheumatol 2014;26(1):31–6. 61. Lin YJ, et al. Genetic variants in PLCB4/PLCB1 as susceptibility loci for coronary artery aneurysm formation in Kawasaki disease in Han Chinese in Taiwan. Sci Rep 2015;5:14762. 62. Onouchi Y, et al. ITPKC and CASP3 polymorphisms and risks for IVIG unresponsiveness and coronary artery lesion formation in Kawasaki disease. Pharmacogenomics J 2013;13(1):52–9. 63. Yeung RS. Kawasaki disease: update on pathogenesis. Curr Opin Rheumatol 2010;22(5):551–60. 64. Gorelik M, et al. Plasma follistatin-like protein 1 is elevated in Kawasaki disease and may predict coronary artery aneurysm formation. J Pediatr 2012;161(1):116–19. 65. Lin IC, et al. Augmented TLR2 expression on monocytes in both human Kawasaki disease and a mouse model of coronary arteritis. PLoS ONE 2012;7(6):e38635. 66. Jia S, et al. The T helper type 17/regulatory T cell imbalance in patients with acute Kawasaki disease. Clin Exp Immunol 2010;162(1):131–7. 67. Rowley AH, et al. The transcriptional profile of coronary arteritis in Kawasaki disease. BMC Genomics 2015;16:1076. 68. Leung DY, et al. Prevalence of superantigen-secreting bacteria in patients with Kawasaki disease. J Pediatr 2002;140(6):742–6. 69. Schulte DJ, et al. Involvement of innate and adaptive immunity in a murine model of coronary arteritis mimicking Kawasaki disease. J Immunol 2009;183(8):5311–18. 70. Cid MC, et al. Immunohistochemical characterization of inflammatory cells and immunologic activation markers in muscle and nerve biopsy specimens from patients with systemic polyarteritis nodosa. Arthritis Rheum 1994;37(7):1055–61. 71. Yalcinkaya F, et al. Prevalence of the MEFV gene mutations in childhood polyarteritis nodosa. J Pediatr 2007;151(6):675–8. 72. Sansonno D, et al. Hepatitis C virus infection, cryoglobulinaemia, and beyond. Rheumatology 2007;46(4):572–8. 73. Varricchi G, et al. Interleukin-5 pathway inhibition in the treatment of eosinophilic respiratory disorders: evidence and unmet needs. Curr Opin Allergy Clin Immunol 2016;16(2):186–200. 74. Craven A, et al. ACR/EULAR-endorsed study to develop Diagnostic and Classification Criteria for Vasculitis (DCVAS). Clin Exp Nephrol 2013;17(5):619–21. 75. Luqmani R, Ponte C. ANCA associated vasculitides and polyarteritis nodosa. In: Hachulla E, Bijlsma JWJ, editors. EULAR Textbook on rheumatic diseases. BMJ Publishing Group Ltd; 2015. p. 717–53. 75a.  Stahelin L, et al. Cocaine-induced midline destruction lesions with positive ANCA test mimicking Wegener’s granulomatosis. Rev Bras Reumatol 2012;52(3):431–7. 76. Flossmann O, Berden A, de Groot K, et al. Long-term patient survival in ANCA-associated vasculitis. Ann Rheum Dis 2011;70(3):488–94. 77. Kemna MJ, Damoiseaux J, Austen J, et al. ANCA as a predictor of relapse: useful in patients with renal involvement but not in patients with nonrenal disease. J Am Soc Nephrol 2015;26(3):537–42. 78. Ntatsaki E, Carruthers D, Chakravarty K, et al. BSR and BHPR Standards, Guidelines and Audit Working Group BSR and BHPR guideline for the

CHAPTER 58  Small- and Medium-Vessel Primary Vasculitis management of adults with ANCA-associated vasculitis. Rheumatology (Oxford) 2014;53(12):2306–9. 79. Ponte C, et al. Optimisation of vasculitis disease assessments in clinical trials, clinical care and long-term databases. Clin Exp Rheumatol 2014;32(5 Suppl. 85):S-118-25. 80. Mukhtyar C, G L, Cid MC, et al. BSR and BHPR Standards, Guidelines and Audit Working Group BSR and BHPR guideline for the management. Ann Rheum Dis 2009;68:310–17. 81. Szpirt WM, Heaf JG, Petersen J. Plasma exchange for induction and cyclosporine A for maintenance of remission in Wegener’s granulomatosis-a clinical randomized controlled trial. Nephrol Dial Transplant 2011;26(1):206–13. 82. Mukhtyar C, Guillevin L, Cid MC, et al. EULAR recommendations for the management of primary small and medium vessel vasculitis. Ann Rheum Dis 2009;68(3):310–17. 83. Walsh M, Casian A, Flossmann O, Westman K, et al. Long-term follow-up of patients with severe ANCA-associated vasculitis comparing plasma exchange to intravenous methylprednisolone treatment is unclear. Kidney Int 2013;84(2):397–402. 84. Flossmann O, Berden A, de Groot K, Hagen C, et al. Long-term patient survival in ANCA-associated vasculitis. Ann Rheum Dis 2011;70(3):488–94. 85. Hoffman GS, Kerr G, Leavitt RY, et al. Wegener granulomatosis: an analysis of 158 patients. Ann Intern Med 1992;116:488–98. 86. Hruskova Z, Stel V, Jayne D, et al. Characteristics and Outcomes of Granulomatosis With Polyangiitis (Wegener) and Microscopic Polyangiitis Requiring Renal Replacement Therapy: Results From the European Renal Association-European Dialysis and Transplant Association Registry. Am J Kidney Dis 2015;66(4):613–20. 87. Suppiah R, Hadden R, Batra R, et al. Peripheral neuropathy in ANCA-associated vasculitis: outcomes from the European Vasculitis Study Group trials. Rheumatology (Oxford) 2011;50(12):2214–22.

807

88. Martinez Del Pero M, Walsh M, Luqmani R, et al. Long-term damage to the ENT system in Wegener’s granulomatosis. Eur Arch Otorhinolaryngol 2011;268(5):733–9. 89. Suppiah R, Judge A, Batra R, et al. A model to predict cardiovascular events in patients with newly diagnosed Wegener’s granulomatosis and microscopic polyangiitis. Arthritis Care Res (Hoboken) 2011b;63(4):588–96. 90. Shang W, Ning Y, Xu X, et al. Incidence of Cancer in ANCA-Associated Vasculitis: A Meta-Analysis of Observational Studies. PLoS ONE 2015;10(5):e0126016. 91. Basu N, McClean A, Harper L, et al. Markers for work disability in anti-neutrophil cytoplasmic antibody-associated vasculitis. Rheumatology (Oxford) 2014;53(5):953–6. 92. Walsh M, Mukhtyar C, Mahr A, et al. Health-related quality of life in patients with newly diagnosed antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Care Res (Hoboken) 2011;63(7):1055–61. 93. Suka M, Hayashi T, Kobayashi S, et al. Improvement in health-related quality of life in MPO-ANCA-associated vasculitis patients treated with cyclophosphamide plus prednisolone: an analysis of 18 months of follow-up data from the JMAAV study. Mod Rheumatol 2012;22(6):877–84. 94. Fujimoto SWR, Kobayashi S, Suzuki K, et al. Comparison of the epidemiology of anti-neutrophil cytoplasmic antibody-associated vasculitis between Japan and the U.K. Rheumatology (Oxford) 2011;50(10):1916–20. 95. Chen M, et al. Propylthiouracil-induced antineutrophil cytoplasmic antibody-associated vasculitis. Nat Rev Nephrol 2012;8(8):476–83. 96. Makino N, Nakamura Y, Yashiro M, et al. Descriptive epidemiology of Kawasaki disease in Japan, 2011-2012: from the results of the 22nd nationwide survey. J Epidemiol 2015;25(3):239–45.

CHAPTER 58  Small- and Medium-Vessel Primary Vasculitis

807.e1

MULTIPLE-CHOICE QUESTIONS 1. Which of the following types of vasculitis are usually associated with the presence of antineutrophil cytoplasmic antibody (ANCA)? A. Microscopic polyangiitis B. Polyarteritis nodosa C. Goodpasture syndrome D. Cryoglobulinemic vasculitis E. Granulomatosis with polyangiitis 2. Which of the following is the least common form of vasculitis in Europe? A. Kawasaki disease B. Polyarteritis nodosa C. Granulomatosis with polyangiitis D. Microscopic polyangiitis E. Leukocytoclastic cutaneous vasculitis

3. Which of the following treatments are not effective for use in patients with newly presenting generalized ANCA-associated vasculitis? A. Colchicine B. Glucocorticoid therapy alone C. Glucocorticoids and methotrexate D. Rituximab and glucocorticoid therapy E. Plasmapheresis with cyclophosphamide and glucocorticoid therapy

59  Large-Vessel Vasculitides Cornelia M. Weyand

Most tissues have compensatory mechanisms that allow them to sustain the damaging effects of acute and chronic inflammation, but medium and large arteries are organs without redundancy and limited regenerative capacity. Life is unsustainable unless the major arteries have uncompromised function. Accordingly, inflammatory damage to such arterial vessels leads to severe clinical consequences, immediately posing a threat for the loss of function of vital organs. When affected by inflammation, the aorta and its branches have two possible response patterns: (i) Inflammatory destruction of the vessel wall leads to dilatation, aneurysm formation, and rupture. Alternatively, the wall layers dissect. (ii) The inflammation initiates a maladaptive response to injury, resulting in luminal occlusion, disruption of blood supply, and ischemic damage of dependent organ structures. In contrast to other vasculopathies, especially those related to atherosclerosis, vasculitides of the larger blood vessels are almost always associated with a syndrome of intense systemic inflammation.1 Recent evidence has challenged the traditional view that systemic inflammation represents a spillover of inflammatory mediators from the vasculitic lesions. Instead, systemic activation of the innate immune system appears to be a pinnacle event that initiates the processes leading to vessel wall inflammation. The coincidence of malaise, fever, wasting, and myalgias, with signs of ischemia caused by vascular failure, remains a critical clue for the physician when diagnosing and treating large-vessel vasculitides (LVVs). The two major forms of LVVs are giant-cell arteritis (GCA) and Takayasu arteritis (TA). In addition, aortitis can infrequently be seen in other diseases, such as infections, connective tissue diseases, sarcoidosis, and inflammatory bowel disease (IBD), and occasionally is diagnosed as an idiopathic syndrome. Polymyalgia rheumatica (PMR) is a condition closely related to GCA; it occurs in the same patient population and often precedes or follows the clinical diagnosis of GCA.2 Patients with PMR do not have typical vascular lesions; consequently, PMR is not classified as a vasculitis. However, patients with PMR do have a systemic inflammatory syndrome indistinguishable from GCA, and about 10% of patients with PMR eventually progress to full-blown vasculitis. Similarities in the vascular lesions of GCA and TA have been interpreted as revealing parallels in immunopathogenesis. Whether similarities in histomorphology and tissue targeting reflect similarities in underlying molecular defects remains unclear. Whether the systemic inflammatory reactions accompanying GCA, TA, and PMR have disease-specific elements also remains unanswered, but this has opened the possibility of developing biomarkers that are urgently needed for clinical

monitoring. Excellent progress has been made in unraveling the pathogenesis of GCA, and this will inevitably lead to improvements in diagnosis, long-term management, and broadening of the therapeutic armamentarium.

EPIDEMIOLOGY GCA may be a very old disease, as suggested by historic evidence that more than 1000 years ago removal of the temporal artery was recommended by a physician in Baghdad. In 1932, Horton et al. at the Mayo Clinic in Minnesota recognized that GCA was an inflammatory vasculopathy when they found dense inflammation in the temporal arteries of two patients who were systemically ill and had severe headaches. The first reports of TA, or “pulseless disease,” in young women surfaced in Japan in the nineteenth century. The syndrome was named after Dr. Takayasu, an ophthalmologist, who, in 1905, described peculiar optic fundus abnormalities caused by ischemia-driven collateral formation. The strongest risk factor for GCA, TA, and PMR is age.3,4 GCA and PMR are essentially absent in individuals 50 of age.2 Iceland, Norway, Sweden, and Denmark are high-risk areas; higher incidence rates are also seen in Scandinavian immigrant populations in the United States. The risk is significantly lower in Hispanics and African Americans. Although TA can afflict all races, a predilection exists for individuals of Asian and Central and South American origins. Japan, Thailand, India, Turkey, and nations in Central and South Americas are considered high-incidence regions. TA is a rare disease with an annual incidence of 1–2 cases/million. The typical patient is a female in her 20s to 30s. In middle-aged men and women, it can be challenging to differentiate TA from rapidly progressing atherosclerotic disease, especially as both disease processes may coexist.6

809

810

Part SIX  Systemic Immune Diseases

Extravascular component intense acute phase response (IL-6, SAA, CRP, ESR, etc) myalgias malaise, anorexia, fever

Vascular component Granulomatous, transmural inflammation dendritic cells, CD4 T cells, macrophages, giant cells, etc

Vessel wall remodeling Aorta: dissection, wall damage, aneurysm, rupture Branches: intimal hyperplasia, luminal occlusion

Giant cell arteritis Age of the host GCA/PMR: >50 yrs TA: 45 minutes Hip pain or limited range of motion Absence of rheumatoid factor (RF) or anti-citrullinated protein antibody (ACPA) Absence of other joint involvement A score of ≥4 is categorized as polymyalgia rheumatica.

required required required 2 1 2 1

Reprinted from Dasgupta B, et al. Provisional classification criteria for polymyalgia rheumatica: A European League Against Rheumatism/American College of Rheumatology collaborative initiative. Arthritis Rheum 2012; 64: 943–954.

820

Part SIX  Systemic Immune Diseases

Histomorphological reports describe mononuclear cell infiltrates penetrating through all layers of the vessel wall (Fig. 59.9).1 Recent discussions have focused on the diagnostic relevance of isolated inflammatory cell clusters in the adventitia or perivascular lymphocytes limited to small blood vessels.36 These findings may not be sufficient to indicate arteritis. Multinucleated giant cells may or may not be found. They tend to lie along the internal elastic lamina, at the junction between the media and the intima. Media destruction is not unusual, but findings of fibrinoid necrosis should prompt a search for a different vasculitic entity. The vessel lumen is more or less compromised by hyperplastic intima formed from proliferating fibroblasts, smoothmuscle cells, and deposition of acid mucopolysaccharides. The histology of TA is similar to that of GCA, making it difficult to dissect both syndromes in tissue samples derived from the aorta or its primary branches. Lymphocytes and plasma cells accumulate around vasa vasorum and form transmural

infiltrates. Marked wall thickening with inflammatory tissue extending into perivascular structures is typical for TA (Fig. 59.10). Destruction of elastic membranes is often extensive and combined with patchy areas of media necrosis. Weakening of the vessel wall can lead to aneurysm formation. Inflammatory lesions may be arranged in a “skipped” pattern, with normal vessel wall segments alternating with stretches of intense destructive inflammation. Physicians may encounter morphological findings of granulomatous aortitis in patients undergoing aortic aneurysm repair without any prior diagnosis of vasculitis. Detailed workup of these patients is necessary to identify those with undiagnosed PMR, GCA, or TA. Rare causes of aortitis, including inflammatory bowel disease, sarcoidosis, syphilis, relapsing polychondritis, and connective tissue disease, should be ruled out. Isolated granulomatous aortitis is diagnosed as idiopathic aortitis. The pathogenesis and prognosis of this condition are essentially unknown.

Diagnostic Imaging TABLE 59.5 American College of

Rheumatology 1990 Criteria for the Classification of Takayasu Arteritis a

Disease onset at ≤40 years Claudication of an extremity Decreased brachial artery pulse >10 mm Hg difference in systolic blood pressure between arms Bruit over the subclavian arteries or the aorta Arteriographic evidence of narrowing or occlusion of the entire aorta, its primary branches, or large arteries in the proximal upper or lower extremities a For purposes of classification, a patient is classified as having Takayasu arteritis if more than three of the six criteria are fulfilled. Reprinted from Arend WP, Michel BA, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis. Arthritis Rheum 1990; 33: 1129–1134, with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. ©1990.

$

Modern imaging modalities have fundamentally changed the diagnostic approach to LVV.26 Indeed, diagnosing TA mostly depends on identifying vascular lesions in typical distribution by imaging.37 Conventional angiography still has its place in preoperative planning and can be combined with intravascular interventions. It provides ideal visualization of the vascular lumen not only for large but also for medium-sized arteries, such as the axillary and brachial arteries (see Fig. 59.5). Ultrasound (US)–based methods are extremely useful for screening carotid arteries, but they have also emerged as the method of choice for initial assessment of the distal subclavian arteries, vertebral arteries, renal arteries, and femoral arteries. US examination is also the optimal method for long-term monitoring of vessel bypasses in patients who have undergone revascularization surgical procedures. Magnetic resonance imaging (MRI), magnetic resonance angiography (MRA),

%

FIG 59.9  Histomorphology of Giant-Cell Arteritis (GCA). (A) Temporal artery cross-section with mononuclear infiltrates throughout all wall layers. The adventitia is infiltrated by round cells with cuffing of vasa vasorum by lymphocytes. The vessel lumen is occluded by intimal hyperplasia. (B) Higher magnification showing intense granulomatous inflammation with multinucleated giant cells in the proximal media and at the media–intima junction.

CHAPTER 59  Large-Vessel Vasculitides

821

been disappointing, cautioning one that edema-weighted MR should not be used as a sole means of measuring disease activity and therapeutic responsiveness.38 Both, CT angiography and MRA are now used routinely to monitor progression or regression of vascular involvement and have an important place in managing the chronic phase of GCA and TA.

THERAPEUTIC MANAGEMENT THERAPEUTIC PRINCIPLES Treatment of Large Vessel Vasculitides

$

%

FIG 59.10  Histopathology of Takayasu Arteritis (TA). (A) Fullthickness section of the aortic wall shows dense mononuclear infiltrates in the adventitia and media. The intima is thickened and wavy; hematoxylin and eosin (H&E). (B) Florid granulomatous inflammation along the media–intima junction with numerous giant cells; H&E.

and computed tomography (CT) are now widely used for evaluating the vascular tree. These methods provide excellent information on abnormalities of the vascular lumen and wall, with increasing resolution, now capturing abnormalities in the more peripheral arterial branches. CT imaging is fast, well tolerated by patients with claustrophobia, and allows excellent assessment of the aorta and its wall (see Fig. 59.7). However, it has the disadvantages of contrast loading and radiation exposure. With its inherent multiplanar imaging capabilities, magnetic resonance is used to examine neck vessels, the aorta, and its primary branches (see Figs. 59.6 and 59.8). Great hope was placed on its potential to measure wall edema and intramural vascularity, which would make MR useful for estimating disease burden and responses to therapy. However, a carefully conducted study comparing imaging results with laboratory parameters of inflammation to results from surgically harvested vessel biopsy specimens has

• To prevent vision loss, patients with giant-cell arteritis (GCA) require immediate treatment. Similarly, with the threat of catastrophic cerebral ischemia in Takayasu arteritis (TA), prompt initiation of therapy is imperative. • Corticosteroids are the immunosuppressive drug of choice for largevessel vasculitides (LVVs). Often, the drugs must be given over a period of several years but may be clinically effective at very low doses. • Clinical trials have failed to show convincing steroid-sparing effects for either methotrexate or tumor necrosis factor (TNF)-α blockade in GCA. • A phase 3 clinical trial has demonstrated steroid-sparing effect of treatment with tocilizumab. • Molecular studies of the inflammatory infiltrate in GCA have shown that early and untreated disease is characterized by two functional T-cell lineages; T helper (Th)1 and Th17 cells. Th17 cells are rapidly responsive to corticosteroids, whereas Th1 cells persist and promote chronic, smoldering vasculitis. • It is not known whether the smoldering activity persisting beyond the acute phase of the disease requires immunosuppressive therapy or whether the benefits of chronic immunosuppressive therapy outweigh the potential risks. • Clinical experience (not evidence-based therapeutic trials) suggests that a combination of methotrexate, mycophenolate mofetil, or TNF-α–blocking agents with corticosteroids may be beneficial in controlling disease in some patients with TA. • Close monitoring for diabetes, hypertension, and hyperlipidemia combined with bone-saving therapy should be part of the treatment regime in patients with LVVs on long-term corticosteroids.

With increasing knowledge of the disease process and refinement of diagnosis and long-term treatment, the prognosis for patients with LVVs has significantly improved. Life expectancy of patients with GCA is preserved. Follow-up studies of Japanese patients with TA have suggested good control of disease activity in about 75% of patients, with only 25% experiencing serious complications and cardiac manifestations dominating long-term outcome. Whether vasculitis predisposes patients to accelerated atherosclerotic disease, given the combination of chronic inflammation and injury to vessel wall structures, is still being discussed. It is not known whether progression of atherosclerosis and its complications require a different management approach or whether standard vasoprotective measures (treating hypertension and hyperlipidemia, smoking cessation, etc.) are sufficient.6 Pathogenic studies have pointed out that the traditional view of GCA as a self-limiting disease is incorrect.11 To the contrary, granulomatous vasculitis has shown surprising resistance to immunosuppression, with vessel wall infiltrates persisting for >12 months in almost 50% of patients despite appropriate immunosuppressive therapy. Based on examination of serial temporal artery biopsy specimens from patients before and after treatment, it is now clear that arteritis persists, albeit sustained

822

Part SIX  Systemic Immune Diseases

ON THE HORIZON • Medium and large arteries in humans sense danger signals through wall-embedded cells; changing the understanding of how the immune system interacts with the vascular system. • The multilineage nature of vasculitic T cells, which display differential therapeutic responsiveness, almost certainly will require more complex therapies, adapted to disease stage and immune status of the host. • The immune system changes profoundly with age. Age-appropriate management of each patient and avoidance of overtreatment of older adults are important. • Current therapies in large-vessel vasculitis induce partial remission. Appropriately designed studies are required to explore whether partial remission is sufficient and whether the risk/benefit ratio is maintained if complete remission is attempted.

by an immune network distinct from that in untreated patients.21 It is currently not known whether this persistent smoldering process needs to be treated and what the risk/benefit ratio is for the older patient population affected by GCA. Unchanged life expectancy in GCA suggests adequacy of current management. Whether intensification of immunosuppressive therapy or chronic maintenance therapy can prevent long-term complications, such as aortic aneurysm/dissection from GCA aortitis, and improve the overall prognosis is unknown. The ultimate decision depends on the cost/benefit analysis comparing the risk from smoldering disease with the risks imposed by long-standing immunosuppression. In that context, it is important to remember the profound impact of the immune aging process, which leaves the patient with an impaired immune system and amplifies the risk of immunosuppression.39 Induction Therapy In newly diagnosed patients with GCA, TA, and PMR, the immunosuppressants of choice are corticosteroids. Patients with GCA are started on a daily prednisone dose of 40–60 mg (about 1 mg/kg body weight). In patients with PMR, a daily dose of 20 mg prednisone is sufficient in almost all patients. The response is usually dramatic, with improvements within 24–48 hours. The promptness of clinical improvement is so exceptional that it has been suggested as a diagnostic criterion for PMR. However, the promptness of response may be limited to extravascular LVVs. Myalgias, fever, malaise, and headaches improve swiftly, in parallel with a fast reduction of acute-phase reactants (CRP, IL-6, ESR). Emerging data suggest that the vascular component is much more resistant to immunosuppression and may require entirely new therapeutic strategies. Once the condition is stabilized, steroid tapering is guided by close monitoring of the clinical presentation as well as laboratory markers of inflammation. In general, steroids should be reduced by 10–20% every 2 weeks. Monthly monitoring of ESR and CRP is mandatory to adjust therapy. Patients frequently return with signs or symptoms of recurrent disease as immunosuppression is lowered. Fortunately, disease exacerbations causing vision loss are infrequent. Disease flare-ups typically present with PMR symptoms or nonspecific manifestations of malaise and failure to thrive. In most patients, a transient small increase in the steroid dose reinstates disease control. Much effort has been invested in identifying steroid-sparing agents. In a small study, treatment with pulse corticosteroids appeared to have long-term beneficial effects, reducing the overall steroid requirement and the rate of disease flare-ups.40 After pulses of 1000 mg methylprednisolone were administered for 3

consecutive days, therapy was continued with oral prednisone, and daily doses were swiftly tapered. Compared with the control arm, patients who received three initial steroid pulses had lower likelihoods of disease flare-ups. Particularly, once they reached prednisone doses close to 10 mg/day, these patients could tolerate steroid withdrawal significantly better, and most were taking 5 mg/ day prednisone at 36 weeks.40 The benefit from initial pulse therapy continued over subsequent months, suggesting the potential benefit of intense immunosuppression during early disease. Several biological agents have been explored or are currently undergoing testing in clinical trials.41 Tumor necrosis factor-α (TNF-α) inhibitors may have a role in TA but had no steroidsparing effect in GCA.42 Preliminary study results suggest that targeting T-cell costimulation with abatacept may prevent disease relapses in GCA. Ustekinumab, a monoclonal antibody (mAb) targeting IL-12 and IL-23, was reported to have potential efficiency in refractory GCA in a small open-label study. The IL-6 receptor blocker tocilizumab has been explicitly effective in reducing acute phase reactants (CRP, ESR) and is being explored in GCA and TA and a phase 3 double-blind trial of tocilizumab given weekly or every other week demonstrated substantial steroid-sparing effect over a one-year period.12 Maintenance Therapy With a major shift in the pathogenic concept about LVVs, especially the realization that the disease process has two, partly independent components (extravascular, vascular) and that vessel wall infiltrates persist chronically, the therapeutic needs for maintenance therapy have become the dominant issue for the treating physician. Patients with PMR are often managed successfully with low-dose corticosteroids (prednisone 5 mg daily) and typically are highly responsive to transient and very small dose increases (1–2 mg prednisone/day). Long-term management of patients with GCA and TA relies on low-dose corticosteroids as well unless there is objective evidence for progressive vascular wall inflammation. Unfortunately no reliable biomarkers can separate the extravascular and vascular disease components, and no evidence that suppressing acute phase response in the periphery will ultimately restrict transmural vasculitis has been presented. Methotrexate is considered to have mild-to-moderate steroidsparing potential in GCA and PMR43 but is more frequently used in TA.44 When given to human artery–severe combined immunodeficiency (SCID) chimeras, acetylsalicylic acid (aspirin) has marked antiinflammatory activities, with suppression of IFN-γ in vascular lesions. Clinical trials are needed to test whether this immunosuppressive action can translate into corticosteroid sparing. Because arteries are the primary targets of LVVs, the use of aspirin as an antiplatelet agent should be routinely recommended. There is no evidence that immunosuppressants, such as azathioprine and cyclophosphamide, lower steroid needs, prevent vascular complications, or shorten the duration of steroid use. Whether any of the biological agents described above has a place to effectively suppress vessel wall inflammation and change the course of chronic disease is currently unknown. An integral part of chronic immunosuppression with prednisone is regular monitoring for diabetes and hypertension. Patients should be encouraged to increase physical activity, as steroid-induced myopathy occurs frequently. A major issue of chronic steroid treatment, particularly in older individuals, is the risk of excessive bone loss, possibly resulting from increased bone resorption and impaired bone formation. Several effective and safe therapies for osteopenia/osteoporosis are available.

CHAPTER 59  Large-Vessel Vasculitides Calcium and vitamin D supplementation should be part of the therapeutic regimen. In many, but not all, patients, immunosuppressive treatment can be discontinued 18–24 months after diagnosis. Markers of systemic inflammation may remain elevated, and continuous monitoring for aortic involvement and recurrence of cranial arteritis is recommended. Most patients with PMR are sufficiently treated with an initial dose of 20 mg of prednisone per day. In some patients, 10 mg of prednisone can induce and sustain a clinical response. Steroids should be titrated to minimally needed doses to avoid side effects; tapering usually needs to be slow, over many months. In TA, long-term management should be tailored to individual patient conditions.44 It has been argued that patients should be maintained on a low dose of corticosteroids, such as 5–7 mg prednisone daily, even after successful control of active disease. Given the age at disease onset in TA, preventive measures to counteract accelerated atherosclerosis and optimize blood pressure control are important aspects of management. It has been suggested that up to 50% of patients with TA may require a second immunosuppressive agent.44 Steroid-sparing effects of methotrexate have been reported for some patients. Similarly, mycophenolate mofetil may have clinical efficiency, although only published data on a small patient cohort are available. Empirically, azathioprine may have a place in maintenance therapy of patients with TA. Finally, there may be a place for agents blocking TNF-α in patients with persistent disease activity. Results from well-designed placebo-controlled treatment trials testing the efficiency of such immunosuppressive drugs are awaited. Detecting and treating hypertension is an essential component of caring for patients with TA. Untreated hypertension leads to acceleration of atherosclerosis and cardiac insufficiency. In patients with upper-extremity involvement, obtaining accurate blood pressure measurements is a challenge and requires education of the patient and caregivers. Revascularization Procedures Besides pharmacological therapy, revascularization procedures— including both surgical and endovascular interventions—have vastly broadened therapeutic options in patients with TA and large-vessel GCA.45 To minimize the risk of complications, such as rapid reocclusion, an effort should be made to suppress vascular wall inflammation, ideally before subjecting the patients to revascularization treatment. Conventional bypass grafts are still considered the method of choice. Percutaneous transluminal angioplasty can be useful in managing renal artery stenosis or other short-segment lesions. Bypass surgery is needed in patients with cerebrovascular ischemia in whom catastrophic strokes may be prevented by bypassing critical stenosis of cervical vessels with grafts originating from the aortic arch. Reestablishing flow in the upper- and lower-extremity arteries can be complicated by multiple and long-segment stenosis, and arterial reconstructions with prosthetic graft materials or veins may be the only alternative to obtain long-term patency. Placing of conventional stents can be complicated by eliciting rapid restenosis, and it is not known whether outcomes can be improved by drug-eluting stents. Occlusive disease of the coronary arteries usually represents a challenging clinical scenario, and most physicians opt for conventional bypass surgery. Depending on symptoms, patients with aortic regurgitation may require repair of the weakened aortic wall.

823

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Weyand CM, Goronzy JJ. Medium- and large-vessel vasculitis. N Engl J Med 2003;349(2):160–9. 2. Salvarani C, Cantini F, Boiardi L, et al. Polymyalgia rheumatica and giant-cell arteritis. N Engl J Med 2002;347(4):261–71. 3. Nordborg E, Nordborg C. Giant cell arteritis: epidemiological clues to its pathogenesis and an update on its treatment. Rheumatology (Oxford) 2003;42(3):413–21. 4. Vanoli M, Bacchiani G, Origg L, et al. Takayasu’s arteritis: a changing disease. J Nephrol 2001;14(6):497–505. 5. Isobe M. Takayasu arteritis revisited: current diagnosis and treatment. Int J Cardiol 2013;168(1):3–10. 6. Numano F, Kishi Y, Tanaka A, et al. Inflammation and atherosclerosis. Atherosclerotic lesions in Takayasu arteritis. Ann N Y Acad Sci 2000;902:65–76. 7. Wagner AD, Goronzy JJ, Weyand CM. Functional profile of tissueinfiltrating and circulating CD68+ cells in giant cell arteritis. Evidence for two components of the disease. J Clin Invest 1994;94(3):1134–40. 8. Baldini M, Maugeri N, Ramirez GA, et al. Selective up-regulation of the soluble pattern-recognition receptor pentraxin 3 and of vascular endothelial growth factor in giant cell arteritis: relevance for recent optic nerve ischemia. Arthritis Rheum 2012;64(3):854–65. 9. Nadkarni S, Dalli J, Hollywood J, et al. Investigational analysis reveals a potential role for neutrophils in giant-cell arteritis disease progression. Circ Res 2014;114(2):242–8. 10. Roche NE, Fulbright JW, Wagner AD, et al. Correlation of interleukin-6 production and disease activity in polymyalgia rheumatica and giant cell arteritis. Arthritis Rheum 1993;36(9):1286–94. 11. Weyand CM, Fulbright JW, Hunder GG, et al. Treatment of giant cell arteritis: interleukin-6 as a biologic marker of disease activity. Arthritis Rheum 2000;43(5):1041–8. 12. Stone JH, Tuckwell K, Dimonaco S, et al. Trial of tocilizumab in giant-cell arteritis. N Engl J Med 2017;377:317–28. 13. O’Neill L, Rooney P, Molloy D, et al. Regulation of inflammation and angiogenesis in giant cell arteritis by acute-phase serum amyloid A. Arthritis Rheumatol 2015;67(9):2447–56. 14. Weyand CM, Goronzy JJ. Immune mechanisms in medium and large-vessel vasculitis. Nat Rev Rheumatol 2013;9(12):731–40. 15. Pryshchep O, Ma-Krupa W, Younge BR, et al. Vessel-specific Toll-like receptor profiles in human medium and large arteries. Circulation 2008;118(12):1276–84. 16. Ma-Krupa W, Jeon MS, Spoerl S, et al. Activation of arterial wall dendritic cells and breakdown of self-tolerance in giant cell arteritis. J Exp Med 2004;199(2):173–83. 17. Krupa WM, Dewan M, Jeon MS, et al. Trapping of misdirected dendritic cells in the granulomatous lesions of giant cell arteritis. Am J Pathol 2002;161(5):1815–23. 18. Weyand CM, Liao YJ, Goronzy JJ. The immunopathology of giant cell arteritis: diagnostic and therapeutic implications. J Neuroophthalmol 2012;32(3):259–65. 19. Weyand CM, Wagner AD, Bjornsson J, et al. Correlation of the topographical arrangement and the functional pattern of tissue-infiltrating macrophages in giant cell arteritis. J Clin Invest 1996;98(7):1642–9. 20. Weyand CM, Schonberger J, Oppitz U, et al. Distinct vascular lesions in giant cell arteritis share identical T cell clonotypes. J Exp Med 1994;179(3):951–60. 21. Deng J, Younge BR, Olshen RA, et al. Th17 and Th1 T-cell responses in giant cell arteritis. Circulation 2010;121(7):906–15. 22. Watanabe R, Hosgur E, Zhang H, et al. Pro-inflammatory and anti-inflammatory T cells in giant cell arteritis. Joint Bone Spine 2016;pii: S1297-319X(16)30124-5. doi:10.1016/j.jbspin.2016.07.005. [Epub ahead of print].

824

Part SIX  Systemic Immune Diseases

23. Seko Y, Minota S, Kawasaki A, et al. Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu’s arteritis. J Clin Invest 1994;93(2):750–8. 24. Wen Z, Shimojima Y, Shirai T, et al. NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs. J Clin Invest 2016;126(5):1953–67. 25. Zhang H, Watanabe R, Berry GJ, et al. Immunoinhibitory checkpoint deficiency in medium and large vessel vasculitis. Proc Natl Acad Sci USA 2017;114(6):E970–9. 26. Weyand CM, Goronzy JJ. Giant-cell arteritis and polymyalgia rheumatica. Ann Intern Med 2003;139(6):505–15. 27. Brack A, Rittner HL, Younge BR, et al. Glucocorticoid-mediated repression of cytokine gene transcription in human arteritis-SCID chimeras. J Clin Invest 1997;99(12):2842–50. 28. Salvarani C, Cantini F, Olivieri I, et al. Proximal bursitis in active polymyalgia rheumatica. Ann Intern Med 1997;127(1):27–31. 29. Caspary L. Inflammatory diseases of the aorta. Vasa. 2016;45(1):17–29. 30. Kobayashi Y, Numano F. 3. Takayasu arteritis. Intern Med 2002;41(1):44–6. 31. Hunder GG, Bloch DA, Michel BA, et al. The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis. Arthritis Rheum 1990;33(8):1122–8. 32. Dasgupta B, Cimmino MA, Kremers HM, et al. 2012 Provisional classification criteria for polymyalgia rheumatica: a European League Against Rheumatism/American College of Rheumatology collaborative initiative. Arthritis Rheum 2012;64(4):943–54. 33. Arend WP, Michel BA, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of Takayasu arteritis. Arthritis Rheum 1990;33(8):1129–34. 34. Kerr GS, Hallahan CW, Giordano J, et al. Takayasu arteritis. Ann Intern Med 1994;120(11):919–29.

35. Achkar AA, Lie JT, Hunder GG, et al. How does previous corticosteroid treatment affect the biopsy findings in giant cell (temporal) arteritis? Ann Intern Med 1994;120(12):987–92. 36. Jia L, Couce M, Barnholtz-Sloan JS, et al. Is all inflammation within temporal artery biopsies temporal arteritis? Hum Pathol 2016;57:17–21. 37. Steeds RP, Mohiaddin R. Takayasu arteritis: role of cardiovascular magnetic imaging. Int J Cardiol 2006;109(1):1–6. 38. Hartlage GR, Palios J, Barron BJ, et al. Multimodality imaging of aortitis. JACC Cardiovasc Imaging. 2014;7(6):605–19. 39. Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to vaccines. Nat Immunol 2013;14(5):428–36. 40. Mazlumzadeh M, Hunder GG, Easley KA, et al. Treatment of giant cell arteritis using induction therapy with high-dose glucocorticoids: a double-blind, placebo-controlled, randomized prospective clinical trial. Arthritis Rheum 2006;54(10):3310–18. 41. Koster MJ, Matteson EL, Warrington KJ. Recent advances in the clinical management of giant cell arteritis and Takayasu arteritis. Curr Opin Rheumatol 2016;28(3):211–17. 42. Hoffman GS, Cid MC, Rendt-Zagar KE, et al. Infliximab for maintenance of glucocorticosteroid-induced remission of giant cell arteritis: a randomized trial. Ann Intern Med 2007;146(9):621–30. 43. Mahr AD, Jover JA, Spiera RF, et al. Adjunctive methotrexate for treatment of giant cell arteritis: an individual patient data meta-analysis. Arthritis Rheum 2007;56(8):2789–97. 44. Liang P, Hoffman GS. Advances in the medical and surgical treatment of Takayasu arteritis. Curr Opin Rheumatol 2005;17(1):16–24. 45. Mason JC. Takayasu arteritis: surgical interventions. Curr Opin Rheumatol 2015;27(1):45–52.

CHAPTER 59  Large-Vessel Vasculitides

824.e1

MULTIPLE CHOICE QUESTIONS 1. A 27-year old female presents with a 6-weeks history of intermittent fever, arthralgias and weight loss. She was admitted after she fainted in the emergency room. On physical examination she has no palpable peripheral pulse in her left upper extremity, and Takayasu arteritis (TA) is added as a differential diagnosis. Which of the following statements is true: A. She should be scheduled for temporal artery biopsy, to rule out giant cell arteritis. B. Laboratory tests, e.g. sedimentation rate will be of no use, as they can be non-specifically abnormal. C. A pregnancy test should be performed. D. A detailed physical examination will not be helpful. E. If a diagnosis of TA is made, it will be a self-limited disease. 2. Giant cell arteritis and Takayasu arteritis have a stringent tissue tropism. Which of the following vascular beds can be involved? Identify all correct answers: A. Aortic arch B. Carotid artery

C. Subclavian artery D. Renal capillaries E. Arteries in the skin 3. Extravascular GCA may result in the following laboratory abnormalities. Identify the incorrect answer: A. Elevated C-reactive protein B. Autoantibodies to nuclear antigens C. Thrombocytosis D. Elevated serum amyloid A E. Elevated alkaline phosphatase

60  Systemic Autoinflammatory Syndromes Catharina M. Mulders-Manders, Jeroen C.H. van der Hilst, Jos W.M. van der Meer, Anna Simon

Autoinflammatory diseases, which are also known as periodic fever syndromes, encompass a group of rare disorders characterized by recurrent or persistent inflammation. Autoinflammation is a term that has been used since the late 1990s to illustrate the difference between autoimmune disorders and diseases characterized by exuberant inflammation. Typically, autoinflammatory diseases do not show features of excess adaptive immune system activation, and autoantigens or auto-antigen specific T-cells are not present in these diseases. It is now recognized that autoinflammation and autoimmunity form two ends of a spectrum of inappropriate immune system activation and share several common features. Located at the autoinflammatory end of this spectrum are the classic monogenic autoinflammatory diseases: familial Mediterranean fever (FMF), cryopyrin-associated periodic syndrome (CAPS), mevalonate kinase deficiency (MKD; also known as hyperimmunoglobulin D and periodic fever syndrome [HIDS]), and tumor necrosis factor (TNF) receptor–associated periodic syndrome (TRAPS). The number of autoinflammatory diseases is increasing rapidly. New monogenic autoinflammatory diseases have been identified in the last decades. For many of the recently described autoinflammatory diseases, no genetic cause has been found yet. It has also become clear that autoinflammation is at least partially involved in the pathogenesis of other, more common diseases, such as gout, Crohn disease, and ulcerative colitis. As it is impossible to discuss all autoinflammatory diseases in detail here, the classic monogenic diseases FMF, CAPS, TRAPS, and MKD have been selected as the main focus of this chapter. Their pathophysiological mechanisms are understood to a much higher degree than in many newer autoinflammatory diseases, and their clinical presentations have been described precisely. In addition, two other autoinflammatory diseases are discussed, one with relatively high prevalence and the other because of its interesting pathophysiological mechanism: (i) periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome and (ii) Schnitzler syndrome. The cornerstone of diagnosing an autoinflammatory disease is the clinical assessment of the patient. This includes a detailed medical and family history and direct observation of an inflammatory episode. The first step in the diagnostic process is to exclude other more common causes of recurrent inflammation, including infections, malignancy and paraneoplastic phenomena, and autoimmune disease.1 A first differential diagnosis can be made on the basis of age of onset, associated signs and symptoms, duration of inflammation, family history, and ethnic background, (Table 60.1), and this can guide targeted diagnostic testing.

KEY CONCEPTS Autoinflammation Versus Autoimmunity • Common features: • Inflammation due to excessive immune activation • Phenotypes characterized by exacerbations and remissions • Distinctive features: • Autoinflammation: dysregulation of innate immunity, no high-titer autoantibodies or autoantigen-specific T cells • Autoimmunity: dysregulation of adaptive immunity, defect in lymphocyte function, autoantibodies may be present. • Autoinflammation and autoimmunity form two ends of a continuous spectrum of excessive immune system activation. • Many diseases show overlapping features between autoinflammation and autoimmunity.

EPIDEMIOLOGY It is important to realize that the incidence of specific diseases varies widely among ethnic groups. With more than 100 000 patients worldwide, FMF is the most prevalent monogenic autoinflammatory disease. It is most common in individuals originating from around the Mediterranean basin, such as Turks, Jews (primarily non-Ashkenazi), Arabs, and in Armenians. In these selected populations, the carrier frequency of mutations in the MEFV gene can be as high as one in three individuals.1 This may indicate a survival benefit for carriers of heterozygous mutations, possibly through protection against certain unknown infectious agents. The first patients with MKD were described in 1984 in The Netherlands2 (then referred to as HIDS). Over 200 patients have now been identified, most of Western European and Caucasian ancestry. This could be partly explained by increased awareness for this disease among physicians in that part of the world. An alternative explanation is a common founder effect with clustering of carriers, illustrated by a carrier rate of 1 : 153 for the most common mutation in the mevalonate kinase gene (MVK) (V377I) in Dutch newborns.3,4 TRAPS is seen in patients from around the world, although most patients originate from northwestern Europe. A few dozen families and over 200 sporadic cases have been reported. The exact prevalence of the CAPS is unknown, but over 130 cases have been recognized. Disease awareness and recognition among clinicians have improved because of the availability of effective treatment for this disease. PFAPA syndrome was first reported at the end of the 1980s.5 It is difficult to estimate the incidence of PFAPA, as the level of

825

826

Part SIX  Systemic Immune Diseases

TABLE 60.1  Hereditary Autoinflammatory Syndromes FMF

CAPS

TRAPS

MKD

Mode of inheritance Age of onset (years)

Autosomal recessive 50,000/ mm3); proteinuria and renal insufficiency occur in patients with thrombotic microangiopathy. Erythrocyte sedimentation rate and hemoglobin and leukocyte count are usually normal in patients with uncomplicated APS, except during acute thrombosis. Complement levels are usually normal or only modestly low.

Imaging Studies MRI studies show vascular occlusion and infarction consistent with clinical symptoms, without special characteristics, except that multiple otherwise unexplained cerebral infarctions in a young person suggest the syndrome. Multiple small hyperintense white matter lesions are common and do not unequivocally imply brain infarction (Fig. 61.2). Occlusions usually occur in vessels below the resolution limits of angiography; hence angiography or magnetic resonance angiography is not indicated unless clinical findings suggest medium- or large-vessel disease. Echocardiography or cardiac MRI may show severe Libman-Sacks endocarditis and intracardiac thrombi.

TREATMENT Treatment recommendations for persistently aPL-positive patients are determined by the specific clinical indication (Table 61.6).

Asymptomatic Individuals The ideal strategy for primary thrombosis prevention in asymptomatic, persistently aPL-positive individuals requires a riskstratified approach based on aPL profile, age, systemic autoimmune diseases, traditional cardiovascular disease, or risk factors for venous thrombosis. Elimination of reversible risk factors for thrombosis (smoking, oral contraceptives) and prophylaxis during high-risk periods (surgical interventions or prolonged immobilization) is crucial for primary thrombosis prophylaxis in persistently aPL-positive individuals. The effectiveness of aspirin is not supported by the literature; in a randomized, double-blind, placebo-controlled trial, low-dose aspirin (81 mg) appeared to be no better than placebo in preventing first thrombotic episodes in persistently asymptomatic aPL-positive patients.15 The generalpopulation cardiovascular disease (CVD) risk prediction tools and prevention guidelines formulated based on risk–benefit calculations should play the primary role in decision making for aspirin therapy. Estrogen and estrogen-containing oral contraceptives are considered unsafe for asymptomatic women serendipitously known to bear high-titer antibody. There is no reliable information regarding the safety of progestin-only contraception, “morning after” contraception, or raloxifene, bromocriptine, or leuprolide in APS patients. However, progestin-only contraception

TABLE 61.6  Treatment Recommendations

in Persistently Antiphospholipid Antibody–Positive Patients

Pathological Studies CLINICAL PEARLS • The clinical manifestations of antiphospholipid antibodies (aPL) represent a spectrum (from asymptomatic to catastrophic antiphospholipid syndrome [APS]); thus patients should not be evaluated and managed as having a single disease manifestation. • Stroke and transient ischemic attack are the most common presentation of arterial thrombosis; deep vein thrombosis, often accompanied by pulmonary embolism, is the most common venous manifestation of APS. • Pregnancy losses in patients with aPL typically occur after 10 weeks’ gestation (fetal loss), but early losses also occur (preembryonic or embryonic losses). • Catastrophic APS is a rare, abrupt, life-threatening complication of APS, which consists of multiple thromboses of medium and small arteries occurring over a period of days. • APS diagnosis should be made in the presence of characteristic clinical manifestations and persistently (at least 12 weeks apart) positive aPL.

Skin, renal, and other tissues show noninflammatory occlusion of all caliber arteries and veins, acute and chronic endothelial injury and its sequelae, and recanalization in late lesions. The finding of inflammatory necrotizing vasculitis suggests concomitant SLE or other connective tissue disease. There are no other diagnostic immunofluorescence or electron microscopical findings.

839

Clinical Circumstance

Recommendation

Asymptomatic Venous or arterial thrombosis Recurrent thrombosis

No treatmenta Warfarin international normalized ratio (INR) 2–3.0 indefinitely Warfarin INR 3–3.5 indefinitely ± low-dose aspirin No treatmenta No treatmenta

First pregnancy Single pregnancy loss, 50 000/mm3 Thrombocytopenia, ≤50 000/mm3 a

Prophylactic-doseb heparin with low-dose aspirin throughout the pregnancy, discontinue heparin 6–12 weeks postpartum Therapeutic-dose heparinc with low-dose aspirin throughout pregnancy, warfarin postpartum Anticoagulation + corticosteroids + intravenous immunoglobulin or plasma exchange No treatment No known effective treatment; full anticoagulation if emboli or intracardiac thrombi are demonstrated No treatment Prednisone and/or intravenous immunoglobulin

Aspirin 81 mg/day may be given. Prophylactic dose such as enoxaparin 30–40 mg subcutaneously (SQ) once daily. Therapeutic dose such as enoxaparin 1 mg/kg SQ twice daily or 1.5 mg/kg SQ once daily. b c

840

Part six  Systemic Immune Diseases

is theoretically safer than estrogen-based contraception. A small retrospective review of women undergoing artificial reproductive technology procedures demonstrated no thrombotic events.

Venous and Arterial Thromboembolism Anticoagulation with unfractionated heparin or low-molecularweight heparin (LMWH) followed by warfarin is the treatment for APS patients with vascular events. Heparin inhibits complement, a fact that makes it theoretically a preferred but impractical agent in most patients. For patients with a positive LA test that prolongs the aPTT, monitoring heparin can be accomplished by measuring antifactor Xa levels. Two prospective controlled studies concluded that recurrence of thromboses in APS patients can be prevented with warfarin to an international normalized ratio (INR) of 2.0–3.0.16,17 Although these studies provide strong evidence for moderate-intensity anticoagulation after an aPL-related venous event, the intensity of the anticoagulation is still debatable for APS patients with arterial events, since such patients constituted a minority in these studies. Although some APS patients may require high-intensity anticoagulation, in the absence of risk-stratified studies the definition of high risk is based currently on clinical judgment. Most aPL-positive patients receive warfarin after ischemic strokes; however, the Antiphospholipid Antibodies and Stroke Study (APASS) concluded that for selected aPL-positive patients who have neither atrial fibrillation nor high-grade arterial stenosis, aspirin (325 mg/day) and warfarin (target INR 1.4–2.8) are equivalent in efficacy and in association with major bleeding complications.18 The generalizability of these results is limited, as the study group had an average age of 60 years (higher than the average for APS populations), the aPL determination was performed only once at study entry, and the cutoff for assigning a patient to the positive aCL group was very low. However, based on APASS results, aspirin is an option for older patients with a single low positive aCL test who present with stroke. Recently, four direct oral anticoagulants (DOACs) that target thrombin (dabigatran) or factor Xa (rivaroxaban, apixaban, and edoxaban) have been approved for the treatment of patients with venous thromboembolism. Use in patients with APS is limited, and cases of therapeutic failure have been reported. A prospective study investigated the use of rivaroxaban compared with standard-intensity warfarin in patients with APS, using the change in endogenous thrombin potential (ETP), a laboratory parameter, as the primary outcome. Although the ETP did not reach the noninferiority threshold, there was no increase in thrombotic events in patients taking rivaroxaban.19 The study was not powered for clinical outcomes, however, and other studies are in progress. Venous thrombosis in aPL-positive patients typically has a high recurrence rate if anticoagulation is discontinued, and lifelong anticoagulation is usually recommended. A recent systematic review of the literature revealed that the available evidence in support of an association between the presence of aPL and risk of recurrence is of low quality, however, suggesting that indefinite anticoagulation may not be needed by all patients.20 For example, it is unknown whether patients whose event was triggered by an acquired, reversible risk factor for thrombosis can discontinue anticoagulation or switch to aspirin when the trigger factor is eliminated. Normalization of the LA or aCL tests is not an indication to discontinue anticoagulation. For well-anticoagulated patients who continue to have thromboses, antiplatelet drugs, hydroxychloroquine, statins, intravenous

immunoglobulin (IVIG), and plasmapheresis have theoretical bases for efficacy and have all been used. There are no systematic studies of treatment for CAPS. Detailed reviews conclude that the most effective treatment combines full-dose anticoagulation, high-dose corticosteroids, plasma exchange, and IVIG.

Pregnancy Morbidity Pregnancy is a prothrombotic state; management strategies in persistently aPL-positive patients should focus on prevention of both pregnancy morbidity and maternal thrombotic complications.9 In pregnancy, heparin and low-dose aspirin combination increases fetal survival rate from 50% to 80% among women who have had a fetal loss and tests positive for aPL. If patients fail this regimen, the next step is to add IVIG, an approach not supported by controlled studies. Most experts in the field use LMWH (e.g., enoxaparin) due to lower risk of thrombocytopenia and osteoporosis—prophylactic doses for women who have had only pregnancy morbidity, or full anticoagulant doses for women who have had prior thromboses (Table 61.6). Treatment begins after confirmation of pregnancy, continues until 48 hours before anticipated delivery (to allow epidural anesthesia), and resumes for 8–12 weeks postpartum (if no prior thromboembolism), or else the patient is transitioned to warfarin for continued therapy. No studies unequivocally justify treatment of women with aPL during a first pregnancy, women with only very early losses, or women whose aPL titers are low or transient. Nonetheless, it is common to offer such patients low-dose aspirin.

Other Clinical Manifestations of APS There is no consensus for the treatment of patients with noncriteria and/or nonthrombotic manifestations of aPL. Corticosteroids and/or IVIG are the first-line treatments for platelet counts less than 50 000/mm3. An open-label phase IIa pilot study of aPL found that rituximab may be effective in controlling some but not all noncriteria manifestations of APS, although aPL profiles did not change with treatment.21

Perioperative Management Serious perioperative complications may occur despite prophylaxis. Patients with APS are at additional risk for thrombosis when they undergo surgery. Thus perioperative strategies should be clearly identified before any surgical procedure or before pharmacological and physical antithrombosis interventions are vigorously employed; in addition, periods without anticoagulation must be kept to an absolute minimum, and any deviation from a normal course must be considered a potential disease-related event.22

Additional Therapeutic Considerations There is experimental and clinical evidence in lupus patients that hydroxychloroquine (HCQ) might decrease the incidence of thrombosis, and recent in vitro studies have demonstrated that HCQ might protect endothelial cells and syncytialized trophoblast cell lines from the disruptive effect of antiphospholipid antibodies.23 In patients with systemic autoimmune diseases (particularly lupus), HCQ is commonly employed for disease control and should be considered independent of patients’ aPL status. However, further controlled studies are needed to determine the effectiveness of HCQ for primary prophylaxis in aPL-positive patients. Although statins have been used in primary and secondary cardiovascular disease prevention, no data exist for their use in

CHAPTER 61  Antiphospholipid Syndrome thrombosis prevention in aPL-positive patients. Experimental evidence in APS mouse models and the clinical trials demonstrating rosuvastatin’s protective effect against a first major cardiovascular event and venous thrombosis in a general population without hyperlipidemia but with elevated high-sensitivity C-reactive protein levels24 justifies clinical studies of statins in nonpregnant aPL-positive patients. Antiplatelet agents other than aspirin or long-term LMWH treatment, empirically used, have not been formally tested in clinical trials. Ongoing prospective, randomized clinical trials investigating the role of the direct oral anticoagulants in the treatment of thromboembolism in patients with APS are eagerly awaited, and other strategies, including complement inhibitors and novel antithrombotic therapies, may be candidates for future clinical trials.

CONCLUSIONS AND TRANSLATIONAL RESEARCH ON THE HORIZON Translational Research in Antiphospholipid Syndrome • A better understanding of the cellular mechanisms of antiphospholipid antibody (aPL) mediated clinical events that will help us design more specifically targeted treatments. • Identification of patients who are at risk for future aPL-related events. • Controlled studies of theoretically useful medications such as hydroxychloroquine, statins, complement inhibitors, or anti-B-cell therapies.

APS is a systemic autoimmune disease consisting of thromboses, pregnancy losses, and persistent high-titer aPL. Inflammation and complement activation are established mechanisms of aPLrelated manifestations in murine models; however, definitive studies in humans do not exist. The disease is too variable clinically, and its mechanisms too diverse, to expect that a single mechanism defined in a single model will apply to all aspects of this disease. Given that the mechanisms of aPL-induced thrombosis are not well understood and that thrombosis is multifactorial and that controversies exist about the strength of association between aPL and clinical events, drug development specific for aPL-positive patients has been challenging. Anticoagulation is the primary treatment today, but a future therapeutic approach will likely include immunomodulatory agents. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. 2. Giannakopoulos B, Krilis SA. The pathogenesis of the antiphospholipid syndrome. N Engl J Med 2013;368:1033–44. 3. The Antiphospholipid Antibodies in Stroke Study (APASS) Group. Anticardiolipin antibodies are an independent risk factor for first ischemic stroke. Neurology 1993;43:2069–73. 4. Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24–9.

841

5. Erkan D, Lockshin MD. What is antiphospholipid syndrome? Curr Rheumatol Rep 2004;6:451–7. 6. Betapudi V, Lominadze G, Hsi L, et al. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway. Blood 2013;122:3808–17. 7. Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J Clin Invest 2003;112:1644–54. 8. Roman MJ, Shanker BA, Davis A, et al. Prevalence and correlates of accelerated atherosclerosis in systemic lupus erythematosus. N Engl J Med 2003;349:2399–406. 9. Ruiz-Irastorza G, Crowther M, Branch W, et al. Antiphospholipid syndrome. Lancet 2010;376:1498–509. 10. Erkan D, Espinosa G, Cervera R. Catastrophic antiphospholipid syndrome: updated diagnostic algorithms. Autoimmun Rev 2010;10:74–9. 11. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/ Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2009;7:1737–40. 12. Galli M, Luciani D, Bertolini G, et al. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature. Blood 2003;101:1827–32. 13. Pengo V, Ruffatti A, Legnani C, et al. Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile: a multicenter prospective study. Blood 2011;118:4714–18. 14. Erkan D, Derksen WJ, Kaplan V, et al. Real world experience with antiphospholipid antibody tests: how stable are results over time? Ann Rheum Dis 2005;64:1321–5. 15. Erkan D, Harrison MJ, Levy R, et al. Aspirin for primary thrombosis prevention in the antiphospholipid syndrome: a randomized, double-blind, placebo-controlled trial in asymptomatic antiphospholipid antibody-positive individuals. Arthritis Rheum 2007;56:2382–91. 16. Crowther MA, Ginsberg JS, Julian J, et al. A comparison of two intensities of warfarin for the prevention of recurrent thrombosis in patients with the antiphospholipid antibody syndrome. N Engl J Med 2003;349:1133–8. 17. Finazzi G, Marchioli R, Brancaccio V, et al. A randomized clinical trial of high-intensity warfarin vs. conventional antithrombotic therapy for the prevention of recurrent thrombosis in patients with the antiphospholipid syndrome (WAPS). J Thromb Haemost 2005;3:848–53. 18. Levine SR, Brey RL, Tilley BC, et al. Antiphospholipid antibodies and subsequent thrombo-occlusive events in patients with ischemic stroke. JAMA 2004;291:576–84. 19. Cohen H, Hunt BJ, Efthymiou M, et al. Rivaroxaban versus warfarin to treat patients with thrombotic antiphospholipid syndrome, with or without systemic lupus erythematosus (RAPS): a randomised, controlled, open-label, phase 2/3, non-inferiority trial. Lancet Haematol 2016;3:e426–36. 20. Garcia D, Akl EA, Carr R, et al. Antiphospholipid antibodies and the risk of recurrence after a first episode of venous thromboembolism: a systematic review. Blood 2013;122:817–24. 21. Erkan D, Vega J, Ramon G, et al. A pilot open-label phase II trial of rituximab for non-criteria manifestations of antiphospholipid syndrome. Arthritis Rheum 2013;65:464–71. 22. Erkan D, Leibowitz E, Berman J, et al. Perioperative medical management of antiphospholipid syndrome: hospital for special surgery experience, review of literature, and recommendations. J Rheumatol 2002;29:843–9. 23. Rand JH, Wu XX, Quinn AS, et al. Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug. Blood 2010;115:2292–9. 24. Glynn RJ, Danielson E, Fonseca FA, et al. A randomized trial of rosuvastatin in the prevention of venous thromboembolism. N Engl J Med 2009;360:1851–61.

CHAPTER 61  Antiphospholipid Syndrome

841.e1

MULTIPLE-CHOICE QUESTIONS 1. Which of the following statements concerning treatment options for a patient with antiphospholipid antibodies is correct? A. Aspirin is an effective thromboprophylactic strategy for asymptomatic individuals with antiphospholipid antibodies. B. Patients with arterial thromboembolism and APS should be treated with aspirin and a direct oral anticoagulant C. Patients with venous thromboembolism and APS should be treated with warfarin with a target INR of 2-3 D. A woman with prior pregnancy loss and APS should receive intravenous immunoglobulin and steroids during subsequent pregnancies 2. A lupus anticoagulant is identified by which of the following laboratory test results? A. Elevated antibody levels to prothrombin B. An antibody that interferes with phospholipid-dependent coagulation reactions C. An antibody that induces an increased thrombin potential D. The presence of a specific factor inhibitor

3. Which of the following patients should be tested for possible APS? A. A 65 year old man with a stroke B. A 22 year old woman taking oral contraceptives with a pulmonary embolism C. A 34 year old woman who presents with fetal demise at 34 weeks gestation D. A 27 year old man new-onset seizures

62  Immunohematological Disorders Jay N. Lozier, Pierre Noel

Congenital (primary) or acquired (secondary) immunodeficiencies, medications, and lymphoproliferative and rheumatological disorders are frequently associated with immune-mediated cytopenias. These processes can affect erythrocytes, leukocytes, and platelets (individually or in combination). In this chapter, we address immune-mediated cytopenias of each hematological component, discussing pathophysiology, clinical presentation, differential diagnosis, and treatment options.

IMMUNE-MEDIATED HEMOLYTIC ANEMIA Immune-mediated hemolysis can be autoimmune, alloimmune, idiopathic, or secondary to drugs or other diseases. Regardless of the underlying cause, immunoglobulin G (IgG) or IgM (rarely IgA) antibodies are directed against antigens on the red blood cell (RBC) membrane (Table 62.1).1,2 These disorders can be categorized on the basis of the underlying cause and the type of antierythrocyte antibody that mediates the process.

Autoimmune Hemolysis Mediated by Warm Antibody Although warm-antibody autoimmune hemolytic anemia is rare, it increases in prevalence above the age of 50 years and tends to be more common in women than in men, as is typical of most autoimmune diseases. In this condition, IgG autoantibodies and complement components are present on the RBC surface (see below). Although the autoantibody target is most commonly the Rh antigen, a variety of other targets are known. The specificity of the antibody, however, does not affect the clinical presentation or management of hemolysis.

Drug-Induced Immune Hemolysis Over 125 drugs have been associated with immune hemolysis.3 Cefotetan, ceftriaxone, and piperacillin currently account for over 80% of cases. The prognosis of drug-related immune hemolysis is much better than that of idiopathic hemolysis because the hemolysis stops once the offending drug has been removed. The drugs most commonly associated with fatal hemolytic anemia are cefotetan (8%) and ceftriaxone (6%).3 Fludarabine and cladribine have been associated with immune hemolysis. Following multiple courses of alkylating agents, fludarabine causes autoimmune hemolytic anemia in 20% of patients with chronic lymphocytic leukemia (CLL). The combination of fludarabine and cyclophosphamide with or without rituximab (FC, FCR) may protect against the development of autoimmune hemolytic anemia.3,4 Although the biochemical mechanisms of drug-related immune hemolysis are not completely clear, several hypotheses are generally accepted. Most commonly, complexes of drug and IgG and/or IgM that adsorb to the RBC surface and fix complement. The resultant intravascular hemolysis is acute and often severe enough

to cause renal failure from toxicities of hemoglobin to renal epithelium. A second less common mechanism develops primarily in patients receiving very high doses of penicillin (rarely used) for at least 1 week. High-titer antipenicillin IgG develops and binds to penicillin that is covalently attached to the RBC membranes. The resultant hemolysis is less acute than that caused by immune complexes but can be life-threatening. In a third mechanism, a drug stimulates the production of an antibody that reacts with the patient’s RBCs independently from the drug. Serologically, this antibody is indistinguishable from an idiopathic autoantibody. This has become rare as the use of the primary causal agent, methyldopa (an antihypertensive medication), has declined. Although these autoantibodies commonly cause positive clinical antibody tests (see below), they rarely cause hemolysis in vivo, and when they do, it usually ceases within 2 weeks of discontinuing the drug.

Cold Agglutinin Diseases This type of hemolytic anemia is mediated predominantly by anti-I or anti-i IgM that agglutinates red cells at temperatures well below 37°C.5 These antibodies engage the complement pathway resulting in C3b-mediated RBC phagocytosis mainly by Kupffer cells, whereas membrane-associated complex is a minor mechanism at low IgM titers. The severity of the clinical illness depends on the concentration of the IgM and its “thermal amplitude.” Thermal amplitude describes the temperature range over which it binds to RBCs: for example, antibodies that exclusively bind at 4°C are only active in vitro, whereas those that bind at >30°C can bind to RBCs as they circulate in the periphery and begin the process of complement fixation, which can persist even as the cells return to body core temperatures. The activity of the IgM is also determined by its relative affinity for the I- and i-antigens, which varies from one individual to the next. Two general types of cold agglutinin disease are recognized: a chronic idiopathic disease presenting in patients over age 50 years, caused by monoclonal anti-I IgM, and a transient disease secondary to certain infections (e.g., mycoplasma, Epstein-Barr virus [EBV], cytomegalovirus [CMV]), caused by polyclonal anti-i and anti-I (see Table 62.1). Avoidance of cold environments is important in both categories of cold agglutinin disease. In addition, cold agglutinin disease can be associated with B-cell lymphoproliferative disorders, and this typically is responsive to rituximab and/or rituximab combined with fludarabine.6

Paroxysmal Cold Hemoglobinuria Paroxysmal cold hemoglobinuria (PCH) is caused by anti-P IgG that is very effective in fixing complement and producing intravascular hemolysis.2 Although rare, it is most common in

845

846

Part Seven  Organ-Specific Inflammatory Disease

TABLE 62.1  Classification of Immune

Hemolytic Disorders Autoimmune

Warm Antibody-Mediated Idiopathic Secondary Drugs, lymphoid malignancies, infections Other autoimmune diseases

Cold Antibody-Mediated Cold agglutinin disease Idiopathic Secondary Infection, lymphoid malignancies

Paroxysmal Cold Hemoglobinuria Idiopathic Secondary to infections

Alloimmune Secondary to red cell transfusions (alloantibodies, isoantibodies) Secondary to fetal-maternal hemorrhage Secondary to transplanted lymphocytes

children following a viral illness and can be managed by avoidance of cold. In the past, it was more commonly associated with syphilis (see Table 62.1). There is also an autoimmune variety of PCH that may require immunosuppression with corticosteroids. Splenectomy is not helpful as a consequence of the fact that the hemolysis is intravascular.

Hemolytic Transfusion Reactions Because individuals with group O RBCs have preformed iso-anti-A and -B, they must only be transfused with group O cells. Similarly, individuals with group A RBCs with preformed anti-B isoantibodies must only receive group A RBCs, and individuals with group B RBCs must only receive group B RBCs. Because of the absence of either group A or B antigens on the surface of group O RBCs, such cells can be used in transfusion of A, B, or AB individuals in emergency situations. Failure to abide by these rules results in acute intravascular hemolysis that can cause renal failure, disseminated intravascular coagulation, and death. Other hemolytic transfusion reactions are caused by alloantibodies, predominantly IgG. Therefore a multiply transfused patient is at risk for hemolysis from alloantibodies if the patient receives incompatible blood. Multiparous women are at similar risk because of exposure to paternal fetal RBC antigens. Fortunately, for unclear reasons, most RBC antigens do not elicit an immune response although the Rh, Kell, Kidd, and Duffy antigens are clearly immunogenic. Exposure particularly to Kidd or Duffy antigens may stimulate alloantibody formation that can rise to sufficient titer to cause hemolysis with an onset typically delayed by approximately 1 week. Such delayed transfusion reactions may be subtle or cause an abrupt drop in hemoglobin with jaundice and hemoglobinuria.

Immune Hemolysis Associated With Transplantation Any transplanted tissue may contain “passenger” lymphocytes from the donor that will survive and proliferate in the recipient if the recipient is sufficiently immunosuppressed.7 When the RBCs of the recipient carry A or B antigen and the donor is ABO incompatible, the transplanted lymphocytes will respond to the recipient RBCs as foreign, and allogeneic anti-A or anti-B

antibodies will be produced and can lead to significant hemolysis. If the transplantation involves hematopoietic stem cells (HSCs), this dilemma will resolve once the donor’s erythropoiesis prevails and the donor lymphocytes are no longer exposed to the recipient RBCs.

Immunopathogenesis The antigens for antierythrocyte IgG are usually proteins, the most clinically important of which are the Rh-associated glycoproteins (RhAG), D, C, c, E, and e.8 In contrast, antierythrocyte IgM is directed at polysaccharides, which include the ABO and I-antigens (I, i) found on the anion and glucose transporter proteins in the RBC membrane.9,10 Antibodies are distinguished by being “warm” and “cold” reactive, respectively, meaning that they bind antigens at core body temperature (warm) or they bind antigens preferentially at lower temperatures (cold) in the peripheral circulation or ex vivo. This distinction results from the different thermodynamics of binding to protein (hydrophobic) and polysaccharide (electrostatic) antigens.10 IgG antibodies typically bind at warm temperatures and IgM antibodies typically bind at cold temperatures, although there can be overlap and exceptions (e.g., the Donath-Landsteiner IgG antibodies that are seen in paroxysmal cold hemoglobinuria). IgG and IgM also differ in their ability to fix complement, and this affects the resulting mechanism of hemolysis. To attach the first component of the classical complement pathway, two IgG molecules must bind in close proximity on the RBC. However, because of its pentameric structure, a single IgM molecule can initiate complement activation. Erythrocyte-bound IgG becomes attached to the Fc receptors of splenic macrophages, which may engulf all or part of the cell or release lysosomal enzymes that digest its membrane (antibodydependent cell-mediated cytotoxicity [ADCC]).11 RBC fragments escaping from this encounter lose more membrane than cytoplasm and become spherical (spherocytes) as a consequence of this change in the surface-to-volume ratio. If IgG has initiated complement activation on the cell surface, binding of C3b to splenic macrophages will augment erythrocyte phagocytosis in the spleen.12 When IgM fixes complement, the process begins in the cooler peripheral circulation, where IgM binds to RBCs. If the amount of IgM bound is relatively high with at least some of it remaining on the cell at 37°C (e.g., anti-A or anti-B isoantibodies), the cascade of complement activation goes to completion. Doughnut-shaped holes are formed in the cell membrane that allow the influx of water and sodium, inducing intravascular osmotic rupture of the cell.11 However, if the IgM elutes from the RBC as it returns to body core temperature, the complement reaction attenuates. In this circumstance, the components remain on the cell but do not cause intravascular hemolysis. Instead, the cells are cleared by hepatic macrophages via complement-binding sites.13 This has important implications for clinical tests for the presence of antibody and complement on the RBC surface during the workup of immune-mediated hemolysis. Antibody-mediated hemolysis causes variable degrees of anemia and reticulocytosis. Intravascular hemolysis releases hemoglobin into the circulation, but this is too small an amount to cause measurable hemoglobinemia, although it will result in consumption of haptoglobin, which is rapidly depleted. In contrast, when hemolysis results from complement-mediated lysis, such as follows an ABO-incompatible blood transfusion, hemoglobinemia becomes massive, overcoming the scavenging capacity of plasma hemoglobin binders

CHAPTER 62  Immunohematological Disorders (haptoglobin, hemopexin, albumin), resulting in hemoglobinuria. Because hemoglobin is toxic to the renal tubular epithelium, renal function may become impaired. RBC membrane fragments released by massive intravascular hemolysis are a rich source of procoagulant phosphatidylserine and can precipitate disseminated intravascular coagulation. In contrast, the consequences of extravascular hemolysis (i.e., via phagocytosis in the reticuloendothelial system of the liver and the spleen) are much less severe. In macrophages, iron is removed from the hemoglobin and recycled to the circulation to support a compensatory reticulocytosis and the heme porphyrin is metabolized to bilirubin. Patients with immune-mediated anemia have an increased incidence of venous thromboembolism, and detection of a lupus anticoagulant in these patients places them at a particularly high risk for this complication.14

Antibody-coated red cells from patient

847

Reagent anti-IgG

+

Visible red cell agglutination

Diagnosis With few exceptions, if the mechanism of hemolysis is immune mediated, an anti-RBC antibody can be demonstrated, either on the RBC surface, in serum, or both.1,2 With autoimmune hemolysis, IgG or IgM and/or complement components can be identified by a direct antibody test (DAT), originally known as a direct Coombs test (Fig. 62.1). For this assay, a patient’s RBCs are washed and suspended in buffer. Surface-bound IgG is detected by adding anti-IgG antibody, which, being divalent, can bind to IgG on adjacent RBCs and agglutinate them into visible aggregates. Because of its pentameric structure, IgM on the cells can cause agglutination without the addition of a second antibody. Even when IgM has been previously eluted from the cell surface as a result of warming in the central circulation, its earlier presence in vivo can be detected by telltale remnants of complement that are fixed to the RBC. In this setting, detection requires the addition of anticomplement (e.g., anti-C3dg) antibody. Alloantibodies can also be detected by the DAT if allogeneic RBCs from a previous transfusion are still circulating. If these have been cleared, however, RBC antibodies can be identified in the patient’s serum by adding the serum to a panel of RBCs carrying different antigens. Agglutination is detected as described above; this constitutes the indirect antibody test.

FIG 62.1  The Direct Antibody Test (DAT). The test is positive when immunoglobulin G (IgG: light blue triangles)–coated red blood cells are cross-linked by anti-IgG antibody (dark blue triangles) to form visible cell aggregates. Cell-bound complement and/or IgM can be detected by using anticomplement or anti-IgM reagent antibodies.

CLINICAL PEARLS In the workup of hemolytic anemia, the following clinical laboratory studies provide important clues as to mechanism and may lead to a diagnosis of an immune hemolytic anemia. Direct antibody test (DAT): The presence of antibodies on the surface of red blood cells (RBCs) suggests an immune-mediated hemolysis. The presence of antibody and complement on the surface of the RBC suggests drug-related hemolysis, whereas the presence of complement alone may suggest an immunoglobulin M (IgM) or cold antibody-related hemolysis. Peripheral blood smear: Examination of the peripheral smear and various RBC indices (chiefly the mean corpuscular volume [MCV]) give mechanistic clues to the etiology of the hemolytic process. Immune-mediated hemolysis is characterized by spherocytosis, even microspherocytosis in severe cases, as antibody-coated RBCs traversing the reticuloendothelial system assume a spherical form, rather than that of a normal biconcave disc. The appearance of other pathological forms, such as schistocytes, sickle cells, targets, or tear drop forms (dacrocytes), suggest other causes of hemolysis, such as thrombotic thrombocytopenic purpura (TTP), or mechanical, shear-induced hemolysis (e.g., aortic stenosis), sickle cell disease, thalassemia, or extramedullary hematopoiesis from bone marrow fibrosis, metastasis, or failure. Nucleated RBCs can be seen in any form of hemolytic anemia if it is severe enough.

Reticulocytes: Evaluation of the reticulocyte count indicates whether bone marrow is capable of making new erythrocytes in response to hemolysis. Lactic acid dehydrogenase (LDH): The LDH is typically elevated with ongoing hemolysis, since LDH is an important housekeeping enzyme found in erythrocytes. LDH is found in cells from all tissues, each having a characteristic isoenzyme form of LDH. It is rarely necessary to distinguish LDH from RBCs from other tissue sources, but LDH isoenzyme 1 is the predominant form found in RBCs. Bilirubin: As heme is released from RBCs, it is metabolized to bilirubin, which is glycosylated and then excreted via hepatic metabolism. Initially, as large amounts of heme are released and metabolized, the bilirubin is predominantly indirect bilirubin (unconjugated), and then it is converted to direct (conjugated) bilirubin. This can be altered by cholestasis, either from biliary obstruction or hepatic disease or Gilbert disease, or hepatic immaturity in the premature infant or newborn. Haptoglobin: Haptoglobin is extremely sensitive to even small amounts of hemolysis. Its absence merely confirms that there is a significant hemolysis, but not its extent. The presence of normal haptoglobin effectively rules out significant hemolysis, and the return of measurable amounts of haptoglobin usually signals the end of hemolysis.

848

Part Seven  Organ-Specific Inflammatory Disease

Therapy The first line of therapy is corticosteroids, and 80% of patients achieve a partial or complete response to 1 mg/kg/day of prednisone (orally). Once a response is achieved, the prednisone dose is tapered slowly. Approximately 50% of patients require prednisone at a dose of 15 mg/day or less to maintain the hemoglobin level >10 g/dL, and it may take up to 3 weeks for patients to achieve a response. Patients who do not respond in 3 weeks should be started on second-line therapy. It is estimated that long-term complete responses not requiring prednisone can be achieved in 20% of patients.15

THERAPEUTIC PRINCIPLES Autoimmune Anemia • Hemolytic anemia induced by “warm” antibody • Acute: corticosteroids, transfusion if severe • Chronic: splenectomy, rituximab, immunosuppression • Hemolytic anemia induced by “cold” antibody • Cold agglutinin disease: avoidance of cold; rituximab +/− fludarabine • Paroxysmal cold hemoglobinuria: avoidance of cold; corticosteroids • Hemolytic disease of the newborn • Neonatal exposure to fluorescent light • Intrauterine transfusion or exchange transfusion

Splenectomy and anti-CD20 antibody (rituximab) are considered second-line therapy. Splenectomy is associated with short-term partial or complete responses in two-thirds of patients. The overall response rate to rituximab is approximately 80%, but rituximab is contraindicated in patients with untreated hepatitis B. The rare, but most severe, long-term complication of rituximab therapy is progressive multifocal leukoencephalopathy.15 Danazol, a synthetic anabolic steroid, has been used as a first-line agent in conjunction with prednisone; its effectiveness appears to be less in relapsed or refractory disease. The role of high-dose intravenous immunoglobulin (IVIG) remains controversial; its effectiveness remains to be determined in larger trials. Third-line therapy consists of immunosuppressive agents (e.g., azathioprine, cyclophosphamide, alemtuzumab, mycophenolate mofetil, cyclosporine).1,15

IMMUNE-MEDIATED NEUTROPENIA Immune neutropenia constitutes a heterogeneous group of acquired diseases in which the immune system responds to circulating neutrophils, selectively reducing their level to below 1500 cells/mm3 (Table 62.2).

KEY CONCEPTS Immune Neutropenia • Immune neutropenia in children is caused by antineutrophil antibodies (ANAs). • Immune neutropenia in adults has a more complex etiology. • Immune complex-mediated neutrophil clearance and cell-mediated suppression of myelopoiesis often play a major role.

TABLE 62.2  Causes of Immune

Neutropenia Primary

Isoimmune neonatal neutropenia Autoimmune neutropenia of childhood Adult autoimmune neutropenia

Secondary Systemic autoimmune disease Rheumatoid arthritis (i.e., Felty syndrome) Systemic lupus erythematosus Sjögren syndrome Lymphoproliferative malignancy Large granular lymphocyte (LGL) leukemia Lymphoma

Drug-Induced Antiplatelets—ticlopidine Inflammatory bowel disease drug—sulfasalazine Antipsychotic—clozapine, phenothiazines Antithyroid medications—propylthiouracil, methimazole Retrovirals Antibiotics—beta-lactams, cefepime, trimethoprim-sulfamethoxazole, vancomycin, rifampicin, quinine/quinidine Diuretics—furosemide, spironolactone Antiepileptic—lamotrigine Rituximab, infliximab, etanercept

antibodies usually disappear within 12–15 weeks, but occasionally it can persist as long as 24 weeks after delivery.

Primary Autoimmune Neutropenia Primary autoimmune neutropenia is an antibody-mediated disease that presents commonly in early childhood.17 Patients typically have normal blood counts at birth and develop neutropenia at 3–36 months of age. Children presenting at 30% reported in some studies. The complex pathophysiology of this disorder is discussed elsewhere in detail (Chapter 52).

have been attempts in the past to classify this pattern as pseudo-Felty syndrome, but the clinical findings and course are often indistinguishable from “classic” Felty syndrome. 2. About half the patients with clinically apparent T-LGL leukemia have circulating rheumatoid factor and immune complexes in their blood; about a third, usually patients expressing HLA-DR4, develop clinically significant arthritis, sometimes requiring antiinflammatory agents. Although the reason why clonal T-LGL disorders and autoimmunity often coexist is unclear, the tendency toward overlap is clear. Since the pathophysiology and therapy of both conditions are similar, these problems in classification usually have little impact on the initial management of neutropenia. When a patient with “Felty syndrome” develops aggressive T-LGL leukemia or a patient with T-LGL manifests severe rheumatological symptoms, the clinician must be prepared to alter therapy, as needed, to fit the clinical picture. Although some clinicians have attempted to develop criteria for distinguishing Felty syndrome from T-LGL with pseudo-Felty syndrome, there is now substantial evidence that clonal T-LGL disorders are commonly found in rheumatology patients and that patients with clonal disorders seldom develop a progressive, neoplastic disorder. Conversely, although patients with T-LGL leukemia have a malignancy, it is typically quite indolent and in these cases, the clinical course is often dominated by rheumatological complication and/or neutropenia and not by progressive neoplastic disease.

T-Cell Large Granular Lymphocyte Leukemia

Drug-Induced Immune Neutropenia

Large granular lymphocytes (LGL) are medium to large lymphocytes recognizable on light microscopy by their distinctive azurophilic granules (Fig. 62.2). These cells normally constitute 0.75 mg/ kg/day confers no advantage. Mild or localized disease can sometimes be managed with potent topical corticosteroids (confirmed in controlled trials).31 When new blister formation has stopped and healing has begun, systemic steroids can be tapered slowly. The speed of tapering is dictated by the severity of the patient’s initial disease and any flare-ups that occur during tapering. Most patients can stop systemic steroids completely within 6–18 months, but recurrence of disease activity is not uncommon. Flare-ups should be managed with the lowest dose of systemic steroids possible; occasionally topical corticosteroids are enough. Usually, BP is self-limiting, lasts between 1.5 and 5 years, and responds promptly to systemic glucocorticoids.31 A minority of patients requires prolonged high-dose systemic glucocorticoids. In these individuals, adding azathioprine, cyclophosphamide, or methotrexate will often allow tapering or discontinuation of systemic steroids, but data on which is best for steroid sparing are limited.31 Other unproven adjunctive therapies that may help in some patients include dapsone, cyclosporine, and rituximab. Our preferred options for additional therapy are azathioprine (1–2 mg/kg/day) or mycophenolate mofetil (1000–2000 mg/day).32 A recent review of IVIG for BP concluded that 70% of patients experienced some improvement but there was no clinical benefit in the remainder. IVIG 2 g/kg over 5 days at monthly intervals for 3 months has been commonly used; typically more than one cycle is needed to prevent recurrence. In some patients, IVIG appears to be steroid sparing. IVIG was ineffective if patients received low-dose IVIG or only a single infusion. Rituximab eliminates CD20+ B cells through complementdependent and antibody-dependent cell-mediated cytotoxicity, as well as by inducing structural changes and apoptosis. The targeted B cells remain absent from the circulation for 6–12 months. Several groups have reported clinical success with the use of rituximab for pemphigus, but it has only had limited success in the treatment of BP.33 An open-label trial of anti-IgE (omalizumab) demonstrated benefit in 5 of 6 patients with BP,34 albeit with varying degrees of success.

KEY CONCEPTS Bullous Pemphigoid Most common autoimmune blistering disease that presents in older adults Severely pruritic with clinical presentations, including urticarial lesions, small vesicles, and/or large tense vesicles Linear immunoglobulin G (IgG) and C3 present at the epidermal basement membrane, binding to the epidermal side of “1 M NaCl split” skin Disease activity correlates best with IgG anti-BP 180 antibodies

CHAPTER 63  Bullous Diseases of the Skin and Mucous Membranes

EPIDERMOLYSIS BULLOSA ACQUISITA EBA is a chronic subepidermal blistering disease that typically presents in the fourth to sixth decades. There are two forms of EBA—a noninflammatory type with blisters on distal extremities and an inflammatory type that closely resembles BP. Patients with non-inflammatory EBA have peripherally distributed blisters that heal with scarring and milia formation. Their skin is extremely fragile, often resulting in numerous erosions in areas of mechanical trauma, such as hands, feet, elbows, and knees. Lesions are often seen on oral mucous membranes, sometimes including the esophagus. Patients with classic EBA may also develop ocular, vaginal, urethral, and rectal mucosal lesions. Ocular changes are common, clinically resembling mucous membrane pemphigoid (MMP). Other cutaneous manifestations include scarring alopecia and variable degrees of nail dystrophy. Patients with nonclassic (inflammatory) EBA often present similarly to those with BP, with widespread tense bullae on an erythematous base, which heal without scarring (Fig. 63.9).35 EBA has been associated with several diseases, particularly inflammatory bowel disease (IBD) and bullous SLE. These associations may partly result from the association of EBA with HLA-DR2.36 Skin biopsies of early lesions from patients with EBA show subepidermal blisters with variable degrees of inflammation. In patients with classic EBA, lesional skin biopsies often have minimal inflammatory cell infiltrate. In contrast, patients with inflammatory EBA may have substantial collections of mononuclear cells, neutrophils, and eosinophils in the superficial dermis. Direct immunofluorescence of perilesional skin biopsies from patients with EBA shows linear deposits of IgG at the DEJ, as

FIG 63.9  Patient with epidermolysis bullosa acquisita showing extremity involvement with tense bullae. Note the similarity to lesions of bullous pemphigoid, with somewhat less inflammation surrounding the base of bullae.

863

in BP. Linear deposits of C3, IgM, IgA, and fibrinogen have also been reported.35 However, in EBA, these deposits are localized exclusively below the lamina lucida. Direct immunofluorescence of saline-split perilesional skin shows IgG in the blister floor in EBA (Fig. 63.10); in BP, IgG is in the blister roof. Indirect immunofluorescence using normal human skin shows circulating anti–basement membrane zone antibodies in 30–50% of patients with EBA. However, IgG antibody can be detected in 85% of patients when using saline-split skin, which is the more sensitive and specific substrate. As expected from the in vivo deposition pattern, IgG from patients with EBA localizes to the blister floor. Sera from these patients recognize the 300-kDa protein, type VII collagen, primarily targeting its immunodominant NC1 domain.

Pathogenesis Immunoelectron microscopy of EBA biopsy specimens has shown immunoglobulin deposits localized to the lamina densa zone of the basement membrane, below the lamina lucida. Transmission electron microscopy of lesional EBA skin shows decreased or absent anchoring fibrils. Anchoring fibrils are implicated in epidermal–dermal adherence via linkage of the hemidesmosome through the basement membrane, and their absence may explain the observed skin fragility. The lack of inflammatory infiltrate in many patients with EBA suggests that autoantibodies may disrupt the interaction between anchoring fibrils and dermal matrix proteins. The target antigen for the IgG autoantibodies present in the sera of patients with EBA is type VII collagen, a 300-kDa glycoprotein composed of a 145-kDa noncollagenous domain (NC1) at the amino-terminal end, an 18-kDa noncollagenous domain (NC2) at the carboxy terminus, and a central collagenous domain. IgG antibodies from patients with EBA appear specific for epitopes within the NC1 noncollagenous domain. Type VII collagen is the major structural component of anchoring fibrils and is produced by both epithelial keratinocytes and dermal fibroblasts. Passive transfer experiments in mice and active immune models

FIG 63.10  Direct immunofluorescence sample from a patient with epidermolysis bullosa acquisita (EBA) after incubation with 1 mol/L sodium chloride (NaCl), showing localization of immunoglobulin G (IgG) immunoreactants to the floor (dermal side) of the blister cavity.

864

Part SEVEN  Organ-Specific Inflammatory Disease

have provided additional support for the critical role of anti-type VII collagen antibodies in the pathogenesis of EBA.35 In experimental mouse-model EBA, induction of specific types of antibodies is linked to the MHC haplotype suggesting future work may identify non-MHC EBA susceptibility genes.37 Utilizing a four-way, autoimmune-prone, advanced mouse intercross line immunized with a COL7 fragment to induce EBA, investigators identified quantitative trait loci (QTLs) on chromosomes 9, 12, 14, and 19 associated with disease development and QTLs on chromosomes 1, 15, and 19 associated with maximum disease severity.4 In this model, gene–microbiota interactions appear to promote disease development.38 Finally, identification of other mediators in inflammatory EBA, such as heat shock protein-90 (HSP-90), granulocyte macrophage–colony-stimulating factor (GM-CSF), chemokine ligand-1 (CXCL1), CXCL2, or IL-1, suggests other possible candidates for therapeutic targeting.38

Treatment Spontaneous resolution is infrequent in EBA, and management is difficult. The main goals of therapy are to minimize blistering and scar formation, with particular attention to ocular and oral mucosal lesions. Systemic glucocorticoids are the mainstay of therapy, especially for patients with inflammatory EBA. Unfortunately, even high-dose systemic steroids do not usually improve skin fragility and trauma-induced blister formation. Mucosal lesions often respond to systemic steroids (prednisone 0.5–1.5 mg/ kg/day) but may recur with tapering and/or discontinuation of steroids. Several adjunctive agents, including azathioprine, cyclophosphamide, colchicine, dapsone, hydroxychloroquine, and plasmapheresis, have been proposed, but none has been consistently effective. Cyclosporine has been used to treat patients with EBA with some success, although toxicity can limit therapy. Photopheresis, IVIG, and rituximab have also been reported to be effective in some patients with refractory EBA.35 Mucous membrane lesions can prove particularly difficult to manage and may require systemic therapy. Patients with ocular lesions need ophthalmologist review and may require systemic glucocorticoids to prevent conjunctival scarring. Oral mucous membrane lesions can sometimes be managed with frequent application of potent topical steroid ointments or gels (0.05% clobetasol propionate, 0.05% fluocinonide). If this fails and the degree of oral erosions inhibits appropriate nutrition, systemic glucocorticoid therapy may be required. Clinicians should also be aware of possible involvement of esophageal mucosa and/or tracheal mucosa and involve appropriate specialists to monitor and treat these potentially severe complications. As well as therapies targeting B cells, novel therapeutic targets addressing autoreactive T cells are being studied in the treatment of EBA. These include inhibitors of the stress-inducible HSP-90.39 Strategies targeting other features of EBA, such as neutrophil recruitment and complement activation, are also being explored. The management of chronic skin wounds is vital in EBA. Protection of skin from trauma and early use of topical and systemic antibiotics are critical to improving the rate of healing. The development of new biological dressings for chronic ulcers has also proven helpful in the management of these wounds.

PEMPHIGOID GESTATIONIS PG is a rare, itchy, blistering disease of pregnancy and the puerperium characterized by linear deposits of IgG and C3 at the DEJ. Formerly known as “herpes gestationis,” PG has no

relationship to herpes virus infections. The term pemphigoid gestationis eliminates this confusion and emphasizes the pathophysiological similarity of PG to BP. PG is rare, occurring in 80% of patients with anti-epiligrin antibodies show laryngeal involvement, something seen in 50% of patients presenting in the first three decades of life. There is a wide spectrum of cutaneous manifestations of psoriasis. Individual lesions vary from pinpoint to large plaques, or even generalized erythroderma. More specifically, the clinical spectrum of psoriasis includes the plaque, guttate, small plaque, inverse, erythrodermic, and pustular variants.1 The most common and well-recognized morphological presentation of psoriasis is that of the plaque type (Fig. 64.1). The disease is characterized by the formation of demarked erythematous plaques with large scaling. The scales are a result of a hyperproliferative epidermis with premature maturation of keratinocytes and incomplete cornification, with retention of nuclei in the stratum corneum (parakeratosis). The mitotic rate of the basal keratinocytes is increased compared with that of normal skin. As a result, the epidermis is thickened (acanthosis), with elongated rete ridges that form finger-like protrusions into the dermis. The granular layer of the epidermis (the starting site of terminal keratinocyte differentiation) is strongly reduced or missing. The epidermis is infiltrated by neutrophils and activated CD8 T lymphocytes, whereas within the dermis there is an inflammatory infiltrate composed mainly of CD3+ T cells, dendritic cells (DCs), macrophages, mast cells, and neutrophils. Elongated and dilated blood vessels in the dermal papillae represent a further histological hallmark of psoriatic skin lesions (Fig. 64.2).1

IMMUNE-RELATED GENETIC FACTORS PREDISPOSING TO PSORIASIS The genetic basis of psoriasis has long been recognized, since family members of patients with psoriasis are at greater risk of developing the disease. The concordance rate of psoriasis is approximately 70% in monozygotic twins and 20% in dizygotic twins, depending on the study and population.2 The mode of inheritance is complex. It is thought that there is no single disease gene, but, rather, a complex set of gene variants, resulting in an aberrant response to environmental factors. At least nine chromosomal loci with statistically significant linkage to psoriasis have been identified, termed psoriasis susceptibility loci 1 through 9 (PSORS1 through PSORS9).2 PSORS1 is the major genetic determinant of psoriasis and is located within the human leukocyte antigen (HLA) complex on chromosome 6p (Chapter 5).

An association with HLA-B13 was first identified and later with other class I molecules HLA-B17, HLA-B37, HLA-B57, HLA-Cw6, and HLA-Cw7, and class II molecules HLA-DR4 and HLA-DR7. Among these, the highest and most consistently reported relative risk is for HLA-Cw6 haplotype. A significantly higher frequency of HLA-Cw6 is associated with early-onset (type I) psoriasis compared with late-onset psoriasis (type II). Current data suggest that HLA-Cw6 is the susceptibility allele within PSORS1, but no disease-specific mutations have been identified, and variants in regulatory sequences potentially affecting several downstream genes cannot be ruled out. HLA-C might be involved in immune responses at the levels of both antigen presentation and natural killer (NK)–cell regulation. Recently, compelling evidence has emerged for an interaction between the HLA-C and ERAP1 (involved in major histocompatibility complex (MHC) class I peptide processing) loci in psoriasis.3 36763ERAP1 variants only influenced psoriasis susceptibility in individuals carrying the HLA-C risk allele. Other predisposing polygenes were found in the PSORS2 region, on chromosome 17q25. Two distinct regions harboring susceptibility loci have been identified: the first contains the genes SLC9A3R1 and NAT9 and the second the gene RAPTOR.4 The SLC9A3R1 gene encodes a PDZ domain-containing phosphoprotein, implicated in several biological processes occurring in T cells. NAT9 encodes an N-acetyltransferase involved in MHC class I antigen-presentation and in immunological processes related to autoimmune diseases. Additionally, between SLC9A3R1 and NAT9 is a polymorphism for the binding site of a transcription factor RUNX1 that may affect regulation of the immune synapse.4 Associations with alleles of interleukin (IL)-12, IL-23 receptor (IL-23R), IL-19/20, and IRF2 have also been described. Interestingly, IL-12 and IL-23R single nucleotide polymorphisms do not have interactions with HLA-Cw6.4 Recently, polymorphisms have been found in two genes, IL-36RN and CARD14, and to be independently associated with psoriasis.4,5 For CARD14 (caspase recruitment domain-containing protein 14) gene, many missense mutations, leading to elevation of CARD14 mRNA in patients, were initially found in pediatric patients with a severe clinical presentation of psoriasis. CARD14 gene mutation protein, in association with an inflammatory stimulus, may induce aberrant activation of nuclear factor (NF)-κb, a transcription factor that controls the expression of many genes, including key chemokines upregulated in psoriasis, such as chemokine (C-X-C motif) ligand (CXCL)8 and CC chemokine ligand (CCL)20. Mutations in IL-36RN gene were described in patients with severe pustular psoriasis. This gene encodes the antiinflammatory protein IL-36Ra, antagonist of IL-36γ, a cytokine highly produced in psoriatic lesions and with important proinflammatory function.

871

872

Part SEVEN  Organ-Specific Inflammatory Disease

FIG 64.1  Clinical Features of Plaque Psoriasis. Scaly, erythematous, sharply demarcated plaques in different sizes and shapes are hallmarks of psoriasis.

KEY CONCEPTS Psoriasis is a common chronic-relapsing immune-mediated skin disease affecting approximately 2% of the general population. There is strong evidence that psoriasis is determined by genetic predisposition. A complex set of gene variants, rather than a single gene, are responsible for an aberrant response to environmental factors. Psoriasis is a disease caused by the infiltration of effector immune cells in both the epidermis and dermis, which determines hyperproliferation of the epidermis with premature maturation of keratinocytes and incomplete cornification. As a result, the epidermis is thickened, with elongated rete ridges forming protrusions into the dermis. Primary effector cells are dermal dendritic cells (DCs), in particular plasmacytoid DCs (pDCs), whose activation can depend on DNA-LL-37 or RNA-LL-37 complexes released by injured keratinocytes and leads to a massive production of interferon (IFN)-α. pDC-released IFN-α or RNA-LL-37 complexes released by keratinocytes activate myeloid DCs (mDCs), which in turn induces type-1 and -17 T-cell responses. T-helper 22 response is also pathogenetically induced. Pathological cytokines include T-cell-derived lymphokines, such as IFN-γ, tumor necrosis factor (TNF)-α, IL-17, IL-22, IL-21, and antigen-presenting cell–derived cytokines, such as IL-12 and IL-23. Intrinsic alterations of keratinocytes in the activation of signal transduction pathways (i.e., STAT3, IKK-2, AP-1, etc.) are fundamental for the amplification of psoriatic processes.

EFFECTOR CELLS AND IMMUNE MECHANISMS OPERATING IN PSORIASIS Even though successful treatment regimens for the therapy of psoriasis are long established, the primary pathogenetic mechanism

and the cell types involved in the onset of the disease are still under debate. Psoriasis is classically responsive to trigger factors that can induce psoriasis de novo or exacerbate skin lesions.6 Trigger factors range from nonspecific stimuli, such as skin trauma (termed the Koebner effect) to more specific triggers, such as pathogens (i.e., streptococci) or drugs (i.e., lithium, interferon (IFN)-α). All of these factors generate a pathogenic cascade culminating in the expansion of lesional and/or circulating T cells in the psoriatic skin (Fig. 64.3). Much effort has been devoted to understanding the link between the trigger stimuli and the pathogenic T-cell cascade that leads to psoriasis. Recent evidence suggests that type-1 IFN may represent the missing link. The prototypical type I IFN, IFN-α, is abundantly produced by plasmacytoid dendritic cells (pDCs) during the early phase of psoriasis development (see Fig. 64.3A).6,7 In turn, IFN-α indirectly stimulates the pathogenic T-cell cascade by promoting the activation and maturation of myeloid DCs (mDCs) or by direct stimulation of IFN-α–sensitive pathogenic T cells (see Fig. 64.3B). The T-cell infiltrate present in active psoriatic skin establishes a cytokine milieu that dictates specific and pathogenic gene signatures in resident skin cells. Thus cytokine-activated keratinocytes overexpress a number of inflammatory mediators that aberrantly amplify and sustain the psoriasiform tissue reactions (see Fig. 64.3C). Intrinsic defects and/or alterations of keratinocytes in their immune response to proinflammatory cytokines are fundamental to the induction of psoriatic processes, as demonstrated in genetically manipulated mouse systems.

CHAPTER 64  Immunology of Psoriasis FIG 64.2  Histological Components of a Mature Psoriatic Plaque. Psoriatic skin lesions are characterized by a hyperproliferative epidermis showing an increased mitotic rate of the basal keratinocytes (A, Ki67 immunostaining). As a consequence, the epidermis is thickened, with elongated rete ridges that form typical finger-like protrusions into the dermis. The epidermis becomes infiltrated by activated CD8 T lymphocytes and neutrophils (B and C, immunostaining for CD8 and CD15, respectively). Within the drmis, an inflammatory infiltrate mainly composed of CD3+ T cells (D), CD11c+ dendritic cells (E), BDCA-2+ plasmacytoid dendritic cells (F), c-kit+ mast cells (G), and neutrophils (C) is observed. Elongated and dilated ICAM-1+ (H) blood vessels in the dermis represent another histological hallmark of psoriatic skin.

A

B

C

D

E

F

G

H

873

874

Part SEVEN  Organ-Specific Inflammatory Disease

STAT3 p38 Erk 1/2

Trigger factors: Pathogens Drugs Stress

PMN PMN

PMN autoantigens K17, K13 LL-37/ ?viral antigens? DNA

LL-37

PMN

LL-37

pDC pDC Chemerin

CD8

Endothelium

TNF-α TNF-α

CX3CL1, CCL17 ICAM-1, VCAM, E-selectin RNA-LL-37 RNA-LL-37 mDC IFN-α IFN-α

HLA-DR

INF-γ

Chemerin NK

Fibroblasts A

Tc1

Th22 Th1 Th17 Th17 Th2

PMN Th17 Th1 PMN

Th17Th1 Th1

Th17 Th22 Th2 Th2

Mast cell PMN

cytokines chemokines TGF-α, GM-CSF, CXCL10-9-11, IL-1, IL-6, CCL2, CXCL8, IL-19, IL-20, IL-36 CCL20 CCL5, CCL19

TNF-α, IFN-γ, IL-17, IL-22

TIP-DC

mDC

IL-1 KGF, FGF10 GM-CSF IL-36

ICAM-1

Th17 Th1

Endothelial cells

Fibroblasts

growth signals TGF-α, KGF IL-19, IL-20 amphiregulin

Tc1

Th1

Th17

PMN

Th1

SlanDC B

C

NF-NB AP1 STAT1

PMN

Tc1

CD8

Fibroblasts

Mast cell

PMN

PMN

VEGF

DNA-LL-37 DNA-LL-37 RNA-LL-37 IFN-α pDC

Chemerin

NF-NB AP1 STAT1 STAT3 p38 Erk 1/2

SlanDC

Fibroblasts Endothelium ICAM-1, VCAM, E-selectin

FIG 64.3  Scheme of Pathogenic Mechanisms Operating in Psoriasis. The psoriatic lesion starts to evolve after keratinocytes are injured, for instance by physical trauma or bacterial products. Thereafter, a cascade of events including the formation of complexes formed by keratinocyte-derived DNA and the cathelicidin LL-37 leads to the activation of plasmacytoid dendritic cells (pDCs), which routinely patrol psoriatic skin (A). Other pDCs are recruited in the early phase psoriasis development by the chemokine chemerin, derived primarily from dermal fibroblasts and, to a lesser extent, from mast cells and endothelial cells, and induced to release high amounts of IFN-α. IFN-α locally activates keratinocytes and participates in the activation processes affecting myeloid DCs. In turn, DCs migrate into draining lymph nodes and induce the differentiation of naïve T cells into effector cells, such as type 17 T-helper (Th) 17 or type 17 T cytotoxic (Tc) cells and type 1 Th1 or Tc1 cells. Effector cells recirculate and proliferate into psoriatic skin and produce massive amounts of proinflammatory cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α (B). The latter cytokine is also abundantly released by dermal DCs, mainly represented by TNF-α and inducible nitric oxide synthase-producing-DCs (TIP-DC). SlanDCs also reinforce immunity in psoriasis by interacting with and potentiating the activity of both neutrophils and natural killer (NK) cells, as well as inducing Th1 and Th17 responses. IFN-γ and TNF-α are responsible for the activation of resident skin cells, in particular keratinocytes, which respond to cytokines with a stereotypical set of genomic responses leading to synthesis of inflammatory mediators (C). Keratinocytes are also targets of T cell–derived IL-22, which induces proliferation and de-differentiation of psoriatic keratinocytes in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Keratinocyte-derived chemokines, cytokines, and membrane molecules have a major role in maintaining the recruitment of leukocytes into inflammatory sites. Because of their intrinsic defects, psoriatic keratinocytes aberrantly respond to cytokines and show altered intracellular signaling pathways, including STAT3 cascade (C). The uncontrolled hyperproliferation and differentiation observed in psoriatic skin could also derive from dysregulated production of tissue growth factors and regulators, such as transforming growth factor (TGF)-α, keratinocyte growth factor (KGF), amphiregulin, granulocyte macrophage–colony-stimulating factor (GM-CSF), fibroblast growth factor-10 (FGF-10), interleukin (IL)-19, IL-20, IL-36 produced by keratinocytes and fibroblasts. Psoriatic keratinocytes produce autoantigens (i.e., keratin (K)17, K13, nucleic acid/LL-37 complexes) capable of inducing clonal T-cell responses. Finally, the inflammatory cytokine milieu also influences the immune functions of fibroblasts and endothelium, with the latter being critical for leukocyte trafficking and extravasation.

CHAPTER 64  Immunology of Psoriasis Plasmacytoid DCs as Inducers of Primary Immune Responses in Psoriasis pDCs are characterized by a plasma cell morphology and a distinctive surface phenotype (CD4+, CD45RA+, CD123+, BDCA-2+, BDCA-4+, CD62L+, cutaneous lymphocyte-associated antigen (CLA)+, and CD11c−). They are considered as key effector cells in antiviral defense because of their ability to produce large amounts of type I IFN.8 Upon viral stimulation, pDCs differentiate into a unique type of mature DC and induce an IFN-α–dependent activation of bystander mDCs with the ability to induce Th1responses (Chapter 16), thus providing a necessary link between innate and adaptive immunity.8 Several studies have demonstrated that pDCs infiltrate psoriatic skin, and that pDC-derived IFN-α initiates the expansion of autoimmune T cells, leading eventually to the skin lesions of psoriasis.6,7,9,10 Blocking of type I IFN signaling with neutralizing antibodies to IFN-α/β receptors inhibited the development of psoriasis in symptomless pre-psoriatic skin engrafted onto immunodeficient AGR129 mice.10 Moreover, inhibition of IFN-α release by pDCs through anti–BDCA-2 antibody prevented the activation and expansion of pathogenetic T cells and the development of a psoriatic phenotype. The detailed mechanisms responsible for the IFN-α-induced expansion of T cells in psoriasis are currently unknown, but it appears that IFN-α favors cross-presentation of sequestered tissue-specific autoantigens by mDCs. pDC-derived IFN-α may also enhance the survival of autoreactive T cells through the induction of IL-15 or by promoting a Th1 cell bias through the induction of T-bet and IL-12Rβ2 expression. The pathogenetic role of IFN-α is also suggested by the observations that its signaling signature is present in resident skin cells of psoriatic plaques11 and that psoriasis is exacerbated if patients with psoriasis are treated with recombinant IFN-α for unrelated conditions (i.e., viral infections or tumors), or with imiquimod, a Toll-like receptor (TLR) agonist that induces production of IFN-α by pDCs.12 In addition, excessive activation of type I IFN signaling in mice deficient for IFN regulatory factor (IRF)-2, a transcriptional repressor of IFN signaling, causes an inflammatory skin disease resembling psoriasis. Recently, a genome-wide analysis conducted on paired lesional and nonlesional psoriatic skin and on the skin of healthy donors revealed a significant overexpression of many components of the IFN-α pathway in patients with psoriasis, including the receptor subunits for type I IFN, IFN-AR1, and IFN-AR2, the transcriptional activators of IFN-α-inducible genes, signal transducer and activator of transcription (STAT) 1, IRF1, and IRF7.11 All of these molecules are master regulators of IFN-αmediated immune responses. The molecular mechanisms leading pDCs to produce type I IFN involve the activation of TLR7 and TLR9, intracellular receptors that recognize viral/microbial nucleic acids within endosomal compartments. pDCs do not normally respond to self-DNA, but this restriction breaks down in some human autoimmune diseases. In psoriatic skin, pDCs can be activated to produce massive amounts of type I IFN in response to extracellular self-DNA fragments.13 However, this process requires the coupling of self-DNA to the endogenous antimicrobial peptide LL-37, known to be overexpressed in psoriatic skin. LL-37 breaks innate tolerance to self-DNA by forming aggregated and condensed structures that can trigger a robust IFN-α induction via TLR9 activation.13 LL-37 can also form complexes with RNA and activate pDCs through TLR7. In parallel, LL-37/RNA can alert myeloid DCs through their TLR8, driving T-cell activation and production of cytokines found in psoriasis.14 This finding

875

suggests a fundamental role for LL-37 in alerting resident skin pDCs of tissue damage associated with cell death and the release of self-DNA. pDCs are typically absent in unperturbed skin and peripheral tissues under homeostatic conditions but can enter secondary lymphoid organs through the expression of CD62L and chemokine receptors (CXCR4, CXCR3, CCR5, and ChemR23). pDCs can also infiltrate inflamed tissue of immune-mediated skin diseases, and, in particular, accumulate in the skin of patients with psoriasis early during disease development.9,10 Many studies have shown an evident IFN-α signature (e.g., increased expression of IRF7 and the presence of MxA, markers for IFN-α activity) in primary psoriatic plaques in the absence of detectable levels of the IFN-α cytokine. Indeed, IFN-α expression was detected early and transiently during the development of the psoriatic phenotype, and its effect persisted until lesions became chronic. Paralleling IFN-α expression, BDCA-2+ pDCs were detected only during the early developmental stages of psoriasis.10 In fact, pDC infiltration in psoriatic skin correlates with the expression of markers typical of early phases of psoriasis (CD15+ neutrophils and c-kit+ mast cells localized in the mid- and papillary dermis, and few CD8 T lymphocytes or ICAM-1+ keratinocytes), whereas they are almost absent in long-lasting lesions.9 Importantly, pDC recruitment in psoriatic skin is strictly associated with the expression of the chemokine chemerin, which is temporally produced by dermal fibroblasts and active during psoriatic plaque development.9 pDC migration toward fibroblast-derived chemerin is completely dependent on the expression of ChemR23 receptor on pDC. Compared with other chemokines potentially active on pDC (CXCL10 and CXCL12), chemerin is the main, if not the only, protein responsible for the pDC chemotactic activity released by fibroblasts in psoriatic skin.9

DC Driving of T-Cell Responses in Psoriatic Skin Although pDCs are responsible for triggering psoriasis, mDCs are the main amplifiers of local inflammation. Dermal mDCs are dramatically increased in psoriasis, and targeted immunotherapy reduces their quantity in patients with psoriasis, supporting the concept that mDCs have a key pathogenetic role.15 Dermal mDCs are found at the dermal–epidermal junction as well as throughout the whole dermis. mDCs are able to capture extracellular antigens for presentation to T cells and also intracellular antigens from adjacent cell types via cross-presentation (Chapter 6). In addition, mDCs within psoriatic lesions are intrinsically stronger stimulators of T-cell proliferation compared with DCs derived from peripheral blood or from the skin of healthy patients. mDCs uniformly express CD11c, and they can be further subdivided on the basis of expression of CD1c (BDCA-1). Steady-state skin has a predominance of CD11c+ CD1c+ resident DCs, whereas CD11c+ CD1c− mDCs predominate in psoriatic inflammation.15 A small fraction of CD11c+ CD1c+ DC bears “maturation” markers, such as DC-LAMP, CD83, and endocytic receptor DEC-205/CD205, suggesting that they could function as conventional DCs and present antigens to T cells to trigger acquired immune responses.15 These rare, phenotypically mature cells, often aggregating in dermal clusters, could be required for rapid antigen presentation to local T cells or for ongoing “micro-” immune responses. During psoriasis development, dermal CD11c+ DC mature and acquire a CD1c- HLA-DR+ CD45+ CD14− DC-specific ICAM-3–grabbing nonintegrin (DC-SIGN)+ phenotype.15 These inflammatory mDCs are CCR7+ and respond to the chemokine CCL19, suggesting that they may migrate to draining lymph nodes for antigen presentation. CD11c+ CD1c− inflammatory DC express very

876

Part SEVEN  Organ-Specific Inflammatory Disease

high levels of tumor necrosis factor (TNF)-α and the enzyme inducible nitric oxide synthase (iNOS) and can be considered the human equivalent of TIP-DC (TNF-α and iNOS-producing DCs), which have been shown in mice to have effector functions in clearing some bacterial infections.15 The proinflammatory nature of TNF-α in psoriasis and other inflammatory diseases is well established, and the receptors TNFR1 and TNFRII are expressed on a wide range of cells in psoriatic skin, including keratinocytes and endothelial cells. TNF-α induces expression of ICAM-1 on keratinocytes, facilitating the adhesion of circulating leukocytes. Moreover, TNF-α can stimulate keratinocytes and dermal fibroblasts to produce the potent neutrophil chemoattractant CXCL8, as well as the proinflammatory cytokines IL-6 and IL-1, which help generate and maintain Th17 cells (Chapters 10 and 16). The role of TNF-α in psoriasis is underlined by the therapeutic success of anti–TNF-α therapies in psoriasis.15 In addition, polymorphisms of the TNF-α promoter region have been associated with psoriasis. However, iNOS production by inflammatory DCs leads to nitric oxide (NO) release, inducing vasodilation, inflammation, and antimicrobial effects in psoriatic skin. Interestingly, NO inhibitors (e.g., statins) can have a beneficial effect on psoriasis, although this has not been studied in a randomized prospective manner. Inflammatory DCs also produce other cytokines (Chapter 9) (e.g., IL-23 and -12), which are linked to psoriasis. IL-12 mainly induces IFN-γ production, whereas IL-23 also stimulates IL-17 and IL-22 release by T cells, as shown in mice.16 Other evidence for a role of IL-23 in psoriasis includes the clinical efficacy of an anti-p40 monoclonal antibody (mAb) against psoriasis and the association of a single nucleotide polymorphism in the IL-23R gene in psoriasis patients. mDCs of psoriatic lesions also release the proinflammatory cytokine IL-15, which induces T-cell proliferation and monocyte activation, as well as skin hyperplasia by protecting keratinocytes from apoptosis. Another population of inflammatory mDCs, defined by the selective expression of the 6-sulfo LacNAc residue on the P-selectin glycoprotein ligand 1 membrane molecule, has been identified in psoriatic skin.17 6-Sulfo LacNAc+ DCs (slanDCs) have a welldefined phenotype (CD1c−, CD11c+, CD16+, CD14−) that clearly distinguishes them from classic CD1c+ blood DC (CD1c+, CD11c+) or pDC (BDCA-2, BDCA-4). SlanDCs produce more TNF-α, IL-23, IL-12, IL-1β, and IL-6 and can thus induce Th1/Th17 cells. SlanDCs also reinforce innate immunity in psoriasis by interacting with and potentiating the activity of neutrophils and NK cells.17 The function of another DC subset, the Langerhans cell, is still controversial. Langerhans cells are only found in the epidermal compartment where they are similar in number and phenotype in lesional and nonlesional skin, but they fail to migrate in response to proinflammatory stimuli18 (Chapter 19). These findings and data from Langerhans cell ablation models suggest that Langerhans cells may help sustain immune tolerance in psoriasis. In psoriatic skin, the epidermis contains an additional DC subset, known as inflammatory dendritic epidermal cells and are distinguishable from Langerhans cells by the expression of the macrophage mannose receptor CD206.

Activation of T Lymphocytes and Establishment of the Cytokine Milieu Influencing Keratinocyte Proliferation and Immune Functions Psoriasis lesional skin shows many inflammatory T cells in both the papillary dermis and the epidermis. Immunophenotyping

of T cells shows that these are mainly activated memory T cells expressing HLA-DR, CD25, CD27, and cutaneous lymphocyte antigen (CLA). Although there is differential T-cell receptor usage in T cells from psoriatic skin, the causative antigens responsible for T-cell activation in psoriasis remain unknown. Exposure of altered autoantigens from keratinocytes could be responsible for the activation and expansion of distinct T-cell subpopulations in psoriatic skin. These autoantigens may include keratin 17 (patients with active psoriasis have an increased frequency of circulating Th1 cells reacting to peptides from keratin 17) and corneodesmosin, an attractive candidate for psoriasis susceptibility based on its putative biological function in keratinocyte adhesion.19 Keratinocytes could also be responsible for the activation of pathogenetic T cells by viral or bacterial products. For example, human papillomavirus 5 (HPV-5) DNA and antibodies to HPV-5 virus-like particles have been found in psoriasis. Streptococcal infections are also frequently associated with psoriasis, and streptococcal superantigens could be presented to T cells by binding to MHC class II molecules expressed by lesional kera­ tinocytes. Putative psoriatic antigens are assumed to be kerati­ nocyte proteins that might share structural homology with streptococcal proteins and might thereby induce cross-reactive T-cell responses against skin components.20 Recently, it has been found that two-thirds of patients with moderate-to-severe plaque psoriasis harbor CD4 and/or CD8 T cells specific for LL-37.21 LL37-specific T cells produce IFN-γ, and CD4 T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and can be tracked in patients’ blood. The presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. T-cell migration from the dermis into the epidermis is a key event in psoriasis. It is controlled by the interaction of α1β1 integrin (very late antigen 1) on T cells with collagen IV in the basement membrane of the epidermis. Blockade of this interaction inhibits the development of psoriasis in clinically relevant models. Based on the analysis of infiltrating cell types, their secreted products, and genetic signatures present in lesional skin, psoriasis has been considered for many years as a type-1 (i.e., Th1)–mediated reaction, with IFN-γ playing a prominent role.22 Consistent with a Th1 pattern of response, CCR10 is preferentially expressed by skin homing CLA+ memory T cells, which secrete TNF-α and IFN-γ, but minimal amounts of IL-10 and IL-4, upon activation. However, other cytokines and T-cell subsets have also been identified during inflammatory responses in psoriasis. These include Th17 and Th22 cells, which produce large amounts of IL-17 and IL-22, cytokines that have relevant effects on epithelial cells.23 Keratinocytes are strongly influenced by IL-17 and upregulate chemokines and immunomodulatory molecules in response to this cytokine.24 A functional role of Th17 cells, and also Tc17 cells, in psoriasis is suggested by their reduction during successful anti–TNF-α treatment or via blockade with neutralizing mAb in a clinically relevant xenotransplantation mouse model.25 IL-22 also acts pathogenetically in psoriatic skin by inducing proliferation and de-differentiation of keratinocytes, as well as promoting their production of antimicrobial peptides and chemokines, including CXCL8 and CXCL1.26 Binding of IL-22 to its receptor, whose expression in the skin is confined to keratinocytes, mediates epidermal acanthosis through the activation of STAT3. These observations may explain the increased STAT3 expression in the epidermal compartment and the pathogenicity of STAT3 overexpression in the epidermis of transgenic mice (see below).27 The demonstration that T cells

CHAPTER 64  Immunology of Psoriasis are the primary regulators of keratinocyte proliferation and differentiation in psoriasis came from studies showing that supernatants from lesional skin-derived T cells transformed β-integrin+ keratin 1/keratin 10− PCNA− stem cells of patients with psoriasis, but not stem cells of healthy subjects, into PCNA+ active cycling cells. T cell–derived supernatants contained high levels of granulocyte macrophage–colony-stimulating factor (GM-CSF) and IFN-γ, and low levels of IL-3 and TNF-α.28 However, among these cytokines, only IFN-γ affects the proliferation of psoriatic stem cells. In vivo, IFN-γ injection into prelesional psoriatic skin triggers keratinocyte proliferation and plaque development. Considering that IFN-γ is an antiproliferative cytokine and an inducer of squamous differentiation, these latter findings are paradoxical. This discrepancy may reflect an intrinsic defect in the response of psoriatic keratinocytes to IFN-γ, and/ or altered localization and expression of the IFN-γ receptor complex in the epidermis of psoriatic skin. Another T cell–derived cytokine that has been shown to regulate keratinocyte proliferation in psoriatic skin is IL-21, which is mainly released by CD4 T cells and natural killer T cells (NKT cells). IL-21 contributes to epidermal hyperplasia, and neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules. Interestingly, IFN-γ is necessary for IL-21–induced epidermal hyperplasia, while abrogation of IL-21 signals reduces IFN-γ expression in psoriatic T cells.22,24 Finally, the importance of T regulatory (Treg) lymphocytes (Chapter 18) in psoriasis has been examined in the peripheral blood and the inflamed skin of patients. The number of Treg cells (defined by expression of the transcription factor FOXP3) is increased in the peripheral blood of individuals with psoriasis, and this increase is positively correlated with the disease activity index. CD4+CD25+FOXP3+ Treg cells are also present in psoriatic lesions, with more in lesional skin biopsy specimens than those from control or uninvolved skin. However, these Treg cells may be less functional in that Treg cells from both the peripheral blood and psoriatic skin lesions showed reduced ability to suppress effector T cells. This impairment may be dependent on IL-6 as blockade of IL-6 reversed the impairment in suppression observed in cocultures of Treg cells and effector T cells from patients with psoriasis.

Intrinsic Defects of Keratinocytes Are Fundamental for the Amplification of Psoriatic Processes Endogenous defects in keratinocytes may be pathogenically relevant for psoriasis, as shown in mice with engineered epidermal phenotypes. Transgenic animals that overexpress the transcription factor STAT3 or that lack the inhibitor of NF-κB kinase-2 (IKK-2) in their epidermis develop skin lesions that closely resemble human psoriasis.27 Similarly, the abrogation of JunB in keratinocytes triggers a skin phenotype with the histological features of psoriasis, including marked hyperplasia of the epidermis and dense dermal inflammatory cell infiltrates. The hyperplasia observed in these models may, in part, depend on overexpression of S100A8 and S100A9, two antimicrobial peptides with chemotactic activity and a recognized role in keratinocyte maturation and proliferation. The development of psoriatic lesions in mice with an epidermal deletion of STAT3 depends on the presence of activated T cells, whereas the inflammatory responses occurring in the skin of IKK2-transgenic mice are mediated by TNF-α. This implicates an intrinsically dysregulated interrelation between keratinocytes and cells of both the innate and acquired immune

877

response in the pathogenesis of psoriasis. Within psoriatic lesions, alterations are observed in the levels of expression of several growth factors, such as insulin-like growth factor, keratinocyte growth factor (KGF), transforming growth factor (TGF)-α, and amphiregulin, all of which stimulate basal cell proliferation in an autocrine fashion.22 Other cytokines aberrantly elevated in psoriasis include members of the inhibitory TGF-β family, and cytokines that stimulate keratinocyte proliferation and inflammation, such as IL-19 and IL-20.22,24 An important pathogenic role has also been uncovered for IL-36γ, a cytokine abundantly induced by IL-17 in keratinocytes, whose overexpression in mouse skin leads to a disease quite similar to human plaque psoriasis, whereas inhibition in human psoriatic skin ameliorates the inflammation.29 Finally, two anti-inflammatory molecules, suppressors of cytokine signaling (SOCS)1 and SOCS3, are dysregulated in psoriatic keratinocytes.30 These efficiently suppress the IFN-γ– and TNF-α–dependent molecular cascades in keratinocytes, and it has been hypothesized that strengthening of the action of SOCS1 in keratinocytes could be a valid therapeutic approach for treatment of IFN-γ- and TNF-α-dependent skin diseases, including psoriasis.

ON THE HORIZON There have been significant advances in therapies for psoriasis during the past 15 years that strongly reduce both symptoms and relapse rates. New therapeutic approaches include the targeting of interleukin (IL)-17A. Personalized therapies based on the employment of antagonists aligned with elements of genetic risk of the patients will be likely more successful. Newer strategies will also need to focus on treatments that do not require continuous, long-term immune suppression (i.e., strategies to restore immune regulation or tolerance). Future work to identify and clarify risk factors and antigenic triggers for psoriasis may lead to strategies for preventing the disease.

CONCLUSIONS A complex interplay between environmental and genetic factors triggers a cascade of events that leads to the expression of psoriasis. Early upstream events occurring in the disease include activation of DCs and the generation of effector T cells that migrate into the psoriatic skin lesions and expand there. Cross-talk between keratinocytes and immune cells amplifies inflammation and is responsible for chronicity. Recent research has implicated many immunological mechanisms in psoriasis progression, and this has led to the development of new, pathogenesis-based therapies. Although this progress is remarkable, much remains unknown, especially regarding prevention of the condition and how to develop drugs with appropriate risk-benefit and long-term profiles. Future work must take into account these aspects to establish therapeutic and preventive approaches that lead to improved patient outcomes. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Griffiths CE, Barker JN. Pathogenesis and clinical features of psoriasis. Lancet 2007;370:263–71.

878

Part SEVEN  Organ-Specific Inflammatory Disease

2. Bowcock AM. The genetics of psoriasis and autoimmunity. Annu Rev Genomics Hum Genet 2005;6:93–122. 3. Strange A, Capon F, Spencer CC, et al. A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet 2011;42:985–90. 4. Tian S, Krueger JG, Li K, et al. Meta-analysis derived (MAD) transcriptome of psoriasis defines the “core” pathogenesis of disease. PLoS ONE 2012;7:e44274. 5. Onoufriadis A, Simpson MA, Pink AE, et al. Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis. Am J Hum Genet 2011;89:432–7. 6. Nestle FO, Kaplan DH, Barker J. Psoriasis. N Engl J Med 2009;361: 496–509. 7. Albanesi C, Scarponi C, Bosisio D, et al. Immune functions and recruitment of plasmacytoid dendritic cells in psoriasis. Autoimmunity 2010;43:215–19. 8. Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity. Nat Immunol 2004;5:1219–26. 9. Albanesi C, Scarponi C, Pallotta S, et al. Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment. J Exp Med 2009;206:249–58. 10. Nestle FO, Conrad C, Tun-Kyi A, et al. Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J Exp Med 2005;202:135–43. 11. Yao Y, Richman L, Morehouse C, et al. Type I interferon: potential therapeutic target for psoriasis? PLoS ONE 2008;3:e2737. 12. Ketikoglou I, Karatapanis S, Elefsiniotis I, et al. Extensive psoriasis induced by pegylated interferon alpha-2b treatment for chronic hepatitis B. Eur J Dermatol 2005;15:107–9. 13. Lande R, Gregorio J, Facchinetti V, et al. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 2007;449: 564–9. 14. Ganguly D, Chamilos G, Lande R, et al. Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 e TLR8. J Exp Med 2009;206:1983–94. 15. Nestle FO, Di Meglio P, Qin JZ, et al. Skin immune sentinels in health and disease. Nat Rev Immunol 2009;9:679–91. 16. Zheng Y, Danilenko DM, Valdez P, et al. Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 2007;445:648–51. 17. Costantini C, Calzetti F, Perbellini O, et al. Human neutrophils interact with both 6-sulfo LacNAc+ DC and NK cells to amplify NK-derived IFN-γ : role of CD18, ICAM-1, and ICAM-3. Blood 2011;117:1677–86.

18. Cumberbatch M, Singh M, Dearman RJ, et al. Impaired Langerhans cell migration in psoriasis. J Exp Med 2006;203:953–60. 19. Gudmundsdottir AS, Sigmundsdottir H, Sigurgeirsson B, et al. Is an epitope on keratin 17 a major target for autoreactive T lymphocytes in psoriasis? Clin Exp Immunol 1999;117:580–6. 20. Prinz JC. Psoriasis vulgaris—a sterile antibacterial skin reaction mediated by cross-reactive T cells? An immunological view of the pathophysiology of psoriasis. Clin Exp Dermatol 2001;26:326–32. 21. Lande R, Botti E, Jandus C, et al. The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis. Nat Commun 2014;5:5621–36. 22. Albanesi C, Pastore S. Pathobiology of chronic inflammatory skin diseases: interplay between keratinocytes and immune cells as a target for anti-inflammatory drugs. Curr Drug Metab 2010;11:210–27. 23. Eyerich S, Eyerich K, Pennino D, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 2009;119:3573–85. 24. Albanesi C, De Pita O, Girolomoni G. Resident skin cells in psoriasis: a special look at the pathogenetic functions of keratinocytes. Clin Dermatol 2007;25:581–8. 25. Di Meglio P, Villanova F, Navarini AA, et al. Targeting CD8(+) T cells prevents psoriasis development. J Allergy Clin Immunol 2016;138: 274–6. 26. Sestito R, Madonna S, Scarponi C, et al. STAT3-dependent effects of IL-22 in human keratinocytes are counterregulated by sirtuin 1 through a direct inhibition of STAT3 acetylation. FASEB J 2011;25: 916–27. 27. Sano S, Chan KS, Carbajal S, et al. Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model. Nat Med 2005;11:43–9. 28. Bata-Csorgo Z, Hammerberg C, Voorhees JJ, et al. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes. J Clin Invest 1995;95:317–27. 29. Gabay C, Towne JE. Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions. J Leukoc Biol 2015;97: 645–52. 30. Madonna S, Scarponi C, Sestito R, et al. The IFN-gamma-dependent suppressor of cytokine signaling 1 promoter activity is positively regulated by IFN regulatory factor-1 and Sp1 but repressed by growth factor independence-1b and Kruppel-like factor-4, and it is dysregulated in psoriatic keratinocytes. J Immunol 2010;185:2467–81.

CHAPTER 64  Immunology of Psoriasis

878.e1

MULTIPLE-CHOICE QUESTIONS 1. The age of onset of psoriasis is most commonly: A. First three decades of life B. Before age 7 years C. Middle age D. Fifth and sixth decades of life 2. Development of psoriatic lesions at sites of skin trauma is known as: A. Koebner phenomenon B. Nikolsky sign C. Auspitz sign D. Dennie-Morgan lines E. Triple response of Lewis

3. Early pathogenic events of psoriasis includes which one of the following: A. Signal transducer and activator of transcription 3 (STAT3) activation in hyperproliferating keratinocytes B. Recruitment of myeloid dendritic cells C. Targeting of keratinocytes by proinflammatory cytokines D. A cascade of events including formation of complexes formed by keratinocyte-derived nucleic acid and the cathelicidin LL-37

65  Myasthenia Gravis Arnold I. Levinson

Myasthenia gravis (MG) is a disease characterized by weakness of striated muscles. The weakness is caused by impaired neuromuscular transmission resulting from a reduction in the number of receptors for the neurotransmitter acetylcholine (ACh) at the postsynaptic myoneural junction. In most cases, this reduction is the result of the action of anti–acetylcholine receptor (antiAChR) antibodies. The disease occurs with a reported prevalence of 0.5–5/100 000 and an incidence of 0.4/100 000/year. Although MG can occur at any age, it typically presents in the second and third decades of life, with a later peak occurring after age 50 years (late-onset disease). A female preponderance (3:1–4:1) has been reported in the first 40 years of life; thereafter, the incidence is comparable between the sexes.

CLASSIFICATION Patients with MG have traditionally been divided into two categories: those with generalized disease and those presenting with disease limited to the ocular muscles.1 Within these two groups, patients can be further subdivided on the basis of age of onset. Neonatal MG affects 10–20% of offspring born to mothers with myasthenia. Disease manifestations are those of generalized MG (see below) but are transient, dissipating with the metabolism of maternal anti-AChR antibodies that had been transmitted across the placenta during the third trimester of pregnancy. Several congenital myasthenic syndromes have been described. For the most part, these manifest during the neonatal period, persist into adulthood, and are not considered to have an autoimmune basis.2 Juvenile MG is said to occur in those patients who present with disease between 1 year of age and puberty. Apart from the age of onset, juvenile myasthenia behaves like adult MG. Adult patients may present with ocular involvement or signs of more generalized disease. The ocular involvement is characterized by impaired ocular muscle motility and lid weakness, manifesting as diplopia and ptosis, respectively. The vast majority of patients with MG will experience ocular involvement, with roughly 50% of patients presenting with ocular signs at the time of diagnosis. Those generally at risk of disease progression are (i) patients with evidence of subclinical disease on electrophysiological testing of limb muscles; and (ii) patients who have markedly elevated titers of anti-AChR antibodies. Typically, patients with ocular symptoms for >2 years will not progress to a more generalized form of disease. In the generalized disease group, patients can be classified into those with mild, moderate, or severe disease on the basis of clinical activity. Any skeletal muscle group can be affected,

but typically the palatal, pharyngeal, and upper esophageal muscles are involved. This results in dysarthria, dysphagia, and difficulty handling secretions. Involvement of the diaphragm and intercostal muscles produces dyspnea and may lead to respiratory failure. Involvement of the muscles of the extremities and trunk occurs in 20–30% of patients at initial presentation and causes difficulties with activities of daily living. The hallmark of all muscle involvement in MG is its variability over time, with weakness usually exacerbated by repetitive use. Furthermore, within the group of patients with generalized disease, patients are further subdivided into subtypes on the basis of age of onset, for example, early-onset MG (EOMG) and late-onset MG (LOMG) with onset before and after age 50 years, respectively. Moreover, patients can be additionally distinguished by their profiles of serum autoantibodies (see discussion of serum autoantibodies) and the presence/absence of thymic pathology (see discussion of thymic pathology).

CLINICAL PEARLS Telltale Signs of Myasthenia Gravis • Variable muscle weakness • Weakness in cranial nerve distribution • Normal reflexes and sensation

DIAGNOSIS The differential diagnosis is extremely broad, encompassing neuropathies, primary and secondary myopathies, muscular dystrophy, demyelinating disorders, degenerative diseases, cerebrovascular accidents, mass lesions, and infectious diseases. The clinical features that point to a diagnosis of MG include the variable nature of the muscle weakness, normal sensation, and normal deep tendon reflexes. The diagnosis can usually be confirmed by pharmacological and electrophysiological testing. A decremental pattern is characteristically seen following repetitive nerve stimulation (Fig. 65.1), and this pattern is normalized following treatment with the anticholinesterase agent tensilon. Further confirmation rests on detecting anti-AChR antibodies, which are found in 85–90% of patients with generalized disease. In the standard assay, sera are reacted with a nicotinic AChR preparation labeled with 125I-α-bungarotoxin, a snake venom polypeptide that binds irreversibly to the receptor. Bound antibodies are immunoprecipitated by an antiimmunoglobulin reagent or staphylococcal protein A, and the quantity of antibodies detected is expressed in terms of the amount of α-bungarotoxin bound.

879

Part SEVEN  Organ-Specific Inflammatory Disease Safety factor of transmission

Amplitude of EPP

880

Failure at single junctions

Muscle action potential

Minimum current for conduction

1 2 Normal response

3s

Reduced safety margin

1 2 3s Myasthenic response (decremental)

FIG 65.1  Neuromuscular Transmission in Normal and Myasthenic Subjects. With repetitive stimulation there is a reduction in the efficiency of acetylcholine (ACh) release, with a subsequent recovery in efficiency as the train of stimuli continues. Although the endplate potential (EPP) fluctuates at the normal junction, sufficient current is generated to stimulate an action potential of constant magnitude. At the myasthenic junction, however, the amplitude of the EPP in response to a given amount of ACh is reduced. Under conditions of inefficient ACh release, for example, repetitive stimulation, the minimum current for conduction is not generated, resulting in a profile of action potentials that shows a progressive decline or “decrement” with subsequent recovery.

β

NH2

δ

α ε or γ

α Main immunogenic region

10 nm C192 C193 Unfolded

COOH

Carbohydrate

Extracellular Junctional membrane

M3

M2

Acetylcholinebinding site

M1

M4 Cytoplasmic

FIG 65.2  The Acetylcholine Receptor. The subunits of the acetylcholine receptor—α, β, δ, and γ or ε—are arranged like barrel staves around the central ion pore. Each subunit winds through the junctional membrane four times (sites M1, M2, M3, and M4). In the unfolded view of the α subunit, the amino-terminal end of the α subunit is extracellular, where it is accessible to acetylcholine, which binds at the site shown (amino acids 192 and 193). In myasthenia gravis, autoantibodies may bind to various epitopes of all subunits, but a high proportion of autoantibodies bind to the main immunogenic region of the α subunit.

ACHR STRUCTURE The nicotinic acetylcholine receptor (nAChR) is a member of a larger family of ligand-gated ion channels. The muscle-type receptor, which is involved in myasthenia, can be subclassified further into mature junctional receptors and immature, extrajunctional, or denervated receptors. The nAChR at a mature

myoneural junction is composed of four subunits, labeled α, β, δ, and ε (Fig. 65.2). In fetal muscle and adult denervated muscle or nonjunctional membrane, a γ subunit replaces the ε subunit found in mature innervated muscle endplates. This form of the receptor differs from the mature junctional form by its lower density (500 receptors/µm2) and its distribution over most of the surface of the sarcolemma. The immature receptor also has

881

CHAPTER 65  Myasthenia Gravis a lower conductance, a longer open time, a more rapid turnover, and a decreased half-life. The genes for the α, δ, and γ subunits are located on chromosome 2 in humans, and subunits β and ε on chromosome 17. The subunits of the AChR are homologous to each other and to their counterparts across species, with the greatest conservation of sequence being in the α subunit. Two α subunits and one of each of the other subunits are assembled to form an asymmetrical hourglass channel spanning the membrane. Each subunit has a large amino-terminus located extracellularly, four transmembrane regions, and a short cytoplasmic tail formed by a loop between the third and fourth transmembrane domains. The receptor appears as a dimer as a result of disulfide bonding between the δ subunits of two receptors. The two α subunits are not contiguous in each receptor but are separated by another subunit. One ACh-binding site is found on each of the α subunits around the pair of cysteines at amino acids 192 and 193. The binding of ACh to the α subunits is believed to engender a conformational change, possibly resulting in rearrangement of charged groups. The binding of ACh to both α subunits increases the probability of transition of the channel to an open conformation. Binding of curare or α-bungarotoxin to the α subunits blocks this channel. In normal innervated neuromuscular junctions, there are two forms of the AChR, the predominant form having a long half-life, and a small subset that is rapidly turned over. The rapidly turned-over receptors are the precursors of the stable receptors. It is not clear how these two types differ or how they are regulated. The receptors are concentrated at the top of the folds in the muscle endplate, adjacent to the nerve terminus, at a density of 10 000/µm2. This localization reflects the action of agrin, a nerve-derived synaptic organizing molecule. The AChRs are organized into clusters by rapsyn, a 43-kilodalton (kDa) cytoplasmic protein. The clustered AChRs are linked to the cytoskeleton by connections between rapsyn and a dystrophin– glycoprotein complex.

NEUROMUSCULAR TRANSMISSION When an impulse is transmitted along an axon terminal, it results in the release of the neurotransmitter ACh across its presynaptic membrane (Fig. 65.3). ACh diffuses across a 50-nm synaptic cleft, where it interacts with AChRs, which are displayed in greatest density at the tops of the junctional folds of the postsynaptic muscle membrane or endplate. This interaction leads to a local depolarization or endplate potential caused by increased membrane permeability to sodium and potassium. The endplate potential is terminated by acetylcholinesterases, which are present in highest concentrations in the synaptic cleft around the junctional folds. If the summation of endplate potentials attains a prescribed threshold, it produces an action potential that depolarizes the surrounding sarcolemma and causes muscle contraction. In a healthy individual, the arrival of an impulse at the presynaptic membrane of a motor nerve releases considerably more ACh than is required to generate an action potential. This reserve, roughly four times the current needed for propagation of the impulse, is referred to as the safety factor of neuromuscular transmission. Because of the severe reduction in receptor number in MG, the electrical threshold for propagation of an action potential cannot be attained and muscle contraction is prevented. With a less severe reduction in receptor numbers neuromuscular transmission may proceed normally unless the efficiency of presynaptic vesicle release is compromised, as occurs with repetitive use of muscles. The combination of decreasing

AChR ACh vesicle Motor neuron

ACh AChe Voltage-gated Ca– channel Rapsyn LPR4 MuSK Voltage-gated Na– channel

Muscle endplate

FIG 65.3  Schematic Representation of the Myoneural Junction. Vesicles of acetylcholine (ACh) release their contents at active zones across from acetylcholine receptors (AChRs) in response to impulses conducted down nerve axons. ACh diffuses across synaptic cleft and binds to AChRs, with opening of the ion channel and the generation of endplate potential. Action potential is propagated to muscle when sufficient amplitude of summated endplate potentials is attained. MuSK, muscle-specific tyrosine kinase.

availability of ACh and the reduced number of receptors accounts for the characteristic decremental nerve conduction pattern seen on electromyograms of patients with MG following repetitive nerve stimulation (see Fig. 65.1).

IMMUNOPATHOGENESIS OF MG KEY CONCEPTS Involvement of Anti–Acetylcholine Receptor (AChR) Antibodies in the Pathogenesis of Myasthenia Gravis (MG) • AChR antibodies are found in the serum of 85–90% of patients with MG. • Infants born to mothers with myasthenia sometimes develop MG. • Immunoglobulin G (IgG) and complement are deposited at the postsynaptic junction. • Transfer of serum IgG from patients with MG to mice induces neuromuscular blockade.

882

Part SEVEN  Organ-Specific Inflammatory Disease

Anti-AChR antibodies are detected in 85–90% of MG patients and are responsible for the impaired neuromuscular transmission.3 There are several lines of evidence to support this contention. Immunoglobulin G (IgG), along with C3 and the terminal attack complex (C5–C9), is deposited at AChR-containing areas of the postsynaptic membrane, and anti-AChR/AChR complexes can be extracted from the muscles of patients with MG. Transfer of myasthenic serum from mother to fetus, or from human to mouse, results in symptoms or signs of myasthenia in the recipient. Plasmapheresis, which decreases anti-AChR antibody levels, is associated with clinical improvement.

Properties of Anti-AChR Antibodies and Characterization of B-Cell Epitopes Anti-AChR antibodies are produced by a small subset of B cells in affected people. The frequency of IgG-producing AChR-specific peripheral blood mononuclear cells is estimated to be 1 in 15 000–70 000. IgG anti-AChR–secreting cells are also found in the peripheral blood of healthy volunteers, albeit in much lower numbers. The proportion of immunoglobulin producing AChRspecific B cells or plasma cells is greater in the germinal centers of hyperplastic thymuses, but still only 1 in 1000–10 000 antibodies produced is AChR specific. Anti-AChR antibodies are predominantly IgG1 and IgG3, but IgG2 and IgG4 isotypes have also been found. IgA and IgM anti-AChR antibodies are present in some patients, but never in the absence of IgG anti-AChR antibodies. The IgA and IgM anti-AChR antibodies tend to appear in patients whose disease is of longer duration and greater severity and in association with high IgG anti-AChR titers. The pathogenic anti-AChR antibodies in MG are thought to be directed to conformationally dependent structures. Immunization of animals with irreversibly denatured AChR leads to the formation of anti-AChR antibodies capable of binding to native AChR, but the antibodies are not capable of causing disease. This observation indicates that conformationally dependent epitope(s) are important in the induction of disease. Many of the anti-AChR antibodies are directed against the α subunit, particularly to a small region on the extracellular portion referred to as the main immunogenic region4 (see Fig. 65.2). Approximately 60% of the anti-AChR antibodies are directed against this region, which encompasses a set of overlapping epitopes clustered around amino acids 67–76 of the α subunit.5 The reason for the predominant role of the α chain in the antibody response in myasthenia is not known. Not all disease-producing antibodies in humans or rats appear to be directed to this region. Many patients also have antibodies recognizing the γ-containing embryonic form of AChR. This observation has spawned speculation about a nonmuscle source of sensitization.

Anti-AChR Antibody Levels and Relationship to Disease Activity The relationship of anti-AChR antibody and disease activity in MG is complicated. In general, serum levels of anti-AChR antibody or anti-AChR/AChR complexes correlate poorly with disease severity. Among patients within one clinical grade, anti-AChR antibody levels can vary by several orders of magnitude. In addition, approximately 10–15% of patients with clinical MG have no anti-AChR antibody by standard assays. Anti-AChR antibodies are more likely to be present if the disease is generalized or severe. In addition, in an individual patient an increase or

decrease in anti-AChR antibody levels often accompanies deterioration or improvement, respectively, in clinical activity. As the quantity of anti-AChR antibodies produced does not fully explain disease severity, studies have also focused on qualitative differences in these antibodies among different patients. These studies, however, have not been able to distinguish properties of anti-AChR antibodies that lead to greater pathogenicity. Differences in specificity and avidity of binding to AChR have not been associated with particular functional effects or disease severity. Anti-AChR antibodies that bind or compete for the same region of AChR can have different functional effects (see anti-AChR blocking antibody discussion). Anti-AChR antibodies show extensive heterogeneity by isoelectric focusing, but this characteristic also does not correlate with pathogenic potential. Patients previously classified as seronegative have been found to have low-affinity AChR-specific IgG antibodies when their sera were tested with very sensitive assays that rely on tissue substrates on which the receptors are aggregated.6,7 Antibody binding was facilitated by the high concentrations of AChRs, which compensate for the low affinity of the autoantibodies. Like the anti-AChR antibodies in classic anti-AChR antibodypositive MG, these antibodies belong largely to the IgG1 subclass. Like the conventional antibodies, the low-affinity antibodies also bind complement. As noted above, 10–15% of MG patients are persistently antibody negative. Approximately 50% of these patients have been discovered to have serum IgG antibodies specific for musclespecific tyrosine kinase (MuSK).7,8 This skeletal muscle receptor tyrosine kinase is activated by agrin and is critical for formation of the neuromuscular junction. Originally, anti-MuSK antibodies were found in patients with marked facial and bulbar weakness, including tongue weakness and respiratory involvement with relative sparing of upper- and lower-extremity muscles. Ophthalmoparesis was also seen but was not a first symptom in at least one series of patients. Subsequently, it has become apparent that patients with anti-MuSK antibodies can also have a more traditional clinical phenotype similar to that seen with anti-AChR patients. Patients often respond poorly to anticholinesterase agents and benefit greatly from plasmapheresis but only modestly from intravenous immunoglobulin (IVIG). They typically lack thymus pathology and, not surprisingly, thymectomy is not typically beneficial. Most anti-MuSK antibodies are of the non–complement-fixing IgG4 isotype. Anti-MuSK antibodies do not cause destruction of the muscle endplate, as might be expected because of their inability to activate the classical complement pathway and reduced capacity to bind activating Fcγ receptors on monocytes and macrophages. However, they have been shown to affect AChR clustering on cultured myotubes. The clinical relevance of these autoantibodies is underscored by the appearance of a myasthenic illness in animals either immunized with MuSK or infused with serum antibodies from patients with anti-MuSK antibody–associated MG. IgG antibodies specific for lipoprotein-related protein 4 (LRP4) have also recently been discovered in the sera of patients with anti-AChR antibody–negative MG.7,9 LRP4 is an important component of the neuromuscular junction, which serves as a receptor for agrin and which is required for agrin-induced activation of MuSK and AChR clustering. The prevalence of reactivity to LRP4 varied from 2–50% of patients who are negative for anti-AChR and MuSK antibodies (double seronegative), with the differences possibly being related to geography or ethnicity.

CHAPTER 65  Myasthenia Gravis Anti-LRP4 antibodies were found in the sera of 3% of patients with anti-MuSK antibodies in one study but otherwise were limited to double-seronegative (anti-AChR−/anti-MUSK−) patients. Anti-LRP4 antibodies were not present in the sera of a large number of patients with other neurological diseases with the exception of neuromyelitis optica spectrum disorder, where 12.5% of patients had anti-LRP4 antibodies. If larger studies demonstrate that anti-LRP4 antibodies are found in patients with MG but without anti-AChR or MuSK antibodies and are rare in other neurological disorders, this test could become important in the diagnosis of MG. MG sera also contain antibodies reacting with the ryanodine receptor.7 These receptors, which are critically involved in muscle contraction, are Ca2+ release channels located in the sarcoplasmic reticulum of striated muscles. Antibodies to ryanodine receptors are found in 50% of patients with MG who have thymoma, and patients with high levels have a worse prognosis than do antibody-negative patients with MG who have thymoma. In vitro studies have suggested a pathogenic role for these autoantibodies in MG. Antirapsyn antibodies have been detected in a small subset of patients with MG.7 Seropositive patients with MG are indistinguishable from seronegative patients with regard to clinical and laboratory features of disease. The presence of antirapsyn is not specific for MG, having been detected in the sera of an occasional patient with MS and a majority of the patients with lupus tested. Antiagrin antibodies have recently been reported, often in MG patients with anti-AChR or anti-MuSK antibodies and occasionally in patients with MG who are “triple seronegative” (AChR− MuSK− LRP4−).7 Studies of larger numbers of patients will be necessary to determine the prevalence of antiagrin antibodies in triple seronegative MG patients and their degree of specificity for MG. Patients with MG associated with thymoma show distinct patterns of antibody production. Almost all patients with thymoma are anti-AChR antibody positive, and most produce antistriational antibodies.9 These latter antibodies react with titin, a giant filamentous protein of striated muscle. Titin filaments are involved in muscle assembly and contribute to the muscle’s ability to recoil following stimulation. Such antistriational antibodies are also found in approximately 50% of the sera of older patients with MG who have thymic atrophy but not thymoma; however, they are not frequently detected in patients with early-onset disease and thymic hyperplasia. The finding of antistriational antibodies in a patient with MG who is less than 40 years old strongly suggests the presence of thymoma. There is no evidence that antistriational antibodies are involved in muscle weakness.

Pathogenic Effects of Anti-AChR Antibodies KEY CONCEPTS • Effects of anti–acetylcholine receptor (AChR) antibodies in myasthenia gravis pathology • Reduced number of receptors • Widening of the synaptic cleft • Distorted geometry of the synaptic membrane • Mechanism of damage • Complement-dependent damage to muscle endplate • Enhanced rate of AChR degradation • Block cholinergic binding sites

883

Complement-Mediated Damage The critical problem in MG is the anti-AChR antibody–mediated reduction in the number of nAChRs at the myoneural junction. There are several possible mechanisms by which the anti-AChR antibodies could lead to impaired neuromuscular transmission.10 Ultramicroscopic studies show marked destructive changes in some endplates, particularly at the peaks of the postsynaptic folds, where AChR is usually present in the greatest concentration. The architecture of the muscle endplate is simplified, with loss of junctional folds and widening of the synaptic cleft that contains membrane debris. C3, C9, and the membrane attack complex are deposited at the muscle endplate, suggesting a role for complement in membrane destruction.11 Indeed, in many patients, anti-AChR antibodies can fix complement in vitro when bound to skeletal muscle and can damage cultured rat myotubes with a resultant decrease in AChR content. Although antibody-directed, complement-mediated destruction is important in the pathophysiology of MG, it is not the entire story. The rapid clinical improvement in MG following certain therapeutic interventions and the lack of destructive changes in many neuromuscular junctions of symptomatic areas despite prominent immunoglobulin deposition suggest that a more readily reversible process is also likely to be involved in the neuromuscular block. Acceleration of AChR Degradation In vitro and in vivo studies have shown that anti-AChR antibodies can accelerate the rate of degradation of extrajunctional and junctional receptors, respectively.12 This reaction is complement independent and results from the endocytosis of AChRs via shallow depressions, presumably clathrin-coated pits. Other membrane receptors are not affected. Both stable and rapidly turned-over receptors appear to be affected, thereby explaining the greater than expected antibody-mediated loss of AChRs observed at neuromuscular junctions. The reaction requires cross-linking of adjacent AChRs, as it can be mediated by F(ab′)2 fragments, but not Fab fragments, of anti-AChR antibodies. The effect of antibody on the synthesis of new AChR is controversial. Receptor Blockade The inhibition of ACh binding has been assessed by studying the effects of MG serum on the binding of the neurotoxin α-bungarotoxin to AChRs. Such blocking antibodies are found in a variable number of MG sera. Blockade has been generally attributed to steric hindrance of the ligand-binding site, rather direct binding to the ACh-binding site.13 The importance of these antibodies in the pathophysiology of MG remains unclear. However, in one study the functional ability of an individual serum to accelerate degradation and cause blockade of AChRs paralleled most closely the clinical status of the patient. The in vivo significance of such antibodies has also been demonstrated by passive transfer of certain rat monoclonal anti-AChR antibodies into chicks. Complete paralysis was observed within 1 hour of the transfer, presumably before there was time for complementmediated damage. It has been reasoned that in patients with MG, such blocking antibodies could further diminish synaptic function already decreased owing to complement-mediated damage or accelerated receptor degradation. This could result in acute clinical deterioration or a rapid clinical improvement after plasmapheresis, before the repair of damaged membrane and regeneration of new AChRs.

884

Part SEVEN  Organ-Specific Inflammatory Disease

Role of T Cells An overwhelming body of data indicates that anti-AChR antibody production in patients with MG and rodents with experimental autoimmune myasthenia gravis (EAMG) is dependent on the activity of CD4 T cells. Researchers have used freshly isolated CD4 T cells, T-cell lines and T-cell clones stimulated with AChR purified from the electric organs of Torpedo californica (T-AChR), recombinant human AChR, human AChR concentrated on immunomagnetic beads, and synthetic AChR peptides. Most of the T cells derived from patients with MG have responded to the complete AChR or the α subunit, but responses to the δ, ε, and γ subunits have also been demonstrated.14 Unlike EAMG, a T cell–immunodominant epitope has not been found in humans. However, T cells from most patients recognize a limited number of AChR sequences. In an individual patient, T cells reactive with a particular AChR epitope can show limited T-cell receptor Vβ (TCR Vβ) usage, but the same TCR Vβ usage has not been observed for different patients. A range of major histocompatibility complex (MHC) class II molecules can present AChR epitopes. This is not surprising, given the degenerate binding capabilities of MHC molecules needed to present an extremely large and diverse group of peptides to the immune system. T cells from normal controls respond to some of the same epitopes to which MG patients’ T cells respond, albeit in smaller numbers and less vigorously.

EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS The serendipitous discovery that rabbits injected with purified AChR developed not only anti-AChR antibodies but signs of MG provided the evidence for the pivotal role of antibodies in this disease and paved the way for the development of an experimental model of human MG.15 EAMG has been studied in a number of different animal species.16 It is generally induced by immunizing animals with AChR purified from the electric organs of T. californica emulsified in adjuvant. Disease can also be passively transferred by immunoglobulin from affected animals or from patients with myasthenia. Animals suffer from fatigue, hypoactivity, weight loss, paralysis, difficulty breathing, and dysphagia; these signs are reversible with anticholinesterases. There is a decremental response to repetitive nerve stimulation on electromyography in EAMG as in MG. Anti-AChR antibodies are present, and most are directed against the main immunogenic region (see Fig. 65.2). They are deposited along with complement at the muscle endplate. Similar to the pathology in humans, there is simplification of the muscle endplate and a loss of AChR content of muscles in the experimental disease. A major difference between the induced rodent models of EAMG and spontaneous human MG is the absence of thymic pathology in the former. In murine models, there are disease-resistant and diseasesusceptible strains. The MHC determines this susceptibility, in part, with specific H-2 alleles being associated with susceptibility or resistance. However, even in the high-responder strains, only 50–70% of immunized animals manifest disease. This lack of concordance remains to be fully explained. Differences in the level of complement activation and the physiological safety factor of neuromuscular transmission may partly account for the variability in disease expression. Most immunized animals demonstrate anti-AChR antibodies, whether or not they develop EAMG. Induction of pathogenic

antibody production requires the integrity of the threedimensional structure of the receptor, as immunization with denatured AChR elicits antibody production but not disease, even in susceptible animals. Anti-AChR antibody levels correlate with anti-AChR/AChR complexes and with the AChR content of the muscle, but not with clinical disease severity. No characteristic feature distinguishing disease-producing and nonpathogenic autoantibodies has been defined. Antibodies in rats with and without disease are predominantly of the same immunoglobulin isotope and subclass, express similar clonotypic heterogeneity, and show similar avidities. The percentage of antibodies that recognize the native receptor (as opposed to the foreign eel receptor immunogen) is low (0.2–2%) but similar between the symptomatic and the asymptomatic mice. The percentage of antibody directed against the main immunogenic region of AChR is also similar. The antibodies that develop in asymptomatic animals can bind and cause a decrease in receptor half-life, as well as those that develop in paralyzed animals. In fact, EMG abnormalities can be demonstrated in mice that appear disease free. An analogous situation in humans may be the high frequency of single fiber EMG abnormalities in asymptomatic family members of patients with MG. Importantly, antibodies from affected animals will cause disease in resistant strains, indicating that disease differences between strains is not caused by differences in the AChR receptor itself. Although EAMG is mediated by autoantibodies, it is a T cell–dependent process. Examination of T-cell responses in rats and mice has shown that EAMG requires the action of CD4 T cells. These cells recognize immunodominant T-cell epitopes located on the α subunit. The T-cell responses to the immunodominant epitope in rats involve a variety of TCRVβ and Jβ gene segments (Chapter 4). However, in susceptible C57BL/6 mice, there is a predominant use of the TCRVβ6 family in the CD4 T-cell response. CD4 T cells in susceptible and resistant strains recognize different immunodominant AChR α-chain epitopes.17 Early reports suggested that AChR-reactive T-cell clones did not fit neatly into T-helper-1 (Th1) and Th2 subsets. However, later studies suggested that interferon-γ production by Th1 cells is essential for the development of disease and that IL-4 may subserve a protective role.16,18 In addition to interferon-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, IL-18, IL-1, IL-5, and IL-10 have been reported to promote the development of EAMG. However, a more recent publication indicated that C57BL/6 mice genetically deficient in IL-12/IL-23 and IFN-γ are susceptible to experimental EAMG, suggesting a pathogenic role of non-Th1 cells. This result led the authors to suggest that AChR-specific Th17 cells may also contribute to the pathogenesis of this experimental autoimmune disease.19 Subsequently, Th17 cells were, indeed, shown to be important in the pathogenesis of EAMG.20 The roles of a number of costimulatory factors have also been investigated in EAMG. CD28/B7 and CD40/CD40L appear to be required for the development of the primary immune response to AChR. An interaction between ICOS and B7RP-1 may play an important role in the secondary response and/or in the maintenance of the immune response to this autoantigen.

THE THYMUS IN MYASTHENIA GRAVIS Several lines of evidence have suggested that the thymus is directly linked to the pathogenesis of MG.

CHAPTER 65  Myasthenia Gravis KEY CONCEPTS Pathogenic Roles of the Thymus in Myasthenia Gravis (MG) • Pathological • 65–75% of patients with MG have follicular hyperplasia with germinal centers • 10% have thymoma • Clinical • Improvement following thymectomy • Immunological • Acetylcholine receptor (AChR) subunits expressed on myoid cells and thymic epithelial cells • AChR-reactive T and B cells localized in the thymus • Increased thymic AChRα expression • Anti-AChR antibody secreted by thymic B-lineage cells • Decreased thymic T regulatory cell (Treg) function • Interleukin (IL)1-β, IL-6, CXCL13, CCL21, and B cell–activating factor (BAFF) overexpressed in thymus

Thymic Pathology Interest in a primary role for the thymus in the pathogenesis of MG has been fueled by pathological, clinical, and immunological lines of evidence, although the nature of its involvement remains to be elucidated.21-24 The thymus is pathologically abnormal in 80–90% of patients with MG. The majority of patients (65–75%) have thymic follicular hyperplasia with germinal center formation. The architecture of the hyperplastic thymi is generally preserved, with well-demarcated cortical and medullary regions. However, the medulla is crowded by numerous germinal centers that display the architectural features and cellular constituents of germinal centers in the secondary follicles of peripheral lymph nodes from normal individuals. Although these germinal centers in patients are generally thought to occupy an intraparenchymal position, some observers feel that they may actually lie extraparenchymally in the perivascular space. Thymomas are seen in approximately 10% of patients with MG who tend to be older than those with hyperplastic thymi.21-24 The thymomas are characterized by a loss of cortico-medullary demarcation and consist largely of neoplastic epithelial cells admixed with thymocytes. The affected epithelial cells belong to the cortical epithelial compartment, and the thymocytes have the immunophenotypic properties of normal immature cortical thymocytes. Further evidence for a pathogenic role of the thymus comes from the results of empiric trials of thymectomy. Despite the absence of controlled clinical trials until recently,25 there was general agreement in the past that removal of the thymus, particularly in young patients with follicular hyperplasia, leads to clinical improvement. The underlying basis of this improvement remains unknown.

Intrathymic Factors Possibly Contributing to Local Anti-AChR Antibody Response The MG thymus, particularly hyperplastic thymus, is characterized by several unique features that strongly suggest its primary role in the immunopathogenesis of MG.21-24 The thymus contains important constituents necessary for and indicative of an immune response directed against nAChRs. There is considerable evidence that resident cells in the thymus, including myoid cells and medullary epithelial cells, express various subunits of AChR

885

including the α subunit. Additional factors found uniquely in hyperplastic MG thymus include increased expression of chemokines that attract immigrant CD4 T and B cells (CXCL13, CCL21); the presence of nAChR-reactive B and CD4 T cells; cytokines that can facilitate B-cell activation, differentiation, and survival (IL1β, IL6, APRIL, BAFF); anti-AChR antibody–secreting plasma cells; and possibly decreased CD4+CD25+ T regulatory cell function.21-24 As yet, it is not known whether or how such perturbations of the thymus lead to a breach in self-tolerance and the induction of anti-AChR antibodies, although there is mounting evidence that an antecedent inflammatory reaction in the thymic medulla may serve as the initiating trigger. An understanding of this enduring mystery likely holds the key to unlocking the immunopathogenesis of this disease. The reader is referred to other works for an in-depth discussion of this topic.22,24,26

ETIOLOGICAL FACTORS Genetic Factors As in most autoimmune diseases, the MHC represents an important genetic susceptibility locus for the development of MG. Studies indicate that the extended human leukocyte antigen (HLA)-A1-B8-DR3 haplotype is associated with EOMG and hyperplastic thymus in Caucasian individuals. This haplotype has been associated with the development of other autoimmune disorders. An association with HLA-B7-DR2, although weaker, has also been described in patients with onset of MG occurring after the age of 40 years and associated with atrophic thymic histology. However, the strongest association, focused on a Norwegian population of patients with late-onset MG, was recently shown to be the DRB1*15:01 allele. A recent study found a strong association between the HLA-DQ5 allele and patients with anti-MuSK antibody–positive MG. Interestingly, the MuSKpositive T-cell repertoire appears to be skewed to the usage of this HLA MHC class II allele. In murine studies, the MHC class II molecules I-Ab and I-Ek have been associated with susceptibility to EAMG.16 In EAMG, the permissive MHC class II molecules are capable of binding AChR peptides that are recognized by antigen-specific CD4 T cells. Studies have also addressed the potential genetic contributions of other immune system–related genes in the pathogenesis of MG. An association has been reported for a particular IL-1β allele and MG and increased serum levels of this cytokine have been reported. This was most pronounced in patients who lacked disease-susceptible HLA genes. Several groups have reported an association between MG and the presence of particular TNF-α polymorphisms. The expression of a high-transcription TNF-α allotype, TNF-α-308 allele 2, correlated with EOMG. In this regard, patients with MG demonstrate increased serum levels of TNF-α and their peripheral blood mononuclear cells display increased expression of TNF-α messenger RNA (mRNA). Polymorphisms in the IL-10 promoter region have been reported to be associated with distinct patterns of thymic histology. No correlations have been made between IL-4 alleles and MG. Allotypic markers on IgG and FcγRIIA receptors have been associated with the coexistence of MG and thymoma. A single nucleotide polymorphism in the gene encoding the intracellular tyrosine phosphatase PTPN22, which has been associated with the risk of developing other autoimmune diseases, has also been identified in both a subgroup of patients with anti-titin antibody– positive nonthymomatous MG and patients with thymomatous

886

Part SEVEN  Organ-Specific Inflammatory Disease

MG.27,28 The risk of developing MG has also been linked to a polymorphism in the promoter region of CHRNA1, the previously mentioned gene that encodes the α subunit of AChR and CHRND, the gene that encodes the δ subunit.22 It has been proposed that such polymorphisms in genes encoding autoantigens cause reduced expression of AChRα on thymic medullary epithelial cells, thereby impairing central deletion of autoantigen-specific thymocytes. Additional MG-associated risk factors include polymorphisms in CTLA4, type II, IFNII, IL12, CD86, AKAP12, VAV1, BAFF, TCF19, and TNIP genes.22,23

Exogenous Factors Whether or not sensitization to AChR occurs in the thymus or the periphery, the stimulus for this autoimmune response remains a conundrum. Moreover, it remains to be determined whether the stimulus is a self-antigen (AChR) or a foreign antigen that mimics the receptor’s molecular structure. In this regard, several examples of molecular mimicry between AChRα chain and other molecules have been reported. Studies carried out with certain monoclonal anti-AChR antibodies demonstrated epitope sharing between the receptor and several bacteria, including Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, and Yersinia enterocolitica.29 However, for the most part, no difference was observed in the binding of polypeptides from these organisms by either sera from patients with MG or control sera. A computer search of protein banks revealed a sequence homology between AChRα chain and a short peptide in herpes simplex glycoprotein D,30 although the significance of this finding is unknown. Finally, similarities were reported between idiotypic determinants on anti-AChR antibodies and antibodies reactive with α1,3-dextran. Interestingly, antidextran antibodies were detected in approximately 13% of patients with MG but rarely in normal controls.31 α1,3-Dextran is found in the cell walls of several common enteric pathogens and thus represents a potential ubiquitous source of immunogen. This type of idiotypic network connectivity led the investigators to postulate that an unregulated antiidiotypic response to anti-α1,3-dextran antibodies might lead, in certain individuals, to an anti-AChR antibody response. Unfortunately, there has been no follow-up to these observations. A striking association has been reported between the development of MG and treatment with the drug penicillamine,32,33 particularly in individuals with HLA-DR1. MG developed in patients with rheumatoid arthritis and patients with Wilson disease treated with this agent. After discontinuation of penicillamine, resolution of MG symptoms was reported in some patients but not others. Penicillamine treatment was associated with the development of anti-AChR antibodies that appeared to have the same type of specificity profile as found in idiopathic myasthenia. Additional evidence suggests that penicillamine may directly interfere with neuromuscular transmission. Although penicillamine has been shown to have diverse effects on the immune response in the normal host and has reactive sulfhydryl groups capable of modifying self-antigens, its role in the development of MG remains to be determined. Currently, considerable attention is being focused on the role of epigenetic mechanisms to explain how environmental factors may promote the development of autoimmune diseases. Although some key epigenomic mechanisms, such as DNA methylation, histone acetylation, and microRNAs, have been demonstrated to possibly play a pathogenic role in several autoimmune disorders, the analysis of these factors is at a rudimentary stage in MG. To date, there is evidence to support an association between miR155

and the development of EOMG and human MG. An interesting story has emerged concerning miR150-P. Increased levels of this miRNA were found in the serum of patients with MG, and these levels fell in association with the clinical improvement that followed thymectomy.34 A decrease in miR320a was observed in the peripheral blood mononuclear cells of a cohort of Chinese patients.22 This finding was associated with increased levels of a number of proinflammatory cytokines. Further studies of the role of miRNAs will need to be conducted to elucidate whether they contribute to the immunoregulatory abnormalities seen in MG.

TREATMENT OF MYASTHENIA GRAVIS THERAPEUTIC PRINCIPLES • Anticholinesterase agents • Corticosteroids • Thymectomy • Plasmapheresis • Immunosuppressive agents • Intravenous immunoglobulin

Therapeutic intervention in MG usually proceeds in a stepwise manner, beginning with anticholinesterase agents.35 Most of the experience dealing with therapeutic modalities is based on treating patients with anti-AChR-associated MG.

Anticholinesterases Anticholinesterases are the mainstay of treatment. These agents protect acetylcholine from hydrolysis by cholinesterase, thereby increasing the amount of neutrotransmitter and the number of contacts with the reduced number of receptors at the postsynaptic junction. This, in turn, raises the probability of attaining the necessary threshold for neuromuscular transmission. In addition, some of the anticholinesterase agents have a direct agonist effect at the postsynaptic junction. The three most popular agents in this group are neostigmine bromide (Prostigmin), pyridostigmine bromide (Mestinon), and ambenonium chloride (Mytelase). Although there are only slight differences between these agents, Mestinon remains the most commonly used. It has an onset of action of 30–60 minutes, peak action at about 2 hours, and loss of activity after 4 hours. Adverse effects of these agents are caused by excessive stimulation of nicotinic and muscarinic receptors. Auxiliary drugs that have been purported to have a salutary effect on neuromuscular transmission are ephedrine and xanthine derivatives (theophylline), which are thought to increase the presynaptic release of ACh. The minimal effect of their added benefit has not warranted their common usage. As mentioned previously, anticholinesterase agents often are not beneficial in patients with anti-MuSK-antibody–associated MG and may even exacerbate weakness. These agents neither induce sustained remission of symptoms nor impede disease progression.

Thymectomy Another mainstay in the therapy of the adult with generalized MG is thymectomy.36,37 The benefit is greatest in younger patients and those with thymic hyperplasia, although many centers include older patients as well. Over many years, despite the absence of a controlled study, there was general agreement that removal of the thymus leads to clinical improvement, particularly in young patients with follicular hyperplasia. In one study, 90% of patients were asymptomatic or in complete remission within a few years of thymectomy, and 46% were off all medications. An international

CHAPTER 65  Myasthenia Gravis multicenter controlled trial completed recently compared clinical outcome in patients with MG treated with corticosteroid versus those treated with corticosteroid plus thymectomy and those receiving thymectomy demonstrated a more favorable clinical outcome.25 With improvements in preoperative care, anesthesia, surgical technique, and postoperative care, thymectomy has become a safe procedure, but its value and safety in children and older patients is less well established. There is still some controversy over what represents the best surgical procedure. The mechanism responsible for the salutary effect of thymectomy remains to be elucidated. No obvious effects on immunoregulatory mechanisms have been demonstrated, although anti-AChR titers tend to fall months after the procedure. Thymectomy is also the recommended treatment for patients of all ages suspected of having thymoma.

Corticosteroids Corticosteroids are used in patients with generalized MG who fail to respond to anticholinesterase agents or thymectomy and in patients needing optimization of their clinical condition in preparation for thymectomy.38 They are generally not used as first-line agents to replace thymectomy but are used in patients with ocular myasthenia who fail to respond to anticholinesterases. Corticosteroids are initially given on a daily basis, with therapy initiated in a hospital. This cautious approach is followed because of the fear of clinical deterioration that may occur in some patients during the introduction of corticosteroids. This concern resulted in some groups advocating initiation of alternate-day therapy, which is not typically associated with clinical deterioration and can be carried out on an outpatient basis. Daily corticosteroids are usually started in patients with generalized MG at a dose >1 mg/kg prednisone. Patients should be continued on this dose until clinical improvement is maintained for several days, then gradually weaned off, and switched to alternate-day therapy. With improvement sustained over several months, an effort should be made to reduce the dose (usually in 5-mg decrements) administered on alternate days. Although a Cochrane review underscored the dearth of controlled trials, the improvement rate is generally estimated to be 60–90%.39 Complete remission is rare, and most patients will require some dose of steroids indefinitely. The physician should be alert to the possibility that anticholinesterase requirements may decrease as the patient responds to corticosteroids.

Plasmapheresis Plasmapheresis has enjoyed popularity since its introduction as an auxiliary treatment modality in patients with generalized MG in 1976, particularly as a temporizing measure.40 It appears to be most beneficial in patients in myasthenic crisis and in those experiencing progressive deterioration despite treatment with anticholinesterases and corticosteroids. Plasmapheresis has also proved to be useful in preparing patients for thymectomy when their course is complicated by involvement of the bulbar and respiratory musculature. Such patients may also require shortterm plasmapheresis during the postoperative period. Plasmapheresis also appears to be particularly efficacious in anti-MuSK antibody–associated disease. There is no long-term benefit of plasmapheresis when added to prednisone. Although there are no hard and fast rules, the average exchange is 1–2 L/day for 7–14 days. Improvement is usually observed within a few days of concluding the treatment course, although patients in crisis often benefit more quickly. The mechanism of

887

action most likely involves the removal of the pathogenic autoantibody, as a reduction in titer of anti-AChR antibody correlates with clinical improvement. However, it is also possible that the removal of other phlogistic humoral factors contributes to clinical efficacy.

Intravenous Immunoglobulin The efficacy of IVIG in the treatment for MG was suggested by several uncontrolled clinical trials. Interest in the use of this biological grew out of its demonstrated efficacy in other autoimmune diseases, most notably autoimmune thrombocytopenia. Subsequent randomized double-blind placebo-controlled trials provided proof of clinical efficacy. Conventional dosing is infusion of 2 g/kg divided over 5 days, although some practitioners prefer to administer this dose over 2 days. In a randomized, controlled trial a total dose of 1 g/kg was found to be as efficacious as a dose of 2 g/kg. IVIG therapy is generally associated with rapid clinical improvement in responsive patients, independent of whether they had undergone thymectomy or were being treated concurrently with corticosteroids or immunosuppressive agents. In some patients, improvement was sustained over a period of several weeks. Improvement has not always been accompanied by a consistent reduction in anti-AChR antibody titers. In general, IVIG and plasmapheresis appear to be equivalent in efficacy.41 However, there is a general impression that IVIG is preferable because of better tolerance and less cost. The mechanism(s) of this apparent salutary effect is unknown, although there is evidence IVIG contains antibodies directed against the idiotypes of anti-AChR antibodies.

Immunosuppressive Agents Immunosuppressive drugs have been tried primarily in patients who have failed treatment with anticholinesterases, thymectomy, plasmapheresis, and corticosteroids.42 Most of the experience has been obtained with azathioprine, which has strong antiinflammatory effects as well as immunosuppressive activity. The dose of azathioprine has varied between 1 and 3 mg/kg/day, with improvement seen between 5 and 20 weeks. The drug is usually started at a lower dose and escalated weekly to achieve the maintenance dose. The patient should be followed with complete blood counts, particularly during the initiation of therapy, as azathioprine has a suppressive effect on the bone marrow. A white blood cell count below 2500 or a neutrophil count below 1500 should prompt a reduction or termination of the dosage. The results of a randomized double-blind placebo-controlled trial indicated that the addition of azathioprine (2.5 mg/kg) to alternate-day prednisolone was associated with a reduction of the prednisolone dose, fewer treatment failures, longer remissions, and fewer side effects.43 There is considerably less experience in the treatment of steroid-unresponsive patients with cyclophosphamide, another powerful immunosuppressive agent. It is associated with more adverse effects and does not appear to offer any significant advantage over azathioprine when used in standard dosing regimens. High-dose intravenous cyclophosphamide therapy for patients with MG refractory to conventional immunosuppressive agents has been investigated as an approach to immunoablate bone marrow and allow for subsequent repopulation by endogenous stem cells. Although durable responses were seen in some patients in an early trial, this regimen is not readily available and should only be utilized in refractory patients under treatment in specialized centers.

888

Part SEVEN  Organ-Specific Inflammatory Disease

Methotrexate has been used in some uncontrolled studies, but there is no information to indicate that it is more efficacious or safer than azathioprine and its onset of action may take as long as several months. As is true for corticosteroid therapy, it is the rare patient who enjoys a permanent remission following institution of immunosuppressive therapy, and those who show some improvement often require treatment indefinitely. Cyclosporine, a potent immunosuppressive agent, has been investigated because it interferes with IL-2–mediated T-cell proliferation and thus would be expected to interfere with the generation of T cells that would “help” the anti-AChR antibody response. A retrospective study suggested that cyclosporine provided benefit in patients whose disease was refractory to corticosteroids and azathioprine. Serious renal toxicity and treatment withdrawal, which plagued earlier studies, were reduced by careful selection of patients. In a 12-month European trial, cyclosporine appeared to be as efficacious as azathioprine in producing clinical improvement. Tacrolimus has a similar mechanism of action to cyclosporine. When used in low dosage, it has proved as effective as cyclosporine as a corticosteroid-sparing agent with fewer side effects. Nevertheless, most practitioners reserve these agents for use in patients whose disease is refractory to the combination of azathioprine and prednisone. Mycophenolate mofetil (MMF), another immunosuppressive agent that affects both T and B cells, was widely touted following the completion of early trials as a steroid-sparing agent in patients with MG. That experience suggested that it was effective 70–75% of the time, although probably less so in refractory MG. It has an acceptable safety profile with adverse effects largely related to gastrointestinal intolerance. Benefit may require many weeks of administration. However, two randomized, placebo-controlled trials have challenged the early optimism. In one, the addition of MMF treatment at the initiation of a 36-week schedule of prednisone tapering was not found to be superior to placebo in maintaining myasthenia control.44 In the second, the coadministration of MMF and prednisone provided no better control of myasthenic weakness than prednisone alone in the initial management of generalized MG.45 However, none of the patients included in these two studies was known to be steroid resistant. Thus whether MMF has long-term benefits with respect to myasthenic weakness or steroid-sparing effects in the population of steroid-resistant patients remains an open question.

POSSIBLE FUTURE THERAPEUTIC OPTIONS Many possible experimental avenues of investigation that have been opened by studies in EAMG. These are aimed at interrupting the sensitization process of helper CD4 T cells, interrupting their effector function, or interdicting the action of downstream proinflammatory molecules. Studies directed at CD4 T cells include impeding their activation by inhibitors of the costimulatory molecules CD28 and ICOS and the induction of anergy or apoptosis of AChR-reactive T cells.34 In addition, inhibition of factors that contribute to AChR antibody production, and against which monoclonal antibodies (mAbs) are now available for human use, make this approach possible and, in some cases, are now under clinical trials. This includes agents that abrogate the action of IL-6, IL-17, and BAFF. Additional studies directed at AChR-specific B cells include the use of AChR/Fcγ fusion proteins to induce apoptosis of AChR-specific B cells by cross-linking their B-cell AChR receptors and inhibitory FcγRIIb receptors. In addition, non–cross-linking IgG4 anti-AChR antibodies have

been used to inhibit pathogenic IgG autoantibody-induced antigenic modulation. Bortezomib, a proteosome inhibitor approved for usage in multiple myeloma and mantle cell lymphoma, has been found to reduce anti-AChR antibody titers, inhibit damage to the postsynaptic muscle membrane, and lead to clinical improvement. Moreover, this agent was shown to suppress anti-AChR antibody production and deplete plasma cells from cultures of cell suspensions obtained from thymus specimens of patients with MG.34 The induction of antigen-specific immune tolerance, the therapeutic holy grail in autoimmune diseases, has received considerable attention in EAMG. Early studies utilized extracellular domain sequences that form epitopes for pathological autoantibodies to induce tolerance to AChR. However, this approach risks provoking autoimmunity rather than suppressing it. To avoid this risk, one group has recently developed a novel vaccine consisting of bacterially expressed human AChR cytoplasmic domains.46 To date, this group has used this vaccine to prevent development of chronic EAMG when administered immediately after the acute phase of EAMG and to rapidly reverse established chronic EAMG when started during the chronic phase of EAMG. Treatment effects are robust and long-lasting. Although the mechanisms of action of this novel approach have yet to be fully elucidated, they may involve a combination of antibodymediated feedback suppression and regulatory T cell–mediated active suppression. Complement inhibition has shown efficacy in the treatment of EAMG and represents a target in patients with MG now that complement inhibitors have demonstrated benefit in the treatment of a number of human disorders. A phase II placebo-controlled cross-over study with a fully humanized monoclonal anti-C5 antibody was initiated only to be terminated because of inadequate recruitment.34 Nevertheless, given the importance of the terminal complement pathway in the pathogenesis of cases of MG and the continued development of a number of complement inhibitors, it is likely that this strategy will be revisited. A B-cell targeting agent that has gotten much attention in patients with MG is rituximab, a mAb specific for CD20, a protein expressed on B lymphocytes. Anecdotal reports and small uncontrolled case series using rituximab have shown improvement of MG. Interestingly, responsive patients do not demonstrate a reduction in anti-AChR antibodies, suggesting an effect on nonantibody functions, such as antigen presentation and proinflammatory cytokine secretion. Although most treated patients have been anti-AChR antibody positive, benefit has also been observed in anti-MuSK-positive/anti-AChR antibody–negative patients, in whom longstanding remission has been seen in association with reduction of anti-MuSK antibody titers. A controlled clinical trial of rituximab in anti-AChR–associated MG is currently underway. A number of other mAbs directed at CD20, including fully humanized ones, are under study in other autoimmune diseases and will likely attract attention with regard to MG.

CONCLUSIONS MG is a prototypic autoimmune disease in which the autoantigen/s are well defined as are the mechanisms that mediate dysfunction of the target tissue, skeletal muscle. EAMG, in which many of the features of MG are faithfully recapitulated, has provided a useful vehicle for elucidating the immunoregulatory abnormalities that underlie the pathogenesis of the human disease, and the

CHAPTER 65  Myasthenia Gravis ON THE HORIZON Development of Immunomodulatory Therapies • Complement inhibitors • Newly developed B-cell lineage inhibitors, including anti–plasma cell monoclonal antibodies • Acetylcholine receptor (AChR)–Fc fusion molecules • Inhibitors that target key immunopathogenic cytokines • AChR-specific immunosuppression

development of novel therapies for use in the humans. MG occupies a unique position in the pantheon of autoimmune diseases because of its strong association with thymic pathology. In MG-associated thymic hyperplasia, thymic events, particularly those leading to antecedent inflammation within the medulla, likely serve as a driving force behind the development of the autoimmune process. Indeed, the overarching idea that tolerance is breached in the thymus may be viewed as antithetical, given the established and critical role this organ plays in T-cell central tolerance. Unravelling the link between the thymus and MG will require further study. However, in the meantime, we can expect that continued exploration of immune perturbations and their underlying molecular mechanisms in animals with EAMG and in patients with MG will lead to safer and more robust therapeutic interventions.

ACKNOWLEDGMENT This work was supported by National Institutes of Health (NIH) grant NS19546. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Lisak RP, Barchi RL. Myasthenia gravis. In: Walton JN, editor. Major problems in neurology, vol. 11. Philadelphia, PA: WB Saunders; 1982. p. 5. 2. Engel A. The investigation of congenital myasthenic syndromes. Ann NY Acad Sci 1993;681:425. 3. Levinson AI, Zweiman B, Lisak RP. Immunopathogenesis and treatment of myasthenia gravis. J Clin Immunol 1987;7:187. 4. Tindal RSA. Humoral immunity in myasthenia gravis: biochemical characterization of acquired anti-receptor antibodies and clinical correlations. Ann Neurol 1981;10:437. 5. Papadouli I, Sakarellos C, Tzartos SJ. High-resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor. Eur J Biochem 1993;211:227. 6. Leite MI, Jacob S, Viegas S, et al. IgG1 antibodies to acetylcholine receptors in “seronegative” myasthenia gravis. Brain 2008;131:1940. 7. Levinson AI, Lisak RP. Myasthenia gravis. In: Detrick B, Schmitz J, Hamilton RG, editors. Manual of molecular and clinical laboratory immunology. 8th ed, ASM Press, 2016. 8. Sanders DB, Juel VC. MuSK-antibody positive myasthenia gravis: questions from the clinic. J Neuroimmunol 2008;201-202:85. 9. Arrli JA, Skeie GO, Mygand A, et al. Muscle striation antibodies in myasthenia gravis. Ann NY Acad Sci 1998;841:505. 10. Drachman DB, Adams RN, Josifek LF, et al. Antibody-mediated mechanisms of ACh receptor loss in myasthenia gravis: clinical relevance. Ann NY Acad Sci 1981;377:175.

889

11. Engel AG, Sahashi K, Fumagalli G. The immunopathology of acquired myasthenia gravis. Ann NY Acad Sci 1981;377:158. 12. Drachman DB, Angus CW, Adams RN, et al. Myasthenic antibodies cross-link acetylcholine receptors to accelerate degradation. N Engl J Med 1978;298:1116. 13. Richman DP, Wollmann RL, Maselli RA, et al. Effector mechanisms of myasthenic antibodies. Ann NY Acad Sci 1993;681:264. 14. Conti-Fine B, Navaneetham D, Karachunski PI, et al. T cell recognition of the acetylcholine receptor in myasthenia gravis. Ann NY Acad Sci 1998;841:283. 15. Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science 1973;180:871. 16. Christadoss P, Poussin M, Deng C. Animal models of myasthenia gravis. Clin Immunol 2000;94:75. 17. Bellone M, Ostile N, Lei S, et al. Experimental myasthenia gravis in congenic mice. Sequence mapping and H-2 restriction of T helper epitopes on the α subunits of Torpedo californica and murine acetylcholine receptors. Eur J Immunol 1991;21:2303. 18. Karachunski PI, Ostlie NS, Okita DK, et al. Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4+ epitope sequences of the acetylcholine receptor. J Neuroimmunol 1999;95:73. 19. Wang W, Milani F, Ostlie G, et al. C57BL/6 mice genetically deficient in IL-12/IL-23 and IFN-γ are susceptible to experimental autoimmune myasthenia gravis, suggesting a pathogenic role of non-Th1 cells. J Immunol 2007;178:7072. 20. Schaffert H, Pelz A, Saxena A, et al. IL-17-producing CD4(+) T cells contribute to the loss of B-cell tolerance in experimental autoimmune myasthenia gravis. Eur J Immunol 2015;45:1339. 21. Levinson AI, Wheatley LM. The thymus and the pathogenesis of myasthenia gravis. Clin Immunol Immunopathol 1995;78:1. 22. Berrih-Aknin S, Lepanse R. Myasthenia gravis: a comprehensive review of immune dysregulation and etiological mechanisms. J Autoimmun 2014;52:90. 23. Marx A, Pfister F, Schalke B, et al. The different roles of the thymus in the pathogenesis of the various myasthenia gravis subtypes. Autoimmunity Rev 2013;12:875. 24. Levinson AI, Zheng Y, Gaulton G, et al. Intrathymic expression of neuromuscular acetylcholine receptors and the immunopathogenesis of myasthenia gravis. Immunol Res 2003;27:399. 25. Wolfe GI, Kaminski HJ, Aban IB, et al. Randomized trial of thymectomy in myasthenia gravis. N Engl J Med 2016;375:511–22. 26. Levinson AI. Modeling the intrathymic pathogenesis of myasthenia gravis. J Neurolog Sci 2013;333:60. 27. Chuang WY, Strobel P, Belharazem D, et al. The PTPN22gain-offunction+1858T(+) genotypes correlate with low IL-2 expression in thymomas and predispose to myasthenia gravis. Genes Immun 2009;10:667. 28. Greve B, Hoffmann P, Illes Z, et al. The autoimmunity-related polymorphism PTPN22 1858C/T is associated with anti-titin antibody-positive myasthenia gravis. Hum Immunol 2009;70:540. 29. Stefansson K, Dieperink ME, Richman DP, et al. Sharing of epitopes by bacteria and the nicotinic acetylcholine receptor: a possible role in the pathogenesis of myasthenia gravis. Ann NY Acad Sci 1987;505:451. 30. Schwimmbeck PL, Dyrberg T, Drachman DB, et al. Molecular mimicry and myasthenia gravis: an autoantigenic site of the acetylcholine receptor α-subunit that has biologic activity and reacts immunochemically with herpes simplex virus. J Clin Invest 1989;84:1174. 31. Dwyer DS, Vakil M, Bradleg RT, et al. A possible cause of myasthenia gravis: idiotypic networks involving bacterial antigens. Ann NY Acad Sci 1987;505:461. 32. Bever CT Jr, Chang HW, Penn AS, et al. Chemical alteration of acetylcholine receptor by penicillamine: a mechanism for induction of myasthenia gravis. Neurology 1982;32:1077. 33. Penn A, Jacques JJ. Cells from mice exposed chronically to D-penicillamine show proliferative responses to D-penicillamine-treated self (macrophage/dendritic cells): a graft-versus-host response? Ann NY Acad Sci 1993;681:319.

890

Part SEVEN  Organ-Specific Inflammatory Disease

34. Guptill JT, Soni M, Meriggioli MN. Current treatment, emergent translational therapies, and new therapeutic targets for autoimmune myasthenia gravis. Neurother 2016;13:118. 35. Kumar V, Kaminski HJ. Treatment of myasthenia gravis. Curr Neurol Neurosci Rep 2011;11:89. 36. Olanow CW, Wechsler AS, Sirotkin-Roses M, et al. Thymectomy as primary therapy in myasthenia gravis. Ann NY Acad Sci 1987;505: 595. 37. Sonnett JR, Jaretzki A III. Thymectomy for Nonthymomatous Myasthenia gravis: a critical analysis. Ann NY Acad Sci 2008;1132:315. 38. Johns TR. Long-term corticosteroid treatment of myasthenia gravis. Ann NY Acad Sci 1987;505:568. 39. Schneider-Gold C, Gadjos P, Toyka K, et al. Corticosteroids for myasthenia gravis. Cochrane Database Syst Rev 2005;(18):CD002828. 40. Seybold M. Plasmapheresis in myasthenia gravis. Ann NY Acad Sci 1987;505:58.

41. Miller RG, Barohn RJ, Dubinsky R. Expanding the evidence base for therapeutics in myasthenia gravis. Ann Neurol 2010;68:776. 42. Sanders DB, Evoli A. Immunosuppressive therapies in myasthenia gravis. Autoimmunity 2010;43:428. 43. Palace J, Newsom-Davis J, Lecky B. A randomized double-blind trial of prednisolone alone or with azathioprine in myasthenia gravis. Myasthenia Gravis Study Group. Neurology 1998;50:1778. 44. Sanders DB, Hart IK, Mantegazza R, et al. An international, phase III, randomized trial of mycophenylate mofetil in myasthenia gravis. Neurology 2008;71:400. 45. The Muscle Study Group. A trial of mycophenylate mofetil with prednisone as initial immunotherapy in myasthenia gravis. Neurology 2008;71:394. 46. Luo J, Lindstrom J. AChR-specific immunosuppressive therapy of myasthenia gravis. Biochem Pharmacol 2015;97:609.

CHAPTER 65  Myasthenia Gravis

890.e1

MULTIPLE-CHOICE QUESTIONS 1. The most frequent pathogenic autoantibodies found in patients with myasthenia gravis (MG) are specific for a conformational epitope on which subunit of the neuromuscular acetylcholine receptor? A. α B. β C. δ D. ε 2. Patients with MG whose serum is positive for anti- musclespecific tyrosine kinase (MuSK) antibodies are most resistant to treatment with which one of the following modalities? A. Corticosteroid B. Intravenous immunoglobulin C. Plasmapheresis D. Pyridostigmine bromide

3. A 35-year-old man presented with diplopia, difficulty swallowing, and weakness in his upper and lower extremities. On laboratory testing of his serum, he was found to have elevated titers of anti–acetylcholine receptor antibodies and antistriational antibodies. He underwent thymectomy, which very likely showed which of the following pathologies? A. Germinal center hyperplasia B. Thymic atrophy C. Cortical thymoma D. Medullary lymphoma 4. An inflammatory process in which of the following anatomical locales is considered to initiate the development of MG? A. Skeletal muscle B. Thymic cortex C. Cervical lymph node D. Thymic medulla

66  Multiple Sclerosis Benjamin M. Segal

Multiple sclerosis (MS), a chronic inflammatory demyelinating disorder of the central nervous system (CNS), is the most frequent cause of nontraumatic neurological disability among young adults in the Western Hemisphere. Although MS is widely considered a disease of North America and Europe, there is increasing evidence that it is more common in other regions of the world, including Asia and the Middle East, than previously appreciated. The median age at presentation is 28–31 years, which is, in part, responsible for the disproportionately high social and economic tolls of the disease. Furthermore, the incidence of MS is increasing for unknown reasons. Fortunately there have been dramatic advances in the treatment of relapsing forms of MS over the past 20 years, spurred by the introduction of 14 disease-modifying agents (DMAs); and more are actively under development. These drugs significantly decrease the risk of relapse and lesion formation. Consequently, the implications of being diagnosed with relapsing-remitting MS have changed considerably in the span of a generation. Despite this success, significant challenges persist. There is a dire need for treatments that slow, or even halt, disability accumulation in patients with progressive forms of MS, and for interventions that restore lost neurological functions across MS subsets.

CLINICAL SUBSETS AND PHENOMENOLOGY Relapsing-Remitting MS (RRMS) In the majority of cases (85–90%), MS presents with a relapsingremitting course, characterized by discrete episodes of neurological dysfunction (relapses or exacerbations) separated by clinically quiescent periods (remissions). The frequency of relapses can vary widely among patients as well as during different periods in an individual patient’s disease course. At present no clinical features or biomarkers that are predictive of relapse rate have been identified. The signs and symptoms that occur during relapses are also diverse and unpredictable, since lesions can form at literally any site in the CNS, spanning the cerebrum, brainstem, cerebellum, optic nerves, and spinal cord. By definition, the peripheral nervous system is spared. MS lesions are readily visualized in CNS white matter via magnetic resonance imaging (MRI) (Fig. 66.1). Symptomatic lesions generally occur in locations where nerve fibers converge to subserve a common function. Hence, typical presentations of RRMS include optic neuritis with monocular visual deficits (secondary to lesions in the optic nerve), myelitis with weakness and numbness in the extremities, sometimes accompanied by incontinence (caused by spinal cord lesions), and brainstem syndromes manifesting as slurred speech, swallowing difficulties,

imbalance, tremor, and/or double vision. Serial MRI studies have demonstrated that the majority of MS lesions are actually clinically silent. This is not surprising considering the abundance of redundant nerve fiber tracts in the CNS and the commitment of large areas of cerebral white matter to subtle personality traits and cognitive skills. Consequently, CNS tissue damage may be inflicted surreptitiously during clinical remissions, making MRI a more sensitive indicator of disease activity compared with the history or the results of neurological examination (see Fig. 66.1). Patients with MS often recover function following a clinical relapse, either partially or fully, particularly during the early clinical course. However, old symptoms can temporarily reemerge when the core body temperature is elevated as a result of infection or strenuous exercise. This unmasking of latent deficits, referred to as the Uhthoff phenomenon, is a consequence of the physiological slowing of axon signal propagation that normally occurs at high core body temperatures. In healthy individuals, the degree of slowing has no clinical consequence, but in MS patients, it may precipitate the decompensation of white matter tracts already compromised by demyelination and axonal drop-out.

Secondary Progressive MS (SPMS) During the course of RRMS, relapses decrease in frequency over time and sometimes disappear completely. However, in the vast majority of cases, they are replaced by an insidious, gradual accumulation of disability, referred to as the secondary progressive (SP) stage. The symptoms and signs that characterize neurological decline during SPMS are diverse. Progressive myelopathy, hemiparesis, and/or gait imbalance are common. Subcortical dementia is increasingly recognized as a feature of the disease. Longitudinal natural history studies conducted before DMAs were widely available found that the majority of patients with RRMS transitioned to the SP stage within 10–20 years of the initial presentation of disease. An epidemiological study of MS patients in British Columbia, published in 2010, found that the median time to SPMS onset was 21.4 years.1 A number of factors, including male gender, the presence of motor symptoms at clinical onset, and a history of poor recovery from relapses, are associated with both a shorter time to, and younger age at, evolution to SPMS. It has not been definitively determined whether optimal management of RRMS with the use of DMAs can delay, or even prevent, the onset of SPMS. Previous longitudinal observational and retrospective cohort studies that investigated whether treatment with first-generation DMAs alters the time to reach SPMS yielded conflicting results. However, a recent prospective study of 517 actively treated patients found that rates of worsening and evolution to SPMS were substantially

891

892

A

Part Seven  Organ-Specific Inflammatory Disease

B

FIG 66.1  (A) T2-weighted fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) scan of the brain of a patient with multiple sclerosis (MS) showing hyperintense lesions located in the periventricular and subcortical white matter. (B) T1-weighted MRI scan showing T1-black holes (arrows), indicative of profound axonal loss, and generalized atrophy with enlarged ventricles.

lower when compared with the findings of earlier natural history studies of untreated patients.2 The cellular and molecular mechanisms underlying the conversion from the RR stage to the SP stage are poorly understood. Some investigators have questioned the relevance of neuroinflammation during the SP stage and have posited neurodegeneration as being primarily responsible for the clinical deterioration that ensues (in the form of neuronal death, mitochondrial dysfunction in axons, Wallerian degeneration, and gliosis).3 Conversely, there is evidence of persistent immune dysregulation in SPMS, although it may differ from RRMS with regard to the cytokine networks and leukocyte subsets involved, as well as the distribution of infiltrating cells within the CNS.4-6 Nonetheless, DMAs that are therapeutically beneficial in RRMS have generally not been found to be effective in slowing disability accumulation in SPMS.7 At present, the management of SPMS involves alleviation of symptoms, optimization of residual functions, and prevention of complications.

Primary Progressive MS (PPMS) PPMS is distinguished from SPMS by the absence of an antecedent RR phase. Otherwise, the clinical features of SPMS and PPMS can be indistinguishable. The most common clinical phenotype is spastic paraparesis, followed by cerebellar dysfunction and hemiplegia. However, there are striking demographic differences between PPMS and RRMS/SPMS. PPMS tends to present at an older age (peaking in the fifth and sixth decades) compared with RRMS (which peaks in the third and fourth decades). Furthermore, RRMS occurs 2–3 times more frequently in females than in males, whereas in PPMS the gender ratio is closer to 50 : 50. Some investigators have noted that the neuropathological and radiological features of PPMS overlap extensively with those of SPMS, leading them to conclude that PPMS and SPMS belong to the same disease spectrum. In support of that viewpoint, familial clusters of MS that include some members with PPMS and others with RR/SPMS have been described. It is possible

that, analogous to SPMS, acute inflammatory lesions form during early stages of PPMS, prior to overt clinical progression, but happen to arise exclusively in clinically silent areas. Conversely, some investigators have argued that PPMS is a distinct disease entity driven primarily by neurodegenerative processes from its inception.3 This viewpoint is supported by the general failure of immunomodulatory agents to attenuate the course of PPMS.8 Conversely, in a randomized double-blind placebo-controlled trial, treatment with a B cell–depleting monoclonal antibody (mAb) delayed disability progression in a subset of patients with PPMS who were younger and/or had inflammatory lesions.9 Collectively, these findings suggest that the pathogenesis of PPMS is multifaceted and heterogeneous and that the relative contribution of inflammation and neurodegeneration to clinical outcomes varies among patients.

CLINICAL PEARLS Clinical Features of MS In the majority of cases (80–85%), multiple sclerosis (MS) presents with a relapsing-remitting course. • The symptoms and signs experienced by patients with MS are diverse because lesions can form at any site in the central nervous system (CNS), including the optic nerves, cerebrum, brainstem, and spinal cord. • The rate, severity, and symptoms of relapses are highly variable and unpredictable. • Most MS lesions form silently; magnetic resonance imaging (MRI) is a more sensitive tool to gauge disease activity compared with history or neurological examination. • Acute relapses tend to decrease in frequency over time and are typically replaced by a gradual accumulation of disability. This later phase of disease is referred to as secondary progressive MS (SPMS).

DIAGNOSIS The most widely used guideline for diagnosing MS is the McDonald criteria, originally proposed by the International Panel on Diagnosis of Multiple Sclerosis in 2001 and subsequently revised in 2005 and 201010 (Table 66.1). RRMS is, by definition, a dynamic multifocal inflammatory demyelinating disease of the CNS. Therefore the demonstration of lesion dissemination in time and space is crucial for its diagnosis. The criteria for dissemination in time can be satisfied by ≥2 distinct clinical exacerbations, by one exacerbation followed by the interval appearance of a new lesion on serial MRI scans, or by the simultaneous presence of asymptomatic gadolinium-enhancing (i.e., acute inflammatory) and nonenhancing MRI lesions at any time. Dissemination in space can be demonstrated by objective clinical evidence of involvement of ≥2 sites in the CNS (based on the neurological examination and/or delayed latencies on evoked potential testing) or by the presence of T2-weighted MRI lesions in at least two of the following areas: periventricular, juxtacortical, and infratentorial, cerebral white matter or spinal cord. In the latest iteration of the McDonald criteria, an MS exacerbation is defined as “patient-reported symptoms or objectively observed signs typical of an acute inflammatory demyelinating event in the CNS, current or historical, with duration of at least 24 hours, in the absence of fever or infection.” There are no clinical features or biomarkers that are pathognomonic for MS. Therefore it is essential to rule out competing diagnoses. The presence of unique oligoclonal bands and/or elevated immunoglobulin G (IgG) index in the cerebrospinal fluid (CSF), indicative of

CHAPTER 66  Multiple Sclerosis TABLE 66.1  McDonald 2010 Criteria for

the Diagnosis of Multiple Sclerosis Clinical Presentation ≥ 2 attacks; objective clinical evidence of ≥ 2 lesions or objective clinical evidence of 1 lesion with reasonable historical evidence of a prior attack ≥ 2 attacks; objective clinical evidence of 1 lesion

1 attack; objective clinical evidence of ≥ 2 lesions

1 attack; objective clinical evidence of 1 lesion (clinically isolated syndrome)

Insidious neurological progression suggestive of MS (PPMS)

Additional Data Needed for an MS Diagnosis None

Dissemination in space demonstrated by ≥ 1 T2 lesion in at least 2 or 4 MS-typical regions (periventricular, juxtacortical, infratentorial, or spinal cord) Simultaneous presence of asymptomatic gadolinium-enhancing and nonenhancing lesions at any time; or a new T2 and/or gadoliniumenhancing lesion(s) on follow-up MRI, irrespective of its timing with reference to a baseline scan; or Await a second clinical attack Dissemination in space and time, demonstrated by: For DIS: ≥ 1 T2 lesion in at least 2 of 4 MS-typical regions of the CNS (periventricular, juxtacortical, infratentorial, or spinal cord); or Await a second clinical attack implicating a different CNS site; and For DIT: Simultaneous presence of asymptomatic gadolinium-enhancing and nonenhancing lesions at any time; or A new T2 and/or gadoliniumenhancing lesion(s) on follow-up MRI, irrespective of its timing with reference to a baseline scan; or Await a second clinical attack 1 year of disease progression (retrospectively or prospectively determined) plus 2 of 3 of the following criteria: 1. Evidence for DIS in the brain based on ≥ 1 T2 lesions in the MS-characteristic (periventricular, juxtacortical, or infratentorial) regions 2. Evidence for DIS in the spinal cord based on ≥ 2 T2 lesions in the cord 3. Positive CSF (isoelectric focusing evidence of oligoclonal bands and/or elevated IgGindex)

From Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011; 69(2): 292–302.

893

(ii) at least two T2 spinal cord lesions; and (iii) positive CSF findings, defined as the presence of oligoclonal bands or elevated IgG index.

RISK FACTORS Genetic Risk Factors A monozygotic twin whose cotwin has MS has a 20–30% risk of developing the disease, whereas the corresponding risk for a dizygotic twin is 2–5%.11 In contrast, the incidence of MS in the general population is approximately 0.1%. Hence, the risk of MS drops with increasing genetic distance, suggesting that genes play a significant role in determining MS susceptibility. The human leukocyte antigen (HLA) region represents the strongest MS susceptibility locus, genome wide, and has been implicated in all ethnic populations thus far studied. It accounts for up to 10.5% of genetic variance underlying risk.12 The primary signal maps to the HLA-DRB1 gene in the class II segment of the locus, implicating a role of CD4 T-cell responses in MS pathogenesis. Genome-wide association studies (GWAS) have revealed over 100 non-HLA susceptibility loci, each of which contributes a small amount to MS risk. Strikingly, most of these map to regions containing genes implicated in immunological, rather than neuronal or glial, pathways. Genes involved in T-helper (Th)–cell differentiation are overrepresented, including the interleukin-2 (IL-2) receptor α chain and the IL-7 α chain, which modulate T-cell proliferation and survival. Over one-third of the MS susceptibility loci overlap with regions previously identified in GWAS of other autoimmune diseases, including celiac disease, type 1 diabetes, rheumatoid arthritis, and/or inflammatory bowel disease. Together these data provide support for a primary immunological, as opposed to neurodegenerative, etiology of MS. An unresolved question is whether genetic variants affect the clinical course of RRMS, including such features as relapse rate or severity and time to conversion to the SP stage. Genetic variants that are predictive of responsiveness to DMAs have yet to be identified. A growing area of interest that warrants further investigation is the potential impact of epigenetic modifications in different cell types on MS susceptibility, clinical course, and/ or therapeutic responsiveness.

Environmental Risk Factors As mentioned above, twin concordance rates in MS have been used to highlight the importance of heredity in MS susceptibility. Paradoxically, the same data can be used to argue for the importance of environmental influences. Hence, over 70% of monozygotic twins of individuals with MS do not develop the disease. Despite arduous attempts, no convincing evidence has been produced for genetic, epigenetic, or transcriptome differences that explain MS discordance among monozygotic twin pairs.13 The risk of MS appears to be predicated on a complex interplay between genetic and environmental factors.

Geographic Prevalence Patterns primary antibody production in the CNS, supports a diagnosis of MS, but those findings are observed in a wide range of neuroinflammatory conditions, including subacute sclerosing panencephalitis, neurosarcoidosis, Lyme disease, and systemic lupus erythematosus (SLE) with CNS involvement. A diagnosis of PPMS requires 1 year of disease progression plus two of the three following criteria: (i) at least one T2 lesion in the periventricular, juxtacortical, or infratentorial cerebral white matter;

One of the most convincing illustrations of the impact of the environment on the development of MS is its geographical distribution. The prevalence of MS is highest in the Scandinavian countries, Canada, and Scotland and lowest in the equatorial regions. Kurtzke et al. were the first to notice a latitudinal gradient in the prevalence of MS across the United States, with the disease being most common in the Northern states and gradually declining toward the South.14 Similar latitudinal gradients have been

894

Part Seven  Organ-Specific Inflammatory Disease

observed in Europe, Australasia, and Japan. Population migration studies indicate that the geographical risk of MS is established prior to adolescence. Prepubescent children who migrate assume the risk of their adopted country, whereas adults carry forward the risk of the location where they spent their childhood. An environmental agent that is encountered in childhood and acts as predisposing factor for the development of MS later in life must be responsible, but this agent has yet to be definitely identified.

Vitamin D The discovery of the protective role of vitamin D in MS may explain, in part, its geographical distribution. Ultraviolet light catalyzes the conversion of vitamin D to its bioactive form, and the prevalence of MS is highest in regions with relatively low annual sunlight exposure. In a large prospective study, the risk of MS decreased with increasing serum levels of 25-hydroxyvitamin D.15 Therefore interventions that raise the level of 25-hydroxyvitamin D levels might have a prophylactic protective effect on healthy individuals who are predisposed to develop MS, such as the first-degree relatives of patients with MS. Indeed, numerous independent prospective studies have found an inverse relationship between dietary or supplemental vitamin D intake in adults and future risk of MS. Furthermore, there is accumulating evidence that low serum 25-hydroxyvitamin D levels and low dietary vitamin D intake during pregnancy increase the risk of MS in the offspring.16 Whether vitamin D levels influence the clinical course of individuals with established MS is more controversial. A randomized controlled trial of vitamin D supplementation in patients with MS is currently underway in 16 academic centers (ClinicalTrials.gov identifier: NCT01490502). The results of that trial should bring clarity to the issue.

Infection In addition to low levels of vitamin D, another prognostic factor for development of MS is primary infection with Epstein-Barr virus (EBV) in adulthood, as indicated by the emergence of EBV-specific IgM antibodies in serum. Analysis of serial serum samples stored in the US Department of Defense Serum Repository revealed that MS risk was extremely low among individuals not infected with EBV but increased sharply in the same individuals following seroconversion to positivity for antibodies against EBV.17 The odds of MS were recently estimated to be >10 times higher among EBV-positive persons than among EBV-negative persons.18 One way in which EBV infection could promote MS pathogenesis is via “molecular mimicry,” which occurs when a microbial epitope shares sequence similarities with a self peptide, in this case a myelin peptide (Chapter 50). T-cell receptors (TCRs) that cross-react with structurally homologous EBV and myelin antigens have been discovered in the peripheral CD4 TCR repertoire of individuals with MS.19 Peripheral CD4 T cells that express such cross-reactive TCRs could be activated during EBV infection, enabling them to cross the blood–brain barrier (BBB), encounter their cognate myelin antigen within CNS white matter, and initiate MS lesion formation. An alternative theory involves infiltration of the CNS by EBV-infected B cells. B cells expressing EBV small RNA and proteins have been detected in meningeal follicle–like structures and inflamed cortical lesions in SPMS brain tissue.20 EBV-driven expansion and activation of meningeal B cells could potentially contribute to the formation of the follicle-like structures, which have been associated with large subpial cortical lesions and a more aggressive clinical course.6

Although infectious agents, such as EBV, have been implicated in MS pathogenesis, other pathogens may have a therapeutic effect. It has been suggested that in addition to high levels of sunlight exposure, endemic helminth infection is one of the factors responsible for the low prevalence of MS in tropical regions. Infection with helminths, including Heligmosomoides polygyrus, Fasciola hepatica, Schistosoma mansoni, and Trichinella, has been shown to be protective in animal models of MS.21 Several small prospective studies showed that patients with MS naturally infected with different species of parasitic worms had a milder disease course and lower MRI inflammatory activity compared with uninfected patients.22 Antiparasite treatment was associated with exacerbation of MS. Mechanistic substudies have suggested that parasites modulate MS disease activity by boosting the frequency of IL-10 and transforming growth factor-beta (TGF-β)–producing regulatory T and/or B cells. A phase I safety study of orally administered Trichuris suis ova (TSO) has been conducted in RRMS, and a number of clinical trials of TSO or dermally administrated hookworm Necator americanus as disease-modifying therapy in MS are planned or underway.

Obesity Observational studies have found that individuals who are obese in early adulthood, as measured by elevated body mass index (BMI), have an approximately twofold increased risk of MS.23 Obesity in girls is associated with an increased risk of pediatric MS or clinically isolated syndrome.24 Furthermore, higher weight in adolescence and young adulthood is associated with an earlier age at onset of MS. A mendelian randomization analysis of large GWAS for MS and BMI, respectively, found that one standard deviation (SD) increase in genetically determined BMI conferred a 41% increase in the odds of MS.25 A number of theories have been proposed for a mechanistic link between obesity and MS risk. First, obesity is known to cause a systemic proinflammatory state, possibly mediated by an adipose-derived hormone that could create a milieu conducive to the differentiation and/or activation of autoimmune effector cells. Alternatively, there is some evidence that genetically elevated BMI decreases 25-hydroxyvitamin D levels.

Modifiable Habits A substantial body of literature indicates that cigarette smoking increases the risk of MS. A recent meta-analysis revealed a dose– response relationship between cigarette pack-years and MS risk.26 On the basis of data collected from the Swedish National MS Registry, it was estimated that each additional year of smoking after diagnosis accelerates the time to conversion to SPMS by 4.7%.27 There is growing evidence that exposure to passive smoking is also a risk factor for MS. Acrolein, a component of cigarette smoke, was found to exacerbate the clinical course of an animal model of MS, in association with enhanced microglial activation.28 Another behavior that has come under scrutiny in relationship to MS is dietary sodium intake. In one study, exacerbation rates were found to be 2.75–3.95-fold higher in patients with RRMS with medium or high sodium intakes, respectively, compared with the low-intake group.29 In contrast, high salt intake was not associated with decreased time to relapse in pediatric-onset MS.30 High-salt conditions promote the induction of highly pathogenic myelin-reactive T cells in vitro and in animal models in vivo.31

CHAPTER 66  Multiple Sclerosis

895

Sex Hormones MS relapse rates decline during pregnancy, particularly in the third trimester, and rebound in the first 3 months post partum before returning to the prepregnancy rate. This has led to the hypothesis that certain female sex hormones may play a protective role in RRMS. Estriol is an estrogen unique to pregnancy. It is synthesized by the fetoplacental unit and reaches its highest levels in the last trimester. A randomized, double-blinded, placebocontrolled phase II trial of estriol in combination with glatiramer acetate (GA) versus placebo plus GA showed a reduction in the annualized relapse rate at 2 years in the estriol-treated group.32 Animal model studies have indicated that estrogens, including estriol, have antiinflammatory and neuroprotective effects through engagement of the estrogen receptors expressed on leukocytes and CNS resident cells, respectively. Testosterone has neuroprotective effects in animal models of MS, and decreased testosterone levels in males with MS were reported to be associated with disability.33 In a small, open-label, phase II clinical trial, testosterone treatment appeared to arrest loss of gray matter (and even to reverse atrophy of gray matter in the right frontal cortex), as quantified by using voxel-based morphology, in 10 male patients with MS.34

FIG 66.2  A post-gadolinium T1-weighed magnetic resonance imaging (MRI) scan of the brain showing Dawson fingers (arrows).

KEY CONCEPTS Risk Factors The risk of multiple sclerosis (MS) is determined by a combination of genetic and environmental factors. • The majority of MS susceptibility loci map to regions containing genes implicated in immunological pathways, including human leukocyte antigen (HLA) class II molecules, the interleukin-2 (IL-2) receptor, and the IL-17 receptor. • Relapse rates decline during the third trimester of pregnancy, in association with high serum levels of estriol. • Environmental risk factors include low vitamin D levels, exposure to the Epstein-Barr virus (EBV) in adulthood, cigarette smoking, and childhood obesity.

PATHOLOGICAL FEATURES OF MS White Matter Lesions The hallmark of MS pathology is the focal demyelinated lesion, or “plaque,” present in the white matter of the optic nerves, brain, and spinal cord. Acute lesions are invariably associated with focal breakdown of the BBB and perivascular inflammatory infiltrates. MS infiltrates are dominated by T cells (with a relatively high CD8/CD4 ratio) and myeloid cells (blood-derived monocytes/ macrophages and activated microglia). Macrophages/monocytes and activated microglia are spatially associated with disintegrating myelin sheaths, and they actively take up myelin debris. Apoptosis and loss of oligodendrocytes vary widely among lesions. Frequent sites of lesion formation include subcortical and periventricular cerebral white matter, middle cerebellar peduncles, and the posterior columns of the cervicothoracic spinal cord. In the brain, infiltrates frequently follow the course of pericallosal venules, resulting in “Dawson fingers,” which are oblong lesions oriented perpendicular to the long axes of the lateral ventricles (Fig. 66.2). Classic actively demyelinating plaques are primarily seen during the RR stage of disease and generally decrease in frequency with increasing disease duration. Lesions more typical of progressive forms of MS have been termed “chronic active,” or smoldering,

and “chronic silent,” or inactive. Chronic active plaques are distinguished by a rim of activated microglia and deposits of complement at the lesion edge, surrounding a hypocellular and gliotic core. They are slowly expansive as a consequence of active demyelination at the lesion edge. In contrast, chronic silent plaques have a sharp border. Other characteristics of silent plaques include prominent loss of oligodendrocytes and axons, pronounced astrogliosis, and a paucity of macrophages and activated microglia. Immunopathological changes in the so-called normal-appearing white matter (NAWM), outside of plaques, are pervasive in progressive MS but have also been observed in RRMS. These changes consist of diffuse axonal injury and microglial activation, as well as scattered lymphocytes. MS is widely classified as a demyelinating disorder. The reason is that a large number of the nerve fiber segments traversing plaques demonstrate myelin loss with relative axonal sparing. However, it is now recognized that axonopathy also occurs and is, in fact, an early and prominent feature of acute MS lesions. Axonal damage results in dysmorphic mitochondria, focal swellings, fragmentation, and frank transections with terminal bulbs at the stumps. Mitochondrial abnormalities and focal swelling have been observed in fully myelinated axons within MS lesions, suggesting that they can occur independent of demyelination.35 In animal models of MS, axons with abnormal mitochondria are restricted to areas of immune infiltration, and progressive axonal changes correlate with the density of infiltrates.35 Hence, it is likely that the axonal damage is directly mediated by direct contact with inflammatory cells. Although demyelination can be reversed to some extent by remyelination, axonal transection is irreversible. Clinicopathological investigations have found that permanent motor disability in MS correlates with loss of corticospinal tract axons more so than with degree of demyelination.

Gray Matter Lesions MS was traditionally considered a white matter disease. It is now established that the gray matter is affected as well. Three types

896

Part Seven  Organ-Specific Inflammatory Disease

of cortical lesions have been described: leukocortical (which span gray and white matter), intracortical, and subpial. All of these lesions show demyelination and oligodendrocyte loss, microglial activation, neuritic transections, neuronal death, and reduced presynaptic terminals. Subpial lesions are the most common. They can cover long distances of the cortical ribbon and usually extend to the cortical layer III or IV. Cortical lesions are not visible on conventional MRI scans and require special staining to be appreciated in CNS tissue sections. This explains why they were not recognized as common features of MS until recently. In fact, cortical demyelination and gray matter atrophy are evident from the earliest stages of disease, even before a clinically definite diagnosis can be made, and continue to advance at an increasing rate throughout the disease course. Extensive cortical demyelination is evident in the forebrain and cerebellum during progressive MS. Gray matter atrophy in individuals with MS correlates strongly with cognitive deficits and clinical disability.36

Meningeal Inflammation White blood cell (WBC) counts tend to be within normal limits or only slightly elevated in the CSF of most patients with MS. Nonetheless, there is growing recognition that low-grade diffuse meningeal inflammation and focal perivascular meningeal inflammation are common. Meningeal inflammation is most prominent in progressive forms of MS but is prevalent in early MS as well. The meningeal infiltrates are topographically associated with cortical lesions. Lymphoid follicle–like structures, composed of proliferating B cells, T cells, and follicular dendritic cells (FDCs), have been observed in the meninges of up to 40% of autopsied brains from individuals with SPMS.6 In almost every case, the follicles were found to reside in deep sulci and abut an underlying subpial lesion, suggesting that toxic factors are released by inflammatory cells in the follicles and diffuse into the brain parenchyma. The presence of lymphoid follicles has been associated with a more severe clinical course, shorter disease duration, and younger age at death.

KEY CONCEPTS Pathology • The hallmark of multiple sclerosis (MS) pathology is the focal demyelinated lesion, or “plaque,” with perivascular inflammatory infiltration and focal blood–brain barrier (BBB) breakdown. • Axonopathy is an early and prominent feature of acute MS lesions. • Central nervous system (CNS) damage includes demyelination, apoptosis and loss of oligodendrocytes, and axonal swellings and transections. • Both gray matter and white matter are affected. • The pathological features of MS are heterogeneous and evolve over time.

IMMUNOPATHOGENESIS Animal Models of MS According to the current dogma, MS is an autoimmune disease mediated by CD4 T cells reactive against myelin antigens. The identification of HLA class II, IL-2Rα, and IL-7Rα as MS susceptibility loci is consistent with a role of CD4 T cells in MS pathogenesis. An autoimmune etiology is further supported by the animal model experimental autoimmune encephalomyelitis (EAE). EAE is a multifocal inflammatory demyelinating disease of the CNS that has striking histological and clinical similarities to MS (Fig. 66.3 and Fig. 66.4). It has been induced in a wide

FIG 66.3  A mouse with experimental autoimmune encephalitis (EAE) (arrow) and a healthy littermate. The mouse with EAE has a limp tail and hindlimb weakness.

variety of mammalian species (including nonhuman primates, but most commonly in rodents) by vaccination against major histocompatibility complex (MHC) class II–restricted myelin epitopes. EAE can be transferred from myelin-vaccinated mice to syngeneic naïve hosts with purified CD4 T-cell lines or clones. These encephalitogenic myelin-specific CD4 T cells invariably fall within the Th1 or Th17 lineage and produce the proinflammatory cytokines interferon-γ (IFN-γ) and IL-17, respectively, in response to antigenic stimulation37(Chapter 16). Both Th1 and Th17 cells produce granulocyte macrophage–colonystimulating factor (GM-CSF), a monocyte mobilizing and growth factor that plays a critical role in many models of EAE. Upon activation in the periphery, myelin-reactive CD4 T cells upregulate adhesion molecules and chemokine receptors, thereby acquiring the ability to cross the BBB. Once having infiltrated the CNS, they are reactivated by local antigen-presenting cells (APCs), such as perivascular macrophages or microglia, which constitutively express MHC class II molecules bound to myelin peptides on their surface. GM-CSF, as well as other Th1 and/or Th17 cytokines, are subsequently released in situ and initiate an inflammatory cascade, resulting in the production of chemokines, mobilizing factors and vasoactive substances, upregulation of adhesion molecules on the cerebrovascular endothelium, and thus the recruitment of myeloid cells and lymphocytes from the circulation to the nascent plaque. GM-CSF may drive the differentiation of infiltrating monocytes and CNS-resident microglia into CD11c+ DCs, which are among the most potent APCs. Adoptive transfer studies with labeled donor T cells have demonstrated that the myelin-specific T cells remain clustered in the perivascular space throughout lesion development. A secondary wave of myeloid cells infiltrate deep into the CNS

CHAPTER 66  Multiple Sclerosis

EAE

897

CONTROL

MBP DAPI

FIG 66.4  Immunofluorescent histology of spinal cord sections from a mouse with experimental autoimmune encephalitis (EAE) (left) and a healthy control (right). White matter tracks were stained with a monoclonal antibody (mAb) specific for myelin basic protein (green). The nuclei of inflammatory cells are stained with DAPI (4’,6-diamidino-2-phenylindole) (blue). The arrows point to areas of demyelination.

white matter, associate with nodes of Ranvier, and directly inflict damage to the myelin sheath and axons35 (see Figs. 66.3 and Fig. 66.4).

Immune Dysregulation in Patients With MS Studies on the frequency of myelin-reactive T cells in patients with MS have reported conflicting results; some investigators found a significantly higher incidence of myelin-specific peripheral blood mononuclear cells (PBMCs) in individuals with MS compared with age- and gender-matched healthy controls (HCs), whereas others found no significant difference.5 Many of the earlier studies that found no differences between patients and HCs used proliferation as a measure of T-cell reactivity and nonhuman myelin proteins for antigenic stimulation. In contrast, several laboratories have found that untreated patients with RRMS have increased frequencies of PBMCs that produce IFN-γ or IL-17 in response to ex vivo challenge with human myelin basic protein (MBP), human proteolipid protein (PLP), or their constituent peptides.4,38,39 IFN-γ responses to PLP peptides were shown to correlate with level of clinical disability.38 T cells that coexpress IL-17 and IFN-γ were identified in the brain tissue of patients with MS, and circulating lymphocytes obtained from patients with MS were found to have an increased propensity to differentiate into IL-17/IFN-γ double producers.40 T cells coexpressing IL-17/IFN-γ, and IL-17–producing T cells that convert into IFN-γ producers (the so-called ex-Th17 cells), have been implicated in the pathogenesis of some EAE models. In a recent study, untreated patients with RRMS had a higher frequency of peripheral blood T cells that expressed intracellular GM-CSF in response to stimulation with phorbol myristate acetate and ionomycin, compared with HCs or IFN-β–treated RRMS patients.41 Approximately 15–20% of both CD4 and CD8 T cells in active MS lesions expressed GM-CSF and the majority coexpressed IL-17 and/or IFN-γ. There is an increasing appreciation that the cytokine dysregulation that occurs during EAE and MS is heterogeneous. Hence, clinically identical forms of EAE can be induced with stable murine Th1 or Th17 cells independently.42 Subsets of patients

with MS who consistently mounted either IFN-γ– or IL-17–skewed responses to human MBP over the course of a year were recently identified, while others exhibited mixed or oscillating responses.37 Diversity in the immune pathways that drive MS pathology holds important implications for the development of a more personalized approach to the management of patients in the future. Indeed, Th1- and Th17-mediated forms of EAE show different patterns of responsiveness to the same immunomodulatory drug.42 Perhaps the strongest evidence of an autoimmune basis of inflammatory demyelination in humans comes from clinical trials of immunomodulatory agents. Patients with RRMS were treated with an altered peptide ligand (APL) of MBP with the intention of tolerizing MBP-reactive T cells or deflecting their differentiation toward an immunosuppressive, regulatory, or innocuous Th2 phenotype. Unexpectedly, administration of the APL was temporally associated with expansion of circulating MBP-reactive Th1 cells and clinical worsening in a subgroup of patients.43 In contrast, as will be discussed in detail below, drugs that impede lymphocyte trafficking to the CNS,44,45 block growth factor signaling into T lymphocytes,46 or deplete lymphocytes from the periphery47 suppress MS relapse rates and the accumulation of MRI lesions.

DISEASE-MODIFYING THERAPIES The approval of IFN-β-1b (Betaseron) by the US Food and Drug Administration (FDA) in 1993 for the management of RRMS marked the beginning of a new era in MS therapeutics. In the intervening 23 years, an additional 13 disease-modifying therapies have been approved, all of which have significantly reduced the annualized relapse rate (in a range from 25% to almost 70%) and the frequency of gadolinium enhancing (acutely inflamed) MRI lesions. Prior to 1993, corticosteroids were the only class of drugs routinely used to treat MS. Although corticosteroids accelerate the rate of recovery from acute exacerbations, there is little evidence that they alter the ultimate clinical outcome or prevent subsequent disease activity. Therefore the introduction

898

Part Seven  Organ-Specific Inflammatory Disease

of disease-modifying therapies has represented a major advance in the treatment of individuals with RRMS and has had a profound impact by mitigating morbidity and enhancing quality of life.

Recombinant IFN-β IFN-β is a type I IFN with potent antiviral properties. It has pleiotropic effects on the innate immune system. Recombinant IFN-β was first tested in MS on the basis of the contemporaneous theory that the disease was caused by an active viral infection of the CNS. Although MS is now believed to be an autoimmune disease, recombinant IFN-β therapy was serendipitously found to significantly reduce annualized MS relapse rates. It is manufactured as four different commercial products, all of which are self-administered via either subcutaneous or intramuscular injection. There are two distinct structural forms. IFN-β-1a is produced in mammalian cells and has the same sequence as the naturally occurring compound, while IFN-β-1b is produced in modified Escherichia coli and has a Met-1 deletion and a Cys-17 to Ser mutation. IFN-β-1a is glycosylated, and IFN-β-1b is nonglycosylated. Studies of the efficacy of IFN-β in RRMS have yielded remarkably consistent results across different formulations of the drug. In multiple randomized, double-blinded placebocontrolled trials, IFN-β therapy reduced annual relapse rates by 20–35%.48 However, approximately 30% of patients with MS do not respond to the drug. The mechanism of action of IFN-β in MS has not been definitively elucidated. Proposed mechanisms include induction of the immunosuppressive cytokine IL-10, inhibition of pathogenic Th17 cells, and stabilization of the BBB via direct effects on the cerebrovascular endothelium.

Glatiramer Acetate GA comprises a mixture of polypeptides of different lengths and sequences, synthesized by the randomized polymerization of four amino acids, namely, glutamic acid, lysine, alanine, and tyrosine. These are the most prevalent amino acids in MBP, a candidate autoantigen in MS. It was originally thought that GA would act as a competitive antagonist of MBP peptides for binding to MHC class II molecules on APCs. Subsequent mechanistic studies did not support that theory. The mechanism of action of GA in MS is currently unknown, but alternative hypotheses that have been proposed include immune deviation of myelinreactive T cells from a destructive Th1 to an innocuous Th2 phenotype, increase in frequency and function of FoxP3+ regulatory CD4 T cells or regulatory CD8 T cells, and induction of antiinflammatory type II monocytes. A pivotal trial of GA versus placebo in 1995 demonstrated a mean reduction in the relapse rate of approximately 30%.49 In contrast, a multinational, multicenter, double-blind, placebo-controlled phase III trial of GA in PPMS was stopped after an interim analysis by an independent data safety monitoring board indicated no discernible treatment effect on the primary outcome measure.8 GA, administered by subcutaneous injection, was approved by the FDA in 1996 for reducing the frequency of relapses, but not for reducing the progression of disability.

Teriflunomide Teriflunomide, an oral pyrimidine synthesis inhibitor, was approved by the FDA in 2012 for relapsing forms of MS. It has broad immunosuppressive effects, including a cytostatic effect on proliferating T and B lymphocytes. In a pivotal phase III trial, teriflunomide reduced annualized relapse rate by 31%

compared with placebo.50 In an independent phase III raterblinded comparator trial, teriflunomide was found to have a similar efficacy to IFN-β-1a in reducing the proportion of participants with at least one relapse over 1 year.51 Teriflunomide is teratogenic in rats and rabbits and is therefore contraindicated in pregnant women and women of childbearing potential not using reliable contraception.

Dimethyl Fumarate Dimethyl fumarate (DMF) is the methyl ester of fumaric acid. Prior to being tested as a DMA in MS, DMF was used as a biocide in furniture and shoes to prevent the growth of molds during storage or transport. A combination of DMF and three other fumaric acid esters was marketed in Germany as a treatment for psoriasis. In two phase III trials conducted in 2012, oral DMF was demonstrated to decrease the annualized relapse rate of adults with RRMS by approximately 34–50% compared with placebo.52,53 An additional cohort of subjects treated with GA was included as a reference comparator in one of the trials.53 There was no significant difference in the effect of twice-daily oral DMF versus daily subcutaneous GA on relapse rate, but significantly fewer new or enlarging hyperintense lesions were noted on T2-weighted MRI images in participants treated with DMF. These results led to FDA approval in 2013. DMF therapy is often associated with lymphopenia. Therapeutic efficacy does not appear to be inversely related to lymphocyte counts. As with IFN-β and GA, the mechanism by which DMF suppresses MS disease activity remains unclear. The most common side effects of DMF are flushing and gastrointestinal upset. In the postmarketing setting, several cases of progressive multifocal leukoencephalopathy (PML), a rare viral infection of the brain, were reported in patients who were taking DMF and had persistent lymphopenia. PML has also been reported as a complication of other DMAs, including fingolimod and, most notably, natalizumab.

Fingolimod During homeostasis, sphingosine 1 phosphate receptor 1 (S1P1) signaling into lymphocytes drives their egress from lymph nodes into the bloodstream. Fingolimod is an orally administered S1P1 receptor modulator that effectively traps myelin-specific T cells in lymph nodes so that they cannot reenter the circulation and gain access to the CNS. In a 24-month randomized, double-blinded, placebo-controlled trial, fingolimod was shown to reduce annualized relapse rates by 50%.54 Furthermore, the probability of disease progression at the 24-month follow-up was lower in the fingolimod-treated subjects. In an independent 12-month, double-blind, double-dummy phase III comparator study, fingolimod reduced the annualized relapse rate to a range of 0.16–0.20, as compared with 0.33 for IFN-β-1a, corresponding to a relative reduction of 38–52%.45 In 2010, fingolimod became the first oral DMA approved by the FDA to reduce relapses and delay disability progression in patients with relapsing forms of MS. However, in a phase III randomized, double-blind, placebocontrolled trial, fingolimod did not slow disease progression in patients with PPMS.11,12 Fingolimod does not distinguish between pathogenic and protective lymphocytes, and a number of opportunistic infections have emerged as complications of the treatment. Patients taking fingolimod are susceptible herpes virus infections, particularly shingles. In the phase III comparator study with IFN-β-1a, two fatal infections occurred among the fingolimod-treated subjects: disseminated primary varicella zoster and herpes simplex encephalitis.45 Consequently, patients are

CHAPTER 66  Multiple Sclerosis screened for immunity to varicella zoster before commencing treatment. In the postmarketing setting, there have been at least three cases of PML in fingolimod-treated patients with no or only distant prior exposure to immunosuppressant drugs. Other potential side effects of fingolimod include macular edema, bradycardia, and atrioventricular block.

Natalizumab Natalizumab is a humanized mAb reactive against the cell adhesion molecule α4 integrin, which is widely expressed on lymphocytes and monocytes. Natalizumab is believed to mediate its ameliorative effects in RRMS by blocking interactions between the very late antigen-4 (VLA-4; a heterodimer composed of the α4 and β1 integrin chains) on leukocytes with its cognate ligand, vascular cell adhesion molecule (VCAM)-1, on cerebrovascular endothelial cells. VLA-4–VCAM-1 interactions are required for the passage of lymphocytes and monocytes past the BBB. Natalizumab was approved by the FDA for relapsing MS in 2004 following a 2-year phase III trial. Natalizumab reduced the relapse rate at 1 year by 68% and the number of gadolinium-enhancing MRI lesions at both 1 year and 2 years by over 90%, compared with placebo.44 Natalizumab also reduced the risk of sustained progression of disability by 42% over 2 years. In an independent placebo-controlled trial, the addition of natalizumab to IFN-β-1a suppressed break through disease activity and reduced the risk of sustained disability in patients who were refractory to IFN-β alone.55 The effects of natalizumab on leukocyte trafficking are not specific to the CNS. In fact, in 2008, the FDA approved natalizumab for the treatment of refractory Crohn disease. The most serious complication of natalizumab treatment is PML; there have been over 400 reported cases. Factors that increase the risk of natalizumab-associated PML include John Cunningham (JC) virus seropositivity, duration of treatment >2 years, and prior exposure to immunosuppressant drugs.

Alemtuzumab Alemtuzumab is a humanized anti-CD52 mAb that globally depletes circulating T and B lymphocytes via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytolysis, and induction of apoptosis. CD52 is primarily expressed on the cell surface of T and B lymphocytes, but it is also expressed at lower levels on macrophages, eosinophils, and natural killer (NK) cells. The function of CD52 is unknown, although there is some evidence that it is involved in T-cell activation. Hematopoietic stem cells (HSCs) do not express CD52, allowing the subsequent repopulation of circulating lymphocyte pools following alemtuzumab treatment, which occurs at variable rates between lymphocyte subsets. Reconstituted circulating T-cell pools have been found to be enriched in CD4+CD25highCD127lowFOXP3+ regulatory T cells (Tregs) that might be responsible, in part, for the long-lasting therapeutic effect of alemtuzumab, that has been observed in some individuals.56 In a 2-year, rater-masked, randomized controlled phase III trial, previously untreated patients with RRMS were randomly allocated to receive intravenous alemtuzumab or subcutaneous IFN-β-1a.57 Alemtuzumab was given once per day for 5 days at baseline and once per day for 3 days at 12 months. Relapse rates were reduced by 54.9% in the alemtuzumab group compared with the IFN-β-1a group. In an independent study, alemtuzumab reduced relapse rates and the risk of sustained accumulation of disability in patients with RRMS refractory to first-line DMAs.47 Although alemtuzumab suppressed new MRI lesion formation and superimposed relapses

899

in patients with SPMS, it did not prevent clinical progression or progressive cerebral atrophy.7 Despite causing a profound lymphopenia, alemtuzumab therapy has only rarely been associated with opportunistic infections and has not been associated an increased incidence of malignancy. Surprisingly, the principal adverse effect of alemtuzumab in MS is antibody-mediated autoimmune disease, most commonly Graves disease. Idiopathic thrombocytopenic purpura, Goodpasture syndrome, and antiglomerular basement membrane disease have been reported less frequently. The underlying mechanism is not fully understood but may be a consequence of homeostatic T-cell proliferation following lymphoablation, leading to the generation of chronically activated oligoclonal CD4 and CD8 T cells capable of producing proinflammatory cytokines.58

Daclizumab Daclizumab is a humanized mAb reactive to CD25, the α subunit of the IL-2 receptor. Daclizumab was originally proposed as a treatment for RRMS based on the hypothesis that it would block IL-2–dependent expansion of effector T cells. However, mechanistic studies have shown that daclizumab does not inhibit T-cell survival, proliferation, or cytokine production. Some patients treated with daclizumab exhibit an expansion of circulating CD56bright NK cells, which correlates with treatment response.59 CD56bright NK cells have been shown to possess immunoregulatory properties and to kill activated autologous T cells in vitro. Daclizumab might also modulate lymphoid tissue inducer cells in patients with MS and thereby suppress the development of meningeal lymphoid follicles.60 In a phase III randomized placebocontrolled clinical trial in RRMS, subcutaneous administration of daclizumab reduced the annualized relapse rate by 45%, the proportion of patients who relapsed by 41%, and new or newly enlarged T2-weighted MRI lesions by 54%, compared with intramuscular IFN-β-1a over 144 weeks.61 It was approved by the FDA in 2016 for the treatment of relapsing MS in adults. Side effects include an increased risk of infections, hepatotoxicity, noninfectious colitis, lymphadenopathy, and cutaneous events, such as rash and eczema. On the basis of its safety profile, daclizumab is generally reserved for patients who have an inadequate response to ≥2 other disease modifying agents.

B Cell–Depleting Monoclonal Antibodies A role of B cells in the pathogenesis of MS has been suspected since the discovery of unique oligoclonal bands in the CSF of the majority of individuals with MS, indicative of intrathecal antibody synthesis. Nonetheless, B cells are not a prominent component of the perivascular infiltrates in MS lesions. This apparent paradox has been resolved, at least in part, by the discovery of lymphoid follicle–like structures, composed of proliferating B cells, antibody-producing plasma cells, Th cells, and FDCs, in the meninges of some individuals with MS.6 Although meningeal lymphoid follicles have primarily been detected in autopsy specimens from persons with progressive MS, it is possible that nascent immune cell aggregates develop in the RR stage but are disrupted during the dissection and processing of CNS tissues. The most direct evidence for a role of B cells as effector cells in MS pathogenesis comes from trials of mAbs directed against CD20, a B cell–specific surface molecule. CD20 is expressed on preB cells and mature B cells. It is not expressed on antibody-secreting plasma cells. Rituximab, a genetically engineered chimeric anti-CD20 mAb, induces a rapid

900

Part Seven  Organ-Specific Inflammatory Disease

depletion of circulating B cells that lasts approximately 6–9 months. In a phase II clinical trial of rituximab in RRMS, subjects in the active treatment arm experienced a significant reduction in clinical relapses and in the formation of new or enhancing MRI lesions compared with subjects in the placebo arm.62 Ocrelizumab is a next-generation fully human recombinant anti-CD20 mAb that binds to a different epitope from rituximab and with higher affinity. In two large phase III studies, ocrelizumab reduced the annualized relapse rate in subjects with RRMS by nearly 50% compared with IFN-β-1a over a 2-year period.63 Additionally, ocrelizumab delayed confirmed disability progression by approximately 40% and the total number of gadoliniumenhancing lesions by >90% compared with IFNβ-1a. Interestingly, some patients with progressive MS might also benefit from the depletion of CD20+ cells, particularly if there is evidence of ongoing activity at the time of treatment initiation. Hawker et al. found that rituximab significantly delayed the time to confirmed disease progression in a subset of patients with primary progressive MS who had gadolinium enhancing lesions on baseline MRI scans of the brain.9 The benefit was most dramatic in subjects aged 90% of these patients 1-7 In contrast, anti-GQ1b antibodies of the IgM class can be found in patients with chronic

905

IgM paraproteinemic polyneuropathies,6 as discussed later. Anti-GQ1b IgG antibodies are also found in postinfectious ophthalmoplegias as well as in GBS with ophthalmoplegia, but not in those without ophthalmoplegia or other autoimmune conditions.1-7 Of interest, anti-GQ1b antibody binds the paranodal regions of oculomotor nerves III, IV, and VI, suggesting that damage to these regions blocks impulse propagation at the nodes of Ranvier, resulting in a conduction block that is characteristic for GBS. Many patients with antibodies to GQ1b also have antibodies to GD1a. The reasons for different clinical syndromes in connection with specific gangliosides remains unclear, but distribution, accessibility, and density or configuration of gangliosides at different sites may be critical factors. For example, there is more GM1 in ventral roots than in dorsal roots, hence the predominantly motor neuropathy seen with anti-GM1 antibodies; there is also more GQ1b in the ocular motor nerves, which may explain the involvement of eyes in MFS. How these antibodies induce disease, however, remains unclear. Current evidence suggests that antibodies against different acidic glycolipids or sulfatides may be related to different viral or bacterial antecedent factors, as discussed below.

Molecular Mimicry: Relationship Between Campylobacter jejuni and Gangliosides in Acute Motor Axonal Neuropathy Antecedent infection with C. jejuni has been commonly associated with AMAN. The strain of C. jejuni associated with AMAN (Penner D: 19 serogroup) is, however, different from those causing common enteritis and is more likely to have the genes for enzymes that synthesize sialic acid in the bacterial wall mimicking ganglioside GM1, GD1a, or GQ1b.1-5 These patients have a higher incidence of anti-GM1 antibodies, which suggests cross-reactivity between epitopes in the lipooligosaccharide in the bacterial wall and the ganglioside.1-5 Further, injection of lipooligosaccharides extracted from C. jejuni into rabbits has been shown to induce acute neuropathy, with development of anti-GM1 antibodies identical to those found in AMAN.1-7 In addition, immunization of mice with these lipooligosaccharides generates a monoclonal antibody (mAb) that reacts with GM1 and binds to human peripheral nerve. This GM1 mAb as well as the anti-GM1 IgG extracted from patients with GBS block muscle action potentials in muscle–spinal cord coculture. Carbohydrate mimicry between the bacterial lipooligosaccharide and human GM1 is therefore an important cause of AMAN. Because C. jejuni is a common cause of a diarrheal illness worldwide, and diarrhea has been an antecedent event in up to 50% of patients with GBS, Campylobacter appears to trigger the disease in many patients, especially in certain parts of the world. Isolation of Campylobacter from stools early in acute GBS varies from 44% to 88% of patients, and IgG or IgM Campylobacter-specific antibody titers are seen in a higher percentage (36%) of patients with GBS than in controls (10%). Molecular mimicry may not be limited to C. jejuni because GM1 and GQ1b epitopes are also found in the bacteria wall of Hemophilus influenzae, which is also a triggering factor in GBS. GBS triggered by CMV infection has been also associated with the presence of IgM anti-GM2 antibodies. Another potential factor for molecular mimicry is M. pneumoniae, which precedes GBS in 5% of cases and is known to stimulate antibodies against human carbohydrate antigens, including galactocerebroside, the main glycolipid antigen in peripheral nerves.1-7

906

Part Seven  Organ-Specific Inflammatory Disease Barrier

Cytokines (INF-γ, IL-1, IL-2, IL-6 TNF)

(Blood–brain) (Blood–nerve) ICAM-1 MHC

Integrins MAC-1

APC

IL-2 MHC

B cell

TCR MHC-II

LFA-1 T cell

T cell

MHC-I, II IL-2

Anti-MAG Anti-GQ1b Anti-glycolipids

Autoantigen

INF-γ IL-1(fever)

TNF-1 CR1 ICAM-1 Viruses

Mφ

Mφ

TNF IL-1

Igs

Fc

Mφ

MHC CR3

MHC

Mφ

O2 Schwann cell

OH

MHC CR3

AG

FIG 67.1  Sequence of events in the mechanisms of immune-mediated demyelinating polyneuropathy. Cytokines lead to increased expression of major histocompatibility complex (MHC) class I and intercellular adhesion molecules, allowing the sensitized T cells and macrophages to exit the endothelial cell wall and traffic to the peripheral nerve. There they recognize myelin antigen and induce a macrophage-mediated demyelination. The antigen-presenting cells (APCs; probably Schwann cells or macrophages), in concert with MHC class II expression, interact with CD4 T cells and lead to clonal expansion of B cells, producing antibodies against various peripheral nerve antigens.

Molecular mimicry may also play a role in the Zika virus– associated GBS as antiglycolipid antibodies were found in 31% of these patients.8 Molecular mimicry between epitopes of viral proteins (which trigger the disease) and myelin components may result in sensitization of cross-reactive T cells that may stimulate B cells to produce specific antibodies directed against myelin components or may recruit macrophages as effector cells. A combination of cellular and humoral factors therefore seems to participate in the cause of the disease.1-8 Circulating cytokines triggered by the initiating event (viruses or bacteria) could also upregulate intercellular adhesion molecule (ICAM)-1 expression on the endothelial cells and facilitate the entrance of activated T cells or antibodies to the endoneurial parenchyma. It is relevant that ICAM-1 is increased in patients with GBS.1-5 A scheme summarizing the immunopathogenic mechanism is shown in Fig. 67.1.

Nodal and Paranodal Alterations and Specific Antinodal Antibodies Antibodies to nodal and paranodal antigens seen in CIDP subsets, as discussed later, have been also detected in a small number of patients with GBS in Europe. Antibodies against neurofascin, ankyrin G, and contactin could potentially explain the acute conduction block or the paranodal axonal degeneration and the rapid reversibility or slow and incomplete recovery seen in several patients with AMAN 1-5,9,10

KEY CONCEPTS Autoimmunity in Guillain-Barré Syndrome (GBS) Cellular Factors • The peripheral myelin or the Schwann cells are targets. • Activated macrophages are the dominant endoneurial cells and lift the outermost myelin lamellae, lysing the superficial myelin sheaths. • Peripheral blood lymphocytes exert myelinotoxic activity in vitro. • Levels of interleukin-2 (IL-2) and soluble IL-2 receptors are increased during the acute phase of the disease and decline during recovery. Humoral Factors • Serum exerts a complement-dependent demyelination in vitro. • Intraneural injections of serum from patients with acute GBS cause demyelination and conduction block. • Immunoglobulin G (IgG), IgM, and membranolytic attack complex are detected immunocytochemically on the patients’ nerves. • High titers of IgG antibodies against peripheral nerve acidic glycolipids (GM1, GQ1b) are detected in the sera of patients with acute motor axonal neuropathy (AMAN) and Miller Fisher syndrome (MFS). The GQ1b ganglioside is a specific antigen for MFS. • There is a high incidence of antibodies to Campylobacter jejuni, and GM1, with molecular mimicry between Campylobacter and nerve gangliosides. • Injection of lipooligosaccharides extracted from C. jejuni cause AMAN and elicit GM1 antibodies in rabbits. • Antiganglioside antibodies extracted from patients with GBS block muscle action potentials in vitro.

CHAPTER 67  Autoimmune Peripheral Neuropathies

CHRONIC INFLAMMATORY DEMYELINATING POLYNEUROPATHY

a relapsing course, with spontaneous remissions necessitating the need to periodically evaluate the usefulness of continuing immunotherapeutic interventions. Because the demyelination is multifocal, affecting spinal roots, plexuses, and proximal nerve trunks,3,13 the clinicopathological picture may be variable, accounting for the different manifestation of symptoms and signs.2,3,11,12 The most notable CIDP variants include the asymmetrical, unifocal or multifocal, motor–sensory form (LewisSumner syndrome); the pure motor form; the pure sensory form; the sensory ataxic form; and the pure distal form.

CIDP is the most common form of chronic APN with prevalence as high as 9/100 000.2,3,11,12 It is also the most gratifying chronic autoimmune neuropathy because it is treatable in the majority of the cases. It can be considered the chronic counterpart of GBS because of the various clinical, electrophysiological, histological, and laboratory similarities. CIDP differs from GBS predominantly by its tempo, mode of evolution, prognosis, and responsiveness to steroids. First described as a “steroid-responding relapsing polyneuropathy,” CIDP shares with GBS a variety of common autoimmune features.

Diagnosis In CIDP, CSF protein is elevated, up to sixfold, without pleocytosis (except if an infection coexists). Nerve biopsy shows demyelination and remyelination, occasional epineurial or endoneurial T cells, and several macrophages that are either scattered or in small perivascular clusters in the endoneurium (Fig. 67.2).2,3,11,12 Electrophysiological testing is fundamental for the diagnosis by demonstrating the following typical features of demyelination in motor and sensory fibers: (i) slow conduction velocity; (ii) prolonged distal motor or sensory latencies; (iii) prolonged F

Clinical Features and Disease Variants The typical CIDP is characterized by a progressive, symmetrical, proximal and distal muscle weakness; paresthesias; sensory dysfunction; and impaired balance that evolve slowly over at least 2 months.2,3,11,12 Tendon reflexes are absent or reduced. Cranial nerves rarely may be affected. The course is often monophasic, with stepwise progression; at times the disease has

B cell

907

T cell Mac

Antibodies

Blood–nerve barrier

Mφ

Mφ

IL-2

TNF-α

INF-γ Mφ

MMPs Normal conduction

MMPs Mφ

TNF-α NO

Mφ

TNF-α NO

Demyelination and conduction block

Axonal degeneration

FIG 67.2  Diagrammatic scheme of the main cellular and humoral factors implicated in the demyelinating process of chronic inflammatory demyelinating polyneuropathy (CIDP) leading to axonal loss. Activated macrophages (Mϕ) and T cells cross the endothelial cell wall of the blood–nerve barrier and reach the myelinated fibers. Activated, TNF-α-positive Mϕ invade the myelin sheath, causing Mϕ-mediated segmental demyelination. Axonal loss secondary to demyelination, probably enhanced by TNF-α and metalloproteinases, may become prominent in the chronic phases of the disease. Other cytokines, T cells sensitized to unidentified antigens, and putative antibodies may participate. IL, interleukin; IFN, interferon; MMP, metalloproteinase; Mϕ, activated macrophage; TNF, tumor necrosis factor.

908

Part Seven  Organ-Specific Inflammatory Disease

waves latencies; and (iv) conduction block with dispersion of the compound muscle action potentials. An associated axonal loss is not unusual in the majority of CIDP cases. A variety of diagnostic criteria have been proposed to capture the most pertinent of the aforementioned features; the revised European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) guidelines seem the most appropriate because they offer 81% sensitivity and 96% specificity to capture patients more likely to respond to therapies.2,3,11,12 Routine CSF testing and nerve biopsy are not mandatory for the diagnosis,2,3,11,12 but they can be helpful when the results of electrophysiological testing are not convincing, or there is a need to exclude hereditary and vasculitic neuropathies. Other diseases causing neuropathy that should be excluded include severe diabetes (although CIDP seems more frequent in patients with diabetes), neoplasms, amyloidosis, IgM paraproteinemia (IgG or IgA monoclonal gammopathy of undetermined significance (MGUS) can be seen in CIDP), myelomas, vasculitis, alcoholism, exposures to neurotoxic drugs, or family history of neuropathy.

KEY CONCEPTS Autoimmunity in Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) • Activated macrophages are the predominant endoneurial cell, displacing the Schwann cell cytoplasm, disrupting myelin, and lysing superficial myelin lamellae. • Complement-fixing immunoglobulin G (IgG) and IgM antibodies are deposited on the myelin sheath. • IgG antibodies to acidic glycolipids LM1, GM1, or GD1b and against the 28-kDa P0 myelin proteins are detected in the sera of some patients. • There is upregulation of DR and B-7 costimulatory molecules in Schwann cells and macrophages. • Serum IgG can induce conduction block when injected into rat nerves. • Up to 25% of patients with CIDP harbor specific antibodies against antigens in the nodes of Ranvier; in 10% of them these antibodies have been identified as directed against neurofascin-155 and CASPR causing conduction block.

Immunopathogenesis Because of immunopathological similarities to GBS and the relapsing–remitting EAN, in CIDP, activated T cells, macrophages, complement, and autoantibodies seem to work in concert with each other to induce an immune attack against peripheral nerve antigens2,3,11,12 (as depicted in Fig. 67.1), but no triggering factors have been identified so far. The predominant endoneurial mononuclear cells in CIDP are the macrophages that constitute the final effector cells associated with demyelination because they express activation markers probably induced by cytokines released by autoreactive T cells in situ or the circulation. These cells sequentially penetrate the basement membrane of the Schwann cell, displace the cytoplasm, and split the myelin lamellae, resulting in focal lysis of the myelin sheath.2,3,11,12 Macrophages as well as Schwann cells may serve as antigen-presenting cells (APCs) because they express human leukocyte antigen (HLA)-DR and costimulatory molecules B7-1 (CD80) and B7-2 (CD86), whereas their counterreceptors cytotoxic T lymphocyte antigen-4 (CTLA-4) and CD28 are expressed on rare endoneurial CD4 T cells.2,3,14 B7-2–deficient mice also develop CIDP.15 Elevated soluble adhesion molecules, chemokines, cytokines, and metalloproteinases are detected in serum and CSF in CIDP, probably facilitating lymphoid-cell

transmigration across the blood–nerve barrier. Although T cells are not prominent overall, the few CD8 and CD4 cells found endoneurially have monoclonal or oligoclonal restrictions in their T-cell receptor (TCR) repertoire, implying an antigen-driven T-cell response.2,3,12,13 Humoral factors seem to play a predominant role, especially with the recent identification of target antigens at the nodes of Ranvier in >10% of patients with CIDP, even though in the majority of the patients, the pathogenic antigens remain elusive. The beneficial effect of plasmapheresis provides indirect evidence that some circulatory factors are pathogenic. The concept that antibodies may be implicated dates back to >30 years when complement-fixing IgG and IgM were found deposited on a patient’s myelin sheath.16 Antibodies to glycolipids LM1, GM1, or GD1b also have been seen in some patients, but less frequently than in GBS and more frequently than in controls.2,3,11,12 Overwhelming evidence accumulated in the last 5 years indicates that molecules associated with saltatory conduction at the nodes of Ranvier may be more meaningful targets3,4,10,17-19 because functional blockade in these regions can best account for the rapid improvement noticeable within days after plasmapheresis or intravenous immunoglobulin (IVIG). Likely antigenic targets include neurofascin-186, moesin, and gliomedin (at the node); neurofascin-155 (NF155), contacting/Caspr 1 (CNTN1), and connexins (at the paranode); and transient–axonal–glycoprotein-1 or potassium channels (at the juxtaparanode).3,4,10,17-19 Evidence from proteomics, transfected cell lines, teased-nerve fibers, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA) has shown that IgG4 antibodies against paranodal NF155 and CNTN1 antigens are the most relevant and seemingly pathogenic antibodies that are currently detected in at least 10% of patients.17-19 Of interest, anti-NF155– and CNTN1-positive patients appear to have a distinct clinical phenotype with more severe disease, axonal involvement, tremors, sensory ataxia, and suboptimal response to IVIG17-19; these patients, however, respond better to anti–B cell therapies, such as rituximab. Overall, the immunopathogenetic scheme proposed for GBS (see Fig. 67.1) is also appropriately applied to CIDP. Molecular mimicry can be implicated in rare cases of CIDP associated with melanoma because the carbohydrate myelin epitopes GM2, GM3, and GD3 are also expressed on melanoma cells, and antibodies against melanoma cells react with myelin glycoproteins.20 The axonal involvement that accompanies demyelination in both CIDP and GBS, either acutely or after long-standing demyelination, is depicted in Fig. 67.2. The relevant nodal target antigens are presented in Fig. 67.3 separately2,3,10 to highlight their recognized importance in the field of antibody-mediated peripheral demyelination.

MULTIFOCAL MOTOR NEUROPATHY WITH CONDUCTION BLOCK MMN is a distinct disease that, although rare with prevalence of 0.6/100 000, should be recognized early because it is treatable. It affects males more than females and is more common in those 50 years of age.22 The incidence increases to 1.7% above age 70 years and reaches up to 6% above age 90 years. Monoclonal gammopathies, however, are 10 times more frequent in patients with polyneuropathy than in an age-matched control population, and almost 10% of patients with acquired polyneuropathy have MGUS.23 If these gammopathies are categorized into subclasses, the incidence of polyneuropathy among patients with IgM monoclonal proteins can be as high as 50%,23,24 implying that almost 50% of patients with IgM MGUS may have or will develop polyneuropathy. At present, polyneuropathies with MGUS comprise 10% of patients with acquired neuropathy,23-25 and paraproteinemic polyneuropathy is a potentially treatable APN.25 Patients with demyelinating polyneuropathy associated with IgG or IgA MGUS are indistinguishable from those with CIDP; the paraprotein in such patients is coincidental and causally unrelated to neuropathy. In contrast, the demyelinating polyneuropathy associated with IgM MGUS is a distinct clinicopathologic entity, and the IgM is considered pathogenic because it is often directed against myelin glycoproteins or glycolipids.2,3,6,24,25 Some patients with paraprotein may have an associated amyloidosis derived from the variable region of the Ig light chain, primarily λ. When amyloidosis is present, the neuropathy is painful, and the sensorimotor deficits are accompanied by autonomic symptoms consisting of orthostatic hypotension, impotence, impaired gastric motility, or frequent diarrhea. Amyloid neuropathy is difficult to treat; apart from symptomatic therapy (mostly opiates), immunosuppressive therapies, especially with the various anti–B-cell agents, and bone marrow transplantation are largely applied.

ANTIBODIES TO MYELIN-ASSOCIATED GLYCOPROTEIN IN PATIENTS WITH IgM M MONOCLONAL GAMMOPATHIES OF UNDETERMINED SIGNIFICANCE POLYNEUROPATHY (ANTI-MAG NEUROPATHY) Most of these patients present with a sensory, large-fiber, demyelinating polyneuropathy that manifests as sensory ataxia.2,3,24,26 Other patients have a sensorimotor polyneuropathy with mixed features of demyelination and axonal loss. CSF protein is often elevated. Nerve conduction studies have demonstrated slow conduction velocity and a rather characteristic prolonged distal motor and sensory latency indicative of distal demyelination. Sural nerve biopsy demonstrates a diminished number of myelinated axons. On electron microscopy, there is splitting of the outer myelin lamellae, linked to the presence of IgM deposits in the same area of the split myelin sheath. Sera from approximately 50% of these patients react with myelin-associated glycoprotein (MAG), a 100-kilodalton (kDa) glycoprotein of the central and peripheral nerve myelin, as well as other glycoproteins or glycolipids that share antigenic determinants with MAG.2,3,24,27,28 The antigenic determinant for the anti-MAG IgM resides in the carbohydrate component of the MAG molecule.3 The anti-MAG IgM paraproteins coreact with

910

Part Seven  Organ-Specific Inflammatory Disease SGPG

GM1 Gal

Gal

cUA -

GlcNAc SO3

Gal Glc

NeuAc

-

GD1a NeuAc GlcNAc Gal Glc

GlcNAc -

GT1b

Gal

Gal

-

NeuAc

GD1b

Gal Glc

NeuAc

GlcNAc

-

NeuAc

-

Gal

NeuAc NeuAc - NeuAc

Glc

Gal

GQ1b NeuAc - Gal NeuAc GlcNAc GlcNAc NeuAc - Gal Gal NeuAc Glc

Glc

FIG 67.4  Glycolipids implicated as antigens in immune-mediated neuropathies. Sulfate-3-glucuronyl paragloboside (SGPG) is the glycolipid sharing a carbohydrate epitope with myelin-associated glycoprotein (MAG), and the terminal sulfated glucuronic acid is a key part of the epitope. GM1 is the ganglioside implicated in motor nerve disorders, and in most cases the terminal Gal (β1-3) GalNAc epitope, which is shared with GD1b, is involved. The disialosyl moiety implicated in sensory neuropathies consists of NeuAca2—8NeuAc—and is present in GD1b and GT1b gangliosides, as well as the simpler GD2 and GD3 gangliosides (not shown). GQ1b ganglioside, which is the target antigen in Miller Fisher syndrome (MFS), has two disialosyl moieties. Although GD1a ganglioside has two sialic acid residues they are not linked to each other, so antibodies to GD1a do not cross-react with anti-GD1b antibodies. The color-coded sugar moieties represent key aspects of the various epitopes, but carbohydrate sequences recognized by the antibodies may include additional sugar residues. GlcUA, glucuronic acid; Gal, galactose; G1cNAc, N-acetylglucosamine; Glc, glucose; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid (sialic acid).

an acidic glycolipid in the ganglioside fraction of human peripheral nerves, identified as a sulfoglucuronyl glycosphingolipid (SGPG).28 In contrast to MAG, which is mostly present in the central nervous system, SGPG is found only in the peripheral nerves. The sera of some patients with IgM-MGUS with sensory ataxia may not react with MAG, but with various gangliosides, most commonly those that contain either a disialosyl moiety, such as GD1b, GQ1b, GT1b, GalNac-GM1b, and GalNAc-GD1a, or two gangliosides that share epitopes with GM2, or a combination of GM2 and GM1, GM1 and GD1b.2,3,6,24,27,28 More than half of the IgM paraproteins recognize MAG and SGPG, and 75% of the rest recognize ganglioside antigens, indicating that acidic glycolipids are the most common antigenic epitopes.2,3,6,24,27,28 The glycolipids implicated as antigens in immune-mediated neuropathies are depicted in Fig. 67.4. Human anti-MAG antibodies can be detected readily in sera with ELISA or preferably with standard Western blot. Because anti-MAG–reacting sera always recognize the SGPG glycolipid, the assay is often performed by using SGPG as antigen instead of purified human MAG. It is preferable, however, to use MAG as the target antigen, rather than SGPG, because the IgM binds to MAG 10–100 times the affinity compared with SGPG—that is, low-affinity anti-MAG antibodies can be missed if SGPG is used as the antigen. The following factors suggest that these antibodies are related to the cause of the neuropathy: 1. IgM and complement are deposited on the myelinated fibers on the patient’s sural nerve biopsy,2,3 suggesting that activated complement may be needed in the induction of demyelination. 2. IgM recognizes neural cell adhesion molecules and colocalizes with MAG on the areas of the split myelin lamellae, suggesting involvement in myelin disadhesion. Skin biopsies from these patients have also confirmed the presence of IgM, complement

C3d, and MAG deposition on the dermal myelinated fibers and the concurrent loss of nerve fibers.29 3. Injection of serum from patients with IgM anti-MAG/SGPG paraprotein supplemented with fresh complement into feline peripheral nerve causes complement-dependent demyelination and conduction block within 2–9 days of the injection.30 The IgM injected intraneurally localizes to the outer layer of the myelin sheath.

KEY CONCEPTS Autoimmunity in Polyneuropathy With Immunoglobulin G (IgM) Monoclonal Gammopathy • In more than 50% of patients IgM is an antibody against two antigens, myelin-associated glycoprotein (MAG) and sulfoglucuronyl glycosphingolipid (SGPG). • In many patients with non-MAG–reacting monoclonal IgM, the IgM recognizes (i) gangliosides containing disialosyl moieties, including GM1, GM2, GD1b, GD1a, and LM1; (ii) sulfatides; and (iii) rarely, chondroitin sulfate. • Overall, in at least 75% of patients the IgM recognizes gangliosides that appear to be the primary antigenic targets. • IgM is deposited on the homologous myelin sheath and fixes complement. • IgM, when deposited on the myelin sheath, results in disadhesion and separation of the myelin lamellae and disruption of normal myelin function. • Intraneural injection of anti-MAG–reacting IgM or passive transfusion into experimental animals causes segmental demyelination, whereas complement-fixing IgM is immunolocalized to the myelin sheath, causing myelin separation. • Immunization of cats with purified SGPG causes an ataxic neuropathy, similar to the one seen in humans, with involvement of the dorsal root ganglia.

CHAPTER 67  Autoimmune Peripheral Neuropathies 4. Systemic transfusion of anti-MAG IgM paraproteins produces segmental demyelination in chickens,31 with deposition of IgM on to the outer lamellae of the myelin along with splitting of the myelin lamellae, similar to that observed in the human neuropathy. 5. Immunization of cats with purified SGPG causes an ataxic neuropathy, similar to the one seen in humans, with involvement of the dorsal root ganglia including inflammation within the ganglionic neurons.32

POLYNEUROPATHY, ORGANOMEGALY, ENDOCRINOPATHY, MYELOMA, AND SKIN CHANGES A subset of patients with malignant IgG or IgA monoclonal proteins have polyneuropathy with osteosclerotic myeloma. Most of them have POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes).33 Not included in the acronym are several features, such as sclerotic bone lesions, giant lymph node hyperplasia (Castleman disease), papilledema, pleural effusion, edema, ascites, and thrombocytosis.34 More than 50% of patients with osteosclerotic myeloma of the IgA or IgG type have a sensorimotor, symmetric polyneuropathy with mixed demyelinating and axonal features and high (usually 200 mg/ dL) CSF protein. Pure axonal neuropathies can be also seen. In POEMS, the neuropathy tends to be associated with edema in the legs, with hyperpigmentation, sclerodermatous thickening, or papular angiomas of the skin, and hypertrichosis with dark hair. Endocrinopathy most often includes gonadal failure, amenorrhea, impotence, gynecomastia, hypothyroidism, diabetes, or elevated prolactin levels. The IgG class is slightly more common than the IgA class, with λ light chain present in the majority of the patients.34 Bone lesions can be sclerotic, solitary, or multiple, sparing the skull and the extremities. Pathological changes in lymph nodes resemble those of Castleman disease, which can also be associated with polyneuropathy.34 In POEMS, there is an imbalance of proinflammatory cytokines with increased IL-1β, IL-6, and tumor necrosis factor-α (TNF-α). Vascular endothelial growth factor (VEGF) may play a major role because it induces a rapid increase in vascular permeability and is a growth factor important in angiogenesis and endothelial cells.34 In some patients, the neuropathy responds to steroids, tamoxifen, or alkylating agents. In others, it may respond to the removal or irradiation of the solitary sclerotic lesion, suggesting that the tumor may secrete neurotoxic factors. IVIG and plasmapheresis are ineffective. The median survival is 165 months.35 Recently, autologous peripheral blood stem cell transplantation has been shown to result in significant improvement with reduction of VEGF, improved nerve conduction velocity, and increased survival.35

Cryoglobulinemic Neuropathy Cryoglobulins are proteins that precipitate in the cold and redissolve when heated (Chapter 58). There are three types of cryoglobulin: type I, which is monoclonal, often of the IgM and IgG class; type II, which is mixed polyclonal, with one monoclonal (often monoclonal IgM with polyclonal IgG); and type III, which is polyclonal (often IgM and IgG). Polyneuropathy occurs most often with mixed cryoglobulinemias and presents as distal, sensory, symmetrical polyneuropathy or as mononeuropathy multiplex. These patients also have purpura, polyarthralgias, cutaneous

911

vasculitis, Raynaud phenomenon, renal involvement with proteinuria, and polyneuropathy. Type II cryoglobulinemia may be associated with an underlying lymphoproliferative process. There is an increased incidence (up to 90%) of hepatitis C virus infection in patients with mixed cryoglobulinemia. The nerve biopsy shows perivascular inflammatory cuffing with axonal degeneration.

PARANEOPLASTIC PERIPHERAL NEUROPATHIES WITH ANTI-HU ANTIBODIES Peripheral neuropathy is not an uncommon complication of cancer, related either to the systemic effects of the tumor or, more often, to various neurotoxic chemotherapeutic agents. It usually affects the small nerve fibers causing numbness or painful dysesthesias. The most distinct immune-related neuropathy in patients with cancer is paraneoplastic sensory neuronopathy (PSN), often associated with small cell lung cancer, and to a lesser degree with breast cancer or other neoplasms, such as lymphoma or thymoma.36 It might be the presenting symptom preceding the discovery of the tumor by months. PSN has a unique clinical picture, characterized by burning or aching paresthesias; sensory loss of the large fibers, causing profound sensory gait ataxia and choreoathetotic movements related to loss of proprioception in the feet and hands; normal strength; and areflexia. Some patients may have autonomic dysfunction, encephalopathic symptoms, or cerebellar disturbances. CSF protein is increased, electrophysiological testing shows an axonal sensory neuropathy, and nerve biopsy demonstrates axonal degeneration with rare mononuclear cell infiltrates. PSN is a ganglionopathy (sensory neuronopathy) caused by a variable degree of inflammation in the dorsal root ganglionic neurons. These patients have specific IgG anti-Hu autoantibodies directed against a closely spaced group of proteins with a molecular weight of 35–40 kDa.36 The antibodies are found in higher titers in CSF, suggesting intrathecal synthesis. The Hu protein is also present in the tumors of patients with PSN. Further, low titers of anti-Hu antibodies can be seen in up to 20% of patients with small cell lung cancer, even without neurological symptoms, suggesting that PSN may be the result of an autoimmune reaction against antigens shared by both the tumor cells and the dorsal root ganglionic neurons. Although the role of anti-Hu antibodies in the causation of PSN is unclear, these antibodies are specific markers to detect an occult small cell lung cancer in patients who present with sensory ataxic neuropathy. Some patients may have other paraneoplastic antibodies, such as collapsing response mediator protein (CRMP-5) often referred to as anti-CV2.36

AUTOIMMUNE AUTONOMIC NEUROPATHIES Autoimmune autonomic neuropathy (AAN) is highlighted by circulating antibodies against the ganglionic nicotinic acetylcholine receptors (AChRs).37 These patients present with a subacute (within 4 weeks) or chronic (within months) onset of neurogenic orthostatic hypotension, defined as a systolic blood pressure reduction of at least 30 mm Hg or mean blood pressure reduction of at least 20 mm Hg that occurs within 3 minutes of head tilting. The subacute onset is often preceded by a viral infection. In addition, patients demonstrate three or four parasympathetic/enteric symptoms: sicca (dry eyes and dry mouth); abnormal pupillary response to light; upper gastrointestinal symptoms (early satiety, postprandial nausea, and vomiting that lead to severe weight loss); and neurogenic bladder.

912

Part Seven  Organ-Specific Inflammatory Disease

Patients with more severe cholinergic impairment have higher-titer antibodies against ganglionic AChRs. Some of these symptoms can be passively transferred to mice injected with the patient’s IgG, suggesting that these antibodies may be pathogenic. Further, rabbits immunized with a fragment of ganglionic AChR protein exhibit autonomic failure, similar to the human disease.37 Because ganglionic AChRs have also been found in small cell lung carcinoma cell lines, cancer may be a potential initiator of ganglionic AChR autoimmunity.

MONONEUROPATHY MULTIPLEX AND LOCALIZED, ISOLATED VASCULITIS OF THE PERIPHERAL NERVES Polyneuropathy is a common manifestation of systemic vasculitis. It occurs in patients with polyarteritis nodosa; connective tissue diseases, such as rheumatoid arthritis or Sjögren syndrome; hypersensitivity vasculitis; Churg-Strauss syndrome; temporal arteritis; and viral infections, such as those with HIV, human T-lymphocyte virus (HTLV)-1, and hepatitis B and C viruses. It classically presents as mononeuritis multiplex affecting several individual nerves with painful weakness and paresthesias caused by ischemia and infarcts caused by inflammation of endoneurial blood vessels. There is, however, a distinct vasculitic entity localized only to the peripheral nerve, known as isolated peripheral nerve vasculitis (PNV). PNV involves the small and medium-sized arteries of the epineurium and perineurium and causes ischemic changes within the peripheral nerve. The presentation is similar to the vasculitic neuropathy seen in systemic vasculitis—the only difference is the lack of systemic organ involvement, slower onset and progression, and negative serology. The diagnosis is confirmed with nerve biopsy of the sural, superficial peroneal, or superficial radial nerve. When nerve biopsy is combined with muscle biopsy, the diagnostic yield is higher. PNV has a better prognosis compared with systemic vasculitides and is a treatable form of neuropathy. An evaluation for PNV should include all the tests needed to exclude systemic vasculitis and cryoglobulinemia, as well as hepatitis B and C infections, which can be associated with PNV.

NEUROPATHY WITH VIRUSES AND HUMAN IMMUNODEFICIENCY VIRUS Neuropathy can be seen in a setting of infectious, viral, or bacterial processes. In patients with Lyme disease, various neuropathies, including GBS and mononeuritis (Bell palsy), have been noted. Other infections, such as CMV, hepatitis, herpes, leprosy, Chagas disease, and diphtheria, can affect the peripheral nerves, triggering autoimmune peripheral neuropathy. Common neuropathies seen today in a setting of a viral infection are those associated with HIV and include GBS, CIDP, acute ganglioneuritis, or mononeuritis multiplex; they occur early in the infection, or they are the presenting manifestation of unsuspected HIV infection.38 HIV has been cultured from the peripheral nerves, and HIV viral RNA has been amplified from sural nerve biopsies of some patients in the author’s laboratory. However, there is no convincing evidence that the neuropathy results from direct infection of the peripheral nerves by the virus. Immunocytochemical studies have shown that HIV is present only in rare endoneurial macrophages, but not within Schwann cells or axons. A strong expression of HLA class I and

A

HLA-DR, Macrophages, CD8 T cells

B

C

FIG 67.5  Serial sections of a nerve biopsy from a patient with human immunodeficiency virus (HIV)–chronic inflammatory demyelinating polyneuropathy stained for (A) human leukocyte antigen (HLA)-DR, (B) macrophages, and (C) CD8 T shows that the majority of the endoneurial cells are macrophages. Only rare CD8 cells are noted.

class II molecules on Schwann cells, endothelial cells, and/or macrophages, but sparse presence of CD8 and CD4 T cells, is also noted (Fig. 67.5). It is possible that systemic viral infection or rare HIV-infected endoneurial lymphoid cells may release cytokines that expose new nerve antigens against which there is no self-tolerance, generating a tissue-specific autoimmune attack. A rare neuropathy seen in later-stage HIV infection is a lumbosacral polyradiculoneuropathy related to CMV infection that affects roots and sensory ganglia. It presents with lowerextremity muscle weakness, sacral and distal paresthesias, areflexia, and atrophy, mostly of the legs, associated with sphincteric dysfunction resembling cauda equina syndrome. CMV inclusions can be found within Schwann cells or endothelial cells (Fig. 67.6). Early recognition is important because anti-CMV therapy with ganciclovir or foscarnet can be helpful. At present, the most common neuropathy in patients with acquired immunodeficiency syndrome (AIDS) is a painful sensory axonal neuropathy that affects up to 70% of adults with AIDS and

CHAPTER 67  Autoimmune Peripheral Neuropathies

FIG 67.6  Cross-section of a root from a patient with human immunodeficiency virus (HIV)–associated Guillain-Barré syndrome (GBS) shows cytomegalovirus inclusions within the Schwann cell.

occurs later in the disease.38 It is caused by a cumulative effect, on the peripheral nerves, of various endogenous or exogenous neurotoxins related to a multisystem disease and dysfunction of many organs along with toxicity from various antiretroviral drugs. Clinical findings include distal painful dysesthesias, sensory loss or hypesthesia, areflexia, and, in advanced cases, distal weakness. Despite the relative lack of motor involvement, the severity of neuropathic pain can be disabling.

TREATMENT APNs are clinically important because they are potentially treatable with various immunosuppressive, immunomodulating, or chemotherapeutic agents. The selection of an effective protocol is based on the results of experimental therapeutic trials, clinical experience, and the risk–benefit ratio of available therapies. The author’s approach to the treatment of these disorders is described below.

Guillain-Barré Syndrome

Supportive Care The dramatic reduction in the mortality of GBS is mainly attributed to the availability of ICUs, improvement of respiratory support, antibiotic therapy, and control of autonomic cardiac dysregulation. A patient with GBS is best monitored in an ICU, even if respiratory compromise is not evident at the time of admission. When forced vital capacity (FVC) drops or bulbar weakness is severe, intubation is necessary. Plasmapheresis In several double-blind controlled studies, plasmapheresis has been shown to be effective if performed within the first week from onset of the illness. A series of five or six exchanges, with one exchange every other day, is sufficient. Early relapses can occur in up to 20% of patients, who may require a second series of plasma exchanges.1-5 Plasmapheresis has been shown to be effective even in mild cases of GBS; two exchanges are sufficient for mild GBS, and four are optimal for moderate cases, but there is no difference between those who receive four plasma exchanges and those who receive six.

913

High-Dose Intravenous Immunoglobulin On the basis of the results from two controlled studies,1,4 IVIG (Chapter 84), given at 2 g/kg over 2–5 days, has been shown to be equally effective as plasmapheresis, with no added benefit when the two procedures were combined. The decision as to which treatment to choose is governed by circumstances, availability of the treatment modality, experience, age of the patient, and other associated conditions. Early relapses can also occur with IVIG, as often as with plasmapheresis. IVIG has become the therapeutic choice worldwide because it is easy to administer and more readily available and because time to initiate treatment is of the essence. Steroids are ineffective in GBS and may even increase the incidence of future relapses. Combining IVIG with IV methylprednisolone has shown no significant added benefit.

Chronic Inflammatory Demyelinating Polyneuropathy

Prednisone CIDP has been originally described as a classic steroid-responsive polyneuropathy. The efficacy of steroids was proven in a controlled study, albeit with inadequate blinding, but reconfirmed in another.2,3,11,12,39 A high-dose regimen of 80–100 mg prednisone daily is preferred, followed by tapering to every-other-day dosing. Azathioprine, cyclosporine, or mycophenolate can be used as steroid-sparing agents, but their efficacy, although not tested in controlled studies, has been disappointing overall. Methotrexate in a controlled study was ineffective.2,3,11,12,39 Intravenous Immunoglobulin In several controlled studies,2,3,11,12,39 IVIG has been effective in the majority of patients with CIDP. The more chronic the disease and more severe the axonal degeneration that has taken place, the lower are the chances that the recovery will be complete or significant. IVIG, in the largest-ever controlled study, has been proven an effective first-line therapy, and maintenance therapy has prevented relapses. Plasmapheresis Plasmapheresis has been also effective in controlled studies.2,3,11,12 After a series of six plasma exchanges, maintenance therapy, with one exchange at least every 8 weeks, may be required if this therapy is beneficial. IVIG has now replaced plasmapheresis, although in the author’s experience, some patients may benefit more from steroids, others more from IVIG, and still others more after plasmapheresis.

Polyneuropathy With Paraproteinemias Patients with benign IgG or IgA demyelinating polyneuropathies respond in a manner similar to CIDP patients. Patients with malignant paraproteinemias should be treated with chemotherapy, as needed for the underlying disease. When the neuropathy is axonal, treatments are generally disappointing. For IgM anti-MAG demyelinating polyneuropathies, treatments with prednisone plus chlorambucil, plasmapheresis, and IVIG2,3,40 has shown a variably marginal benefit. Rituximab, an mAb against CD20, is the most promising therapy,41 providing efficacy in almost 40% of the patients in a small double-blind study, as confirmed later with a larger study,33 even though both did not reach significance. Additional, uncontrolled series with many patients, have confirmed that rituximab is effective in 30–40% of these patients.

914

Part Seven  Organ-Specific Inflammatory Disease

MULTIFOCAL MOTOR NEUROPATHY MMN responds very well only to IVIG, which is the treatment of choice based on controlled trials. In difficult cases, cyclophosphamide or rituximab may be promising, but no controlled studies have been carried out.2,3,21

Paraneoplastic Anti-Hu Neuropathy Anecdotally, some of these patients have responded to plasma exchange or IVIG, but overall this neuropathy has not been consistently responsive to available therapies.

Vasculitic Neuropathies For isolated peripheral nerve vasculitis, a combination of prednisone 1.5 mg/kg/day with cyclophosphamide 2 mg/kg/day orally, or 1 g/m2 intravenously monthly for 6 months, are the treatments of choice; specific treatment protocol may vary from patient to patient. The administration of cyclophosphamide may not be necessary for >6 months, as in systemic vasculitis. Plasmapheresis has been tried in cryoglobulinemic neuropathies, with variable results.

Human Immunodeficiency Virus Neuropathies The two demyelinating neuropathies, GBS and CIDP, are treated with the same immunomodulatory therapies as used in HIVnegative patients with IVIG being the preferable choice. Ganciclovir may be helpful in CMV-related polyradiculoneuropathy. Painful sensory neuropathy can be quite disabling because of intractable pain. Tricyclic antidepressants (TCAs), nonsteroidal antiinflammatory drugs (NSAIDs), anticonvulsants (carbamazepine, gabapentin, topiramate), and topical capsaicin in various combinations provide some relief from neuropathic pain.

ON THE HORIZON • Identification of common causes that break tolerance, either in connection with the gut microbiome or exogenous agents, and expansion of the current limited data on molecular mimicry between nerve glycoproteins and viral or bacterial antigens • Development of neuroimaging capabilities to image peripheral nerves and dorsal roots in vivo and quantify inflammation, demyelination and axonal degeneration, providing an accessible and reliable tool for diagnosis and monitoring response to therapies, in a model similar to magnetic resonance imaging (MRI) in multiple sclerosis • Performance of proteome studies aimed to identify biomarkers of nerve autoimmunity that may lead to specific therapies • Trials with target-specific therapies for acute and chronic demyelinating neuropathies, applied in combination with the existing therapies, targeting early in the disease process complement activation, regulatory T cell (Treg) functions, key proinflammatory cytokines, and B cells • Identification of agents and trophic factors that could prevent axonal degeneration from the outset, promote remyelination, prevent axonal loss, or trigger axonal regeneration and reverse, early in the disease process, a seemingly “permanent” clinical deficit

ACKNOWLEDGMENTS The author thanks the various Neurology Fellows who provided excellent care to his patients; and Drs. Amjad Ilyas and Richard Quarles for help with immunochemistry of MAG and SGPG and gangliosides.

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Yuki N, Hartung H-P. Guillain-Barre syndrome. N Engl J Med 2012;366:2294–304. 2. Dalakas MC. Pathophysiology of autoimmune polyneuropathies. Presse Med 2013;42(6 Pt 2):e181–92. 3. Dalakas MC. Pathogenesis of autoimmune neuropathies. Biochim Biophys Acta 2015;1852:658–66. 4. Willison HJ, Jacobs BC, van Doorn PA. Guillain-Barré syndrome. Lancet 2016 Feb 29. pii: S0140-6736(16). 5. Kuwabara S, Yuki N. Axonal Guillain-Barre syndrome: concepts and controversies. Lancet Neurol 2013; 12; 1180-8. 6. Dalakas MC, Quarles RH, editors. Autoimmune ataxic neuropathies (sensory ganglionopathies): are glycolipids the responsible autoantigens? Ann Neurol 1996;39:419–22. 7. Illa I, Ortiz N, Gallard E, et al. Acute axonal Guillain–Barré syndrome with IgG antibodies against motor axons following parenteral gangliosides. Ann Neurol 1995;38:218–24. 8. Cao-Lormeau V-M, et al. Guillain-Barre Syndrome outbreak associated with Zika virus infection in French Polynesia: a case controlled study. Lancet 2016;387(10027):1531–9. 9. Uncini A, Kuwabara S. Nodopathies of the peripheral nerve: an emerging concept. J Neurol Neurosurg Psychiatry 2015;86:1186–95. 10. Stathopoulos P, Alexopoulos H, Dalakas MC. Autoimmune antigenic targets at the node of Ranvier in demyelinating disorders. Nat Rev Neurol 2015;11:143–56. 11. Dalakas MC. Advances in the diagnosis, pathogenesis and treatment of CIDP. Nat Rev Neurol 2011;7(9):507–17. 12. Koller H, Kieseier BC, Jander S, et al. Chronic inflammatory demyelinating polyneuropathy. N Engl J Med 2005;352:1343–56. 13. Schneider-Hohendorf T, Schwab N, Uceyler K, et al. T cell immunity in CIDP. Neurology 2012;78:402–8. 14. Murata K, Dalakas MC. Expression of the co-stimulatory molecule BB-1, the ligands CTLA-4 and CD28 and their mRNAs in chronic inflammatory demyelinating polyneuropathy. Brain 2000;123:1660–6. 15. Salomon B, Rhee L, Bour-Jordan H, et al. Development of spontaneous autoimmune peripheral polyneuropathy in B7-2-deficient NOD mice. J Exp Med 2001;194:677–84. 16. Dalakas M, Engel WK. Immunoglobulin deposits in chronic relapsing polyneuropathies. Arch Neurol 1980;37:637–40. 17. Devaux JJ, Miura Y, Fukami Y, et al. Neurofascin-155 IgG4 in chronic inflammatory demyelinating polyneuropathy. Neurology 2016;86:800–7. 18. Miura Y, Devaux JJ, Fukami Y, et al. Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia. Brain 2015;138:1–8. 19. Dalakas MC, Gooch C. Close to the node but still far: What antibodies tell us about CIDP and its therapies. Neurology 2016;86:796–7. 20. Weiss MD, Luciano CA, Semino-Mora C, et al. Molecular mimicry in chronic inflammatory demyelinating polyneuropathy and melanoma. Neurology 1998;51:1738–41. 21. Van Asseldonk JT, Franssen H, Van den Berg-Vos RM, et al. Multifocal motor neuropathy. Lancet Neurol 2005;4:309–19. 22. Kyle RA, Therneau TM, Rajkumar SV, et al. Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med 2006;354:1362–9. 23. Kelly JJ, Kyle RA, O’Brien PC, et al. The prevalence of monoclonal gammopathy in peripheral neuropathy. Neurology 1981;31:1480–3. 24. Latov N, Hays A, Sherman WH. Peripheral neuropathy and anti-MAG antibodies. Crit Rev Neurobiol 1988;3:301–32. 25. Dalakas MC. Pathogenesis and treatment of anti-MAG neuropathy. Curr Treat Options Neurol 2010;12:71–83. 26. Dalakas MC, Engel WK. Polyneuropathy and monoclonal gammopathy: studies of 11 patients. Ann Neurol 1981;10:45–52.

CHAPTER 67  Autoimmune Peripheral Neuropathies 27. Ilyas AA, Quarles RH, McIntosh TD, et al. IgM in a human neuropathy related to paraproteinemia binds to a carbohydrate determinant in the myelin-associated glycoprotein and to a ganglioside. Proc Natl Acad Sci USA 1984;81:1225–9. 28. Ilyas AA, Quarles RH, Dalakas MC, et al. Polyneuropathy with monoclonal gammopathy: glycolipids are frequently antigens for IgM paraproteins. Proc Natl Acad Sci USA 1985;82:6697–700. 29. Lombardi R, Erne B, Lauria G, et al. IgM deposits on skin nerves in anti-myelin-associated glycoprotein neuropathy. Ann Neurol 2005;57:180–7. 30. Willison HJ, Trapp BD, Bacher JD, et al. Demyelination induced by intraneural injection of human antimyelin associated glycoprotein antibodies. Muscle Nerve 1988;11:1169–76. 31. Tatum AH. Experimental paraprotein neuropathy; demyelination by passive transfer of human IgM anti-MAG. Ann Neurol 1993;33:502–6. 32. Ilyas AA, Gu Y, Dalakas MC, et al. Induction of experimental ataxic sensory neuronopathy in cats by immunization with purified SGPG. J Neuroimmunol 2008;193:87–93. 33. Leger JM, Guimaraes-Costa R, Muntean C. Immunotherapy in peripheral neuropathies. Neurother 2016;13:96–107. 34. Dispenzieri A, Kyle RA, Lacy MQ, et al. POEMS syndrome: definitions and long-term outcome. Blood 2003;101:2496–506.

915

35. D’Souza A, Lacy M, Gertz M, et al. Long-term outcomes after autologous stem cell transplantation for patients with POEMS syndrome (osteosclerotic myeloma): a single-center experience. Blood 2012;120:56–62. 36. Muppidi S, Vernino S. Paraneoplastic neuropathies. Continuum (Minneap Minn) 2014;20:1359–72. 37. Lennon VA, Ermilov LG, Szurszewski JH, et al. Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease. J Clin Invest 2003;111:907–13. 38. Dalakas MC, Pezeshkpour GH. Neuromuscular diseases associated with human immunodeficiency virus infection. Ann Neurol 1988;23:38–48. 39. Hughes RA, Donofrio P, Bril V, et al. Intravenous immune globulin (10% caprylate-chromatography purified) for the treatment of chronic inflammatory demyelinating polyradiculoneuropathy (ICE study): a randomised placebo-controlled trial. Lancet Neurol 2008;7:136–44. 40. Dalakas MC, Quarles RH, Farrer RG, et al. A controlled study of intravenous immunoglobulin in demyelinating neuropathy with IgM gammopathy. Ann Neurol 1996;40:792–5. 41. Dalakas MC, Rakocevic G, Salajegheh M, et al. Placebo-controlled trial of rituximab in IgM anti-myelin associated glycoprotein antibody demyelinating neuropathy. Ann Neurol 2009;65:286–93.

CHAPTER 67  Autoimmune Peripheral Neuropathies

915.e1

MULTIPLE-CHOICE QUESTIONS 1. Which of the following findings predicts an incomplete recovery in patients with Guillain-Barré syndrome (GBS)? A. Slow initial progression B. Absence of bulbar findings C. Presentation in the third decade D. Requirement of mechanical ventilation

3. Which of the following tests is the most useful in establishing the diagnosis of GBS? A. Sural nerve biopsy B. Cerebrospinal fluid (CSF) protein levels C. Nerve conduction study D. Brain magnetic resonance imaging (MRI)

2. Which is the most common form of GBS? A. Acute inflammatory demyelinating polyneuropathy B. Acute motor axonal neuropathy C. Acute motor–sensory neuropathy D. Miller Fisher syndrome

4. Which of the following finding is characteristic of chronic inflammatory demyelinating polyneuropathy (CIDP)? A. Asymmetrical weakness B. Sensory dysfunction C. Normal peripheral reflexes D. Cranial nerve involvement

68  Immunological Renal Diseases Meryl Waldman, Howard A. Austin III, James E. Balow

Compelling clinical, pathological, and experimental data indicate that most forms of glomerular diseases manifest some element of immune-mediated injury.1 Major advances in our understanding of the etiology and pathogenesis of the various glomerular diseases have occurred in the past decade. However, the primary events or inciting factors that trigger the host immune responses and secondary pathways that are directly or indirectly pathogenic to the kidney are still not fully elucidated. In some conditions, normal or allergic immune responses to exogenous infectious agents or drugs can lead to incidental nephropathic injury (e.g., postinfectious glomerulonephritis, serum sickness). In the case of autoimmune diseases, nephropathic processes range from loss of self-tolerance to specific constitutive renal tissues (e.g., antiglomerular basement membrane [GBM] disease) or to extrarenal tissues and cells (e.g., antineutrophil cytoplasmic autoantibodies [ANCAs]). Other mechanisms include excessive and unregulated polyclonal immune responses that produce nephritogenic immune complexes (e.g., lupus nephritis).1,2 Recent advances have begun to uncover candidate antigens involved in the pathogenesis of immune-mediated renal diseases (e.g., some subsets of membranous nephropathy [MN]). But it has become increasingly apparent that the immune system does not operate in isolation and that there are extraordinarily complex interactions among vast networks involving components of native and adaptive immune systems, humoral and cell-mediated immune systems, small-molecular-weight mediators derived from lymphoid and other cell types, complement factors and their regulatory proteins, other inflammatory and coagulation system pathways, microvascular biology, and tissue repair, as well as sclerosis and fibrosis reactions. Underlying this constellation are associated genetic risk factors that modulate these responses and predispose to a nephritogenic response. Rapidly evolving molecular and genetic technologies with the tools of modern systems biology and bioinformatics will continue to unravel these complex interactions.3 Appropriate evaluation of patients with immune-mediated kidney diseases requires particular attention to the findings of urinalysis, assessment of the type and amount of proteinuria, tests of renal functions, and renal biopsy. Urine microscopy also plays a pivotal role in the assessment of urinary disorders.

HEMATURIA Red blood cells (RBCs) that emanate from lesions of the calices, ureters, bladder, or urethra tend to maintain their normal morphology; when nondysmorphic RBCs are present, it is usually appropriate to refer the patient to urology for further evaluation.

Urinary RBCs that result from glomerular or tubulointerstitial pathology are more likely to appear dysmorphic (abnormal shapes and sizes, fragmented); when these dysmorphic RBCs, called acanthocytes, are present, it is usually appropriate to refer the patient to nephrology for further evaluation. Erythrocyte and/or leukocyte casts are indicative of glomerulonephritis (or interstitial nephritis). Cellular casts can be formed from erythrocytes and/ or leukocytes that enter the tubular lumen because of glomerular or tubular inflammation (Fig. 68.1).

PROTEINURIA Glomerular proteinuria results from a loss of the size-selective and/or charge-selective properties of the glomerular capillary wall and disruptions of the glomerular epithelial cells (podocytes), allowing plasma proteins (especially albumin) to leak into the filtrate. Tubulointerstitial nephropathy often leads to impaired tubular absorption of other normally filtered low-molecularweight proteins; this so-called tubular proteinuria exhibits a characteristic pattern on urine protein electrophoresis (low fraction of albumin), rarely exceeds 2 g/day, and is often associated with other manifestations of tubular dysfunction. Estimates of proteinuria are based on 24-hour urine collections or on the protein/creatinine or albumin/creatinine concentration ratios in random urine samples. Normally, these ratios should be 3.5 g/day or protein to creatinine ratio >2.5) resulting in hypoalbuminemia, edema, hyperlipidemia, and lipiduria. The degree of proteinuria can offer a helpful diagnostic clue because some immune-mediated conditions with typically diffuse glomerular disease (e.g., MN, systemic lupus erythematosus [SLE]) are more likely than others with typically focal disease (e.g., IgA nephropathy, ANCA-associated glomerulonephritis) to be associated with nephrotic syndrome.

917

918

Part Seven  Organ-Specific Inflammatory Disease

FIG 68.1  Red Blood Cell Cast. Cast present in situ within the lumen of a distal renal tubule. (Periodic acid–Schiff [PAS] stain.)

FIG 68.3  Normal Glomerular Architecture. The glomerular capillary loops are patent and have normal thickness. Neither increased endocapillary cells nor expanded mesangial matrix encroach upon the patency of the capillary lumina. (Periodic acid–Schiff [PAS] stain).













TABLE 68.1  Indications for Renal Biopsy

















1. Active “nephritic” urine sediment • Dysmorphic erythrocytes: >10 per high-power field • Cellular casts: erythrocyte or leukocyte 2. Proteinuria >2 g/day 3. Abnormal renal function • Associated with the above features of active nephritis • Particularly important if the duration of renal disease and/or rate of change are unknown 4. Document indications for use of high-risk therapeutic interventions

63(

,J*

,J$

,J0

κ

λ

FIG 68.2  Immunofixation Electrophoresis of Urinary Protein. Proteins in a concentrated urine protein are separated in six replicate lanes by standard protein electrophoresis. Separated proteins are identified by overlaying specific antisera to immunoglobulin G (IgG), IgA, IgM, κ and λ. In this example, a monoclonal paraprotein composed of λlight chain is identified as an intense narrow band in the far right hand lane.

clarify the precise type of renal involvement, to formulate a prognosis, and to direct therapy. Some of the more important indications for renal biopsy are listed in Table 68.1. Light microscopy appearance of a normal glomerulus is illustrated in Fig. 68.3.

ACUTE NEPHRITIC SYNDROME

MINIMAL CHANGE DISEASE

Acute nephritic syndrome is characterized by hematuria (dysmorphic cells), erythrocyte casts, abnormal proteinuria, fluid retention, azotemia, and hypertension. Histologically, this constellation of clinical findings is due to proliferative glomerulonephritis. A variant of this syndrome, called rapidly progressive glomerulonephritis (RPGN), is defined by ≥50% loss of glomerular filtration rate over 3 months, along with characteristically >50% of glomeruli showing cellular crescents on renal biopsy..

CHRONIC GLOMERULONEPHRITIS Broad and waxy casts are features of chronic renal disease that are not likely to be seen in acute or subacute glomerulonephritis.

RENAL BIOPSY After extensive clinical and laboratory evaluations, renal biopsy may be indicated to establish or confirm a tissue diagnosis, to

KEY CONCEPTS Minimal Change Nephropathy • • • • •

Most common cause of nephrotic syndrome in children High rate of response to glucocorticoids Cyclophosphamide is useful for frequent relapsers Renal prognosis is characteristically excellent Subset may evolve to focal segmental glomerulosclerosis

Nephrotic syndrome of childhood is mainly caused by minimal change disease (MCD). The frequency of MCD in adults with nephrotic syndrome is low compared with those of other entities discussed below.4

Clinical Features Minimal change nephropathy characteristically presents with a rather precipitous onset of severe nephrotic syndrome in the absence of signs of a systemic disease. There are no specific tests

CHAPTER 68  Immunological Renal Diseases other than kidney biopsy to establish a diagnosis of minimal change nephropathy, although proteinuria exclusively comprising albumin is characteristic; standard immunological screening tests are usually normal.

Etiology and Pathogenesis The cause of MCD is largely unknown. There is evidence of immune dysregulation, mainly involving cell-mediated immunity, which is supported by the tendency of the disease to manifest and relapse after viral infections or an allergic reaction, the association of some cases with Hodgkin lymphoma, and its characteristically favorable response to immunosuppressive drugs. Another hypothesis is that a systemic circulating factor of immune origin results in increased glomerular permeability5 (discussed in the section on “Focal Segmental Glomerulosclerosis”) The basic lesion of MCD is loss or neutralization of the normal high density of anionic proteoglycans in the glomerular capillary loops. Dissipation of the negative-charge barrier allows anioncharged albumin to pass freely. Interdigitating foot processes normally joined by intercellular bridges, called slit diaphragms, form a second barrier to the passage of protein into the urinary space. Characteristically, the podocyte foot processes and slit diaphragms are also disrupted in minimal change nephropathy.

919

KEY CONCEPTS Focal Segmental Glomerulosclerosis • • • • •

Nephrotic syndrome with progressive renal insufficiency Glomerular permeability factor in plasma of some cases Unpredictable responses to glucocorticoids or cyclophosphamide Cyclosporine effective, but relapses common upon withdrawal Moderately high relapse rate in renal allografts

a poorer response to immunosuppressive drug therapies, and a higher risk of progression to end-stage renal failure. The incidence of FSGS is clearly increasing as a cause of nephrotic syndrome, particularly among black patients.

Etiology and Pathogenesis

With the use of light microscopy, renal biopsy results are found to be essentially normal. Electron microscopy shows the characteristic pathological lesion, where there is fusion of the foot processes of the epithelial cells (podocytes) diffusely around glomerular capillaries.

Diverse etiologies may disrupt the podocyte structure and function leading to the histological expression of FSGS. Genetic mutations of key podocyte proteins, such as podocin and nephrin play a role in a subset of FSGS that occurs in children or young adults. Circulating permeability factors have been proposed as mediators of podocyte injury in FSGS (i.e., soluble urokinase-type plasminogen activator receptor). In certain situations, it appears that podocytes have increased surface expression of the transmembrane protein B7-1 (CD80) that stimulates interactions with T cells and leads to foot process effacement.7 “Secondary” forms of FSGS may result from toxic effects of drugs (i.e., pamidronate), viral infections (e.g., human immunodeficiency virus [HIV]) or maladaptive hemodynamic stress (i.e., resulting from obesity or reduced nephron mass).

Treatment

Pathology

MCD is characteristically exquisitely sensitive to glucocorticoids (remitting within the first few weeks of therapy in >90% of children). The response rate to glucocorticoids in adults is somewhat lower and more delayed. A substantial portion of patients with minimal change nephropathy face long-term difficulties because of the disease and the therapy: Some are steroid-resistant from the start; others are steroid-dependent for control of nephrotic syndrome; still others become frequent relapsers and suffer substantial steroid toxicity with repeated treatments. Controlled trials have shown that alkylating agents (i.e., cyclophosphamide) increase response rates and reduce rates of relapse. Cyclosporine (CSA) and tacrolimus are alternatives to prolonged and repeated courses of steroid therapy, but relapses frequently occur with drug withdrawal. Rituximab (a monoclonal antibody [mAb] to CD20 on B cells) has been used with variable success, particularly in those who are steroid-dependent or frequent relapsers. Also, rituximab may have “off target” effects on the podocyte cytoskeletal structure.6 The risk of progression to end-stage renal failure is extremely low in true MCD. Renal biopsy sampling errors account for a mistaken diagnosis of MCD in a proportion of cases that are, in fact, focal segmental glomerulosclerosis (FSGS). It is debated whether MCD and FSGS represent different manifestations of one disease (with MCD potentially progressing to FSGS), or if they represent two different diseases.

The characteristic renal pathology includes segmental areas of podocyte damage and detachment, irregular foot process fusion, and collapse of glomerular capillaries associated with marked local increase in matrix and collagen accumulation. Segmental sclerotic areas typically stain nonspecifically with antisera to IgM and C3 (but not to IgG or IgA), particularly in areas of glomerular tuft hyalinosis (representing trapped plasma constituents), but none of these collections should be considered to represent classic immune complexes. Various histological subtypes of FSGS have been described on the basis of the type and location of lesions. One classification scheme divides FSGS into five variants: tip, perihilar, cellular, collapsing, and not otherwise specified (NOS)8 (Fig. 68.4). The collapsing variant of FSGS, which has been associated with viral infections, notably HIV, tends to follow a particularly aggressive course.

Pathology

FOCAL SEGMENTAL GLOMERULOSCLEROSIS Patients with FSGS have a higher frequency of microscopic hematuria, higher frequency of persistent nephrotic syndrome,

Treatment Patients with primary FSGS generally have hypoalbuminemia and nephrotic range proteinuria, whereas those with secondary FSGS typically have subnephrotic proteinuria, a normal or near normal serum albumin concentration, no peripheral edema, and focal rather than diffuse foot process effacement on kidney biopsy. The possibility of genetic forms of FSGS should be considered before beginning immunosuppressive therapy, but steroid resistance may also serve as a clue to genetic forms of FSGS. Treatment of genetic and hyperfiltration-induced forms of FSGS is focused mostly on renoprotection with angiotensin antagonists and lipid-lowering agents (statins). The treatment of other forms of primary FSGS is basically similar to that of MCD and includes immunosuppression with prednisone,

920

Part Seven  Organ-Specific Inflammatory Disease

$

%

&

'

FIG 68.4  Focal Segmental Glomerulosclerosis (FSGS). Several forms of glomerular lesions are seen in FSGS, often within the same biopsy: A, minimal abnormality (periodic acid–Schiff [PAS] stain); B, tip lesion manifested by segmental glomerular tuft lesion near the origin of the proximal tubule (PAS stain); C, classical perihilar lesion (PAS stain); and D, collapsing glomerulopathy; the glomerulus is globally contracted with wrinkling of the basement membranes; this is associated with hyperplasia of podocytes surrounding the glomerular capillaries (methenamine silver stain).

cyclophosphamide, calcineurin inhibitors, or mycophenolate. There are mixed outcomes with use of rituximab for FSGS, but it appears less effective for steroid resistant cases.9,10 Complete remission of proteinuria is less commonly achieved in FSGS than in MCD. Relapses of the disease and progression to end-stage kidney disease (ESKD) remains a major concern despite immunosuppression, particularly in steroid-resistant and frequently relapsing patients. Several novel therapies that target various immunological, inflammatory, and costimulatory pathways (i.e., blockade of CD 80 with cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) are currently under investigation for FSGS.11,12

MEMBRANOUS NEPHROPATHY MN is identified in approximately 20% of adults who undergo renal biopsy for nephrotic syndrome. It is a less frequent cause of nephrotic syndrome in children. Primary MN is usually a diagnosis of exclusion after considering secondary causes, such as medication reactions, infections, neoplasms, and systemic illnesses (i.e., SLE)

KEY CONCEPTS Membranous Nephropathy (MN) • Common cause of idiopathic nephrotic syndrome in adults • Autoantibodies to phospholipase A2 receptor (PLA2R) detected in large percentage of patients with primary MN • Several secondary causes: systemic lupus erythematosus (SLE), drugs, chronic hepatitis, certain malignancies • One-quarter of patients have spontaneous remission • One-quarter of patients develop end-stage renal disease within a decade • Protracted nephrotic syndrome confers risks of cardiovascular and thromboembolic events • Therapies: steroids, alkylating agents, calcineurin inhibitors, rituximab, lipid-lowering drugs, angiotensin antagonists

Etiology and Pathogenesis MN is characterized by subepithelial (epimembranous) immune deposits containing IgG and complement components. The leading hypothesis based on experimental models is that immune

CHAPTER 68  Immunological Renal Diseases

921

$

FIG 68.5  Membranous Nephropathy. Capillary walls are nearly uniformly thickened but remain widely patent. Cellularity of the glomerulus is normal (Periodic acid–Schiff [PAS] stain).

complexes are formed in situ by the interaction of a pathogenic antibody with a constitutive glomerular antigen or with an antigen that had been ectopically planted in the glomerulus. Major advances have been made with the identification of several constitutive podocyte surface antigens, which are the target of autoantibodies in some subsets of patients with primary MN.13,14 The majority (70-80%) of patients with primary MN have circulating autoantibodies to M-type phospholipase A2 receptor (PLA2R), a transmembrane receptor that is expressed in glomerular podocytes.13 Antibodies to thrombospondin type-1 domain containing 7A protein (THSDA7A) and neutral endopeptidase have been identified in smaller subsets of patients.14

Clinicopathological Features Patients with MN characteristically present with nephrotic syndrome. Renal biopsy is usually required to establish the diagnosis, although serological testing for relevant autoantibodies (i.e., anti–PLA2R) may be informative if renal biopsy is contraindicated. In most cases, light microscopy shows uniform thickening of the glomerular capillary walls without endocapillary cell proliferation (Fig. 68.5). Characteristic subepithelial (epimembranous) and/or intramembranous deposits are seen on electron microscopy (Fig. 68.6). Identification of PLA2R in glomerular immune deposits (by immunofluorescence or immunohistochemistry) favors the diagnosis of primary MN; mesangial deposits are often present in secondary MN. But a diagnosis of secondary MN depends on concurrent abnormalities in clinical and laboratory data.

Natural History The clinical course of primary MN is highly variable. On average, about one-quarter of adult patients progress to ESKD within 10 years. Another quarter experiences a spontaneous remission of proteinuria. The majority is likely to have persistent proteinuria and moderately impaired renal function over an extended period of observation. Baseline characteristics, such as severe nephrotic syndrome, hypertension, and azotemia, have been associated with poor outcomes. Protracted high-grade nephrotic-range proteinuria

%

FIG 68.6  Membranous Nephropathy (Ultrastructure). A, Electron micrograph demonstrates heavy, dark-staining immune complex deposits along the outer surface of the glomerular basement membrane and beneath the epithelial foot processes (hence the terms subepithelial or epimembranous deposits). Note the thickening and projections of the gray-staining basement membrane between the electron-dense deposits. B, Ultrastructure of a normal glomerular capillary wall for comparison.

is a relatively strong predictor of an adverse renal outcome. Some studies suggest that the titer of autoantibody to PLA2R at baseline correlates with disease activity such that higher titers may be associated with greater risk of renal function decline, whereas low titers may be associated with greater probability of spontaneous remission.15

Treatment Management of patients with MN usually includes diuretics to reduce edema, lipid-lowering drugs (for severe hyperlipidemia), anticoagulant therapy for thromboembolic complications, and

922

Part Seven  Organ-Specific Inflammatory Disease

FIG 68.7  Membranoproliferative Glomerulonephritis (MPGN). Glomerulus exhibits the typical lobulated appearance of this disease. Markedly increased mesangial cells and matrix in all of the lobules. Mesangium extends outward into the capillary loops and forms double contours with the glomerular basement membrane. (Periodic acid–Schiff [PAS] stain).

antihypertensive agents. Angiotensin antagonists have been shown to have a substantial antiproteinuric effect. Immunosuppressive treatment is generally reserved for patients with persistent high-grade proteinuria (>4 g/day. First-line immunosuppressive therapy consists of cytotoxic drugs (usually cyclophosphamide) in combination with glucocorticoids or a calcineurin inhibitor (CSA or tacrolimus) with or without low-dose glucocorticoids.16 The most compelling results from controlled trials have shown that patients with MN treated with alternating monthly courses of pulse methylprednisolone and chlorambucil or cyclophosphamide were more likely to experience a remission of the nephrotic syndrome and achieve stable renal function compared with controls. Rituximab may be a useful alternative for treatment of MN based on encouraging results from pilot studies and a good safety profile compared with other immunosuppressive regimens.16–19

MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS KEY CONCEPTS Membranoproliferative Glomerulonephritis (MPGN) • Histologically classified into immune complex–mediated glomerulonephritis or complement-mediated glomerulonephritis based on immunofluorescence staining pattern • C3 glomerulopathy characterized by C3 accumulation in the glomeruli in the form of electron-dense deposits. • Genetic or acquired abnormalities in the activation of the alternate alternative complement pathway complement pathway associated with C3 glomerulopathy • Response to immunosuppressive drug treatment generally poor • Tends to recur in renal allografts

Membranoproliferative glomerulonephritis (MPGN) is a morphological entity that encompasses a heterogeneous group of diseases with a similar appearance at the light microscopic level. The

pattern of injury is characterized by mesangial matrix expansion and increased cellularity and thickening of the glomerular capillary walls with a double contour appearance. These changes give a lobulated appearance to the glomerulus (Fig. 68.7).

Etiology and Pathogenesis Until recently MPGN was classified into three types based on the location and type of electron dense deposits: type 1 characterized by subendothelial deposits; type II by intramembranous electron dense deposits in a ribbon-like pattern (also referred to as dense deposit disease [DDD]); and type III by subendothelial and subepithelial deposits (Fig. 68.8). This older classification scheme also made a distinction between secondary causes when it was possible to identify an etiology New insight into the pathogenesis of MPGN has recently led to a major paradigm shift in the classification of this disease. Of great importance is the recognition that some cases of MPGN result from the deposition of Igs with secondary complement activation, whereas others arise from primary abnormalities in the control of complement activation.20 The new classification system now broadly categorizes MPGN into Ig-mediated or complement-mediated disease based on the pattern and composition of the deposits, as assessed by immunofluorescence staining. The presence of both Ig and complement (C3) indicates an Ig-mediated process in which immune complexes are deposited and then secondarily activate the classical complement pathway. In contrast, the presence of complement staining (C3) without significant Ig deposition implies that an antibody-independent means of complement activation has been triggered and suggests a primary problem with regulation of the alternative complement pathway (Fig. 68.9). Such diseases are now grouped under the umbrella term, “C3 glomerulopathy” (C3G), which encompasses both C3 glomerulonephritis (C3GN) and DDD.21 A diagnosis of C3G should prompt an evaluation for inherited mutations of complement regulatory proteins (i.e., factor H or factor I) or acquired autoantibodies to regulatory proteins (i.e., C3 nephritic factor or anti-factor H), which lead to dysregulation of the alternative pathway. C3 nephritic factor, an autoantibody to C3

CHAPTER 68  Immunological Renal Diseases

$

923

%

FIG 68.8  Membranoproliferative Glomerulonephritis (MPGN) Ultrastructure. A, Capillary wall is markedly thickened and contains heavy, dark-staining, electron-dense immune complexes in the subendothelial space. Mesangium (lighter material) extends into the capillary loop, where it is interposed between the basement membrane and the endothelium; the process gives the appearance of a massively thickened capillary loop on hematoxylin and eosin (H&E) staining, and the split appearance by periodic acid–Schiff (PAS) and silver stains. B, Dense deposit disease; capillary loops contain smooth, continuous, linear dense material within the basement membrane.

convertase, is frequently detected in C3G. C3 nephritic factor stabilizes the convertase, rendering it resistant to control by factor H, thus leading to persistent C3 activation and deposition of alternative pathway activation products in glomeruli.

Pathology In addition to endocapillary proliferation and findings on immunofluorescence, a double contour appearance to the glomerular basement membrane (GBM) best seen with silver stain represents synthesis of GBM-like material from capillary wall remodeling. Irregular capillary wall subendothelial and mesangial deposits are seen by electron microscopy in Ig- mediated MPGN; often small intramembranous and subepithelial deposits are also present in C3GN. The hallmark of C3GN is dominant bright staining for C3 by immunofluorescence in the mesangium and glomerular capillary walls (see Fig. 68.9). Double contours of the GBM are also present in conditions associated with chronic endothelial injury, including thrombotic microangiopathy, transplant glomerulopathy, and preeclampsia, and show a histological appearance of MPGN by light microscopy. However, there are no associated immune deposits and immunofluorescence is negative for Ig and C3.

Clinical Presentation Although it is characteristically a chronic low-grade nephropathy, some patients experience nephrotic syndrome and even rapidly progressive and crescentic disease. Hypocomplementemia is common in all types of MPGN. In immune complex-mediated MPGN, serum C4 levels are characteristically low, consistent with classical pathway activation. Serum C3 concentration may be normal or mildly decreased. In complement-mediated MPGN, serum C4 levels are normal, and C3 is typically low, consistent with alternative pathway activation, but a normal serum C3 does not exclude the diagnosis. The renal outcome in C3 glomerulopathy

is variable with up to 30% of patients progressing to ESKD. The prognosis of DDD is worse, with almost half the patients progressing to kidney failure. Idiopathic MPGN and MPGN resulting from complement dysregulation tend to recur in renal allografts; recurrences of both types of MPGN have a detrimental effect on graft survival.

Treatment It is important to recognize that existing treatment trials for MPGN predate the new classification system and our appreciation of the different pathogenesis. Thus their relevance is questionable because of the heterogeneity of patient populations. It is anticipated that the new diagnostic categories will better define groups of patients with similar pathogenesis, thus enabling a more rational approach to targeted therapy in the future. There is no consensus regarding treatment of “idiopathic” immune complex–mediated MPGN, but various immunosuppressants have been used with variable success, including prednisone alone or in combination with either mycophenolate or cyclophosphamide. There are limited data on the role of rituximab in this disease. Optimal treatment of C3GN remains unclear but will likely need to be individualized on the basis of proper identification of the defect in the alternative complement system (i.e., genetic vs acquired autoantibodies). Chronic infusions of fresh frozen plasma to replace missing complement factors may be beneficial in some cases. Immunosuppression with corticosteroids, rituximab, and mycophenolate is of theoretic benefit in cases resulting from pathogenic antibodies to complement regulatory proteins but is unproven. A more targeted approach using eculizumab, a mAb that blocks activation of C5 complement (C5b-9), has been tested in small series, but the data are inconclusive. Additional new complement blocking agents may also hold promise.22

924

Part Seven  Organ-Specific Inflammatory Disease

$

&

%

'

FIG 68.9  C3 Glomerulopathy. A, Glomerulus exhibiting the characteristic membranoproliferative pattern (hematoxylin and eosin [H&E] stain). B, Immunofluorescence showing prominent glomerular staining for C3 complement. C and D, Electron microscopy ultrastructure showing mesangial interposition giving a double contour of the glomerular capillary wall, as well as interposed electron dense deposits in the subendothelial space and the mesangium.

POSTINFECTIOUS NEPHROPATHIES An ever-expanding list of infectious agents has been implicated in the pathogenesis of certain forms of immune-mediated nephropathies.

Viral Infections

Hepatitis B Hepatitis B–associated nephropathy most frequently presents as nephrotic syndrome accompanied by microscopic hematuria; renal biopsy most commonly shows MN. Immunofluorescence studies usually show Ig, C3, and some IgM staining in the subepithelial region along the glomerular basement membranes and in some cases viral antigens can be detected within the glomerulus. Electron microscopy shows subepithelial and intramembranous deposits, but there may also be mesangial and even some subendothelial deposits. Therapy for hepatitis B renal disease has focused on antiviral drugs because glucocorticoids and cytotoxic agents may promote viral replication. Treatment generally consists of interferon-α (IFN-α) or nucleoside/nucleotide analogues, such as lamividine,

entacavir, adefovir, tenofovir, and telbivudine. Treatment with nucleoside/nucleotide analogues are generally used for at least 1 year and continued for at least 6 months after HBeAg seroconversion. Immunosuppression (corticosteroids with or without cyclophosphamide or rituximab) may cautiously be considered in patients with rapidly progressive glomerulonephritis, but concomitant antiviral therapy is required. Hepatitis C Hepatitis C virus (HCV) is a major cause of both transfusionassociated and sporadic chronic hepatitis. The most common HCV-associated renal disease is usually, but not invariably, MPGN in the context of type II mixed cryoglobulinemia. The pathogenesis of the renal disease is caused by deposition within glomeruli of immune complexes containing HCV, anti-HCV antibody and virus-related (or unrelated) cryoglobulins. Treatment of HCV infection has greatly improved in the last few years. Historically, treatment was mainly based on IFN-α and ribavirin. These agents are now being replaced by HCV-specific antiviral drugs that are used in combination (i.e., sofosbuvir plus ledipasvir), which are highly effective and often curative.23 The

CHAPTER 68  Immunological Renal Diseases evidence for antiviral treatment in HCV-related kidney disease is based mostly on IFN-based regimens, which have reported remission of proteinuria and hematuria and improvement of renal function.24,25 There are limited data regarding use of the newer antiviral agents in HCV-associated glomerulonephritis, but these hold considerable promise.26 For patients with progressive renal disease and/or nephrotic-range proteinuria, treatment with rituximab or pulse intravenous steroids and cyclophosphamide may be warranted in conjunction with antiviral therapy. Treatment with rituximab and plasma exchange may provide additional benefit in patients with HCV-associated severe cryoglobulinemia refractory to antiviral therapy.27,28 Human Immunodeficiency Virus HIV-associated nephropathy (HIVAN), the first kidney disease to be associated with HIV infection, is a collapsing form of focal segmental glomerulosclerosis accompanied by associated tubular microcysts and interstitial inflammation. It was first described in patients with acquired immune deficiency syndrome (AIDS) but also is occasionally diagnosed in less advanced cases of HIV infection and before acute HIV seroconversion has been identified. HIVAN classically presents with significant proteinuria and rapidly progressive kidney disease. HIVAN displays a striking racial predilection for black patients, which may indicate the importance of genetic influences.29 A spectrum of other renal abnormalities have also been described in patients with HIV infection, including IgA nephropathy, lupus-like glomerulonephritis, postinfectious glomerulonephritis, MPGN, MN, cryoglobulinemic glomerulonephritis, fibrillary and immunotactoid glomerulopathy, and thrombotic microangiopathy.30,31 Tubulointerstitial changes related to drug toxicity, acute interstitial nephritis, or superimposed viral, fungal, or mycobacterial infections may also be present. The mainstay of treatment of HIVAN is combination antiretroviral therapy (cART) regardless of the CD4 lymphocyte count. Highly effective modern therapies for HIV have reduced the frequency of developing HIVAN and have greatly improved the otherwise dismal renal prognosis of HIVAN. This has greatly improved the dismal renal prognosis of HIVAN. The evidence for initiating cART in other HIV-associated immune complex glomerular diseases is inconclusive, but use of cART is a rational approach. The use of standard immunosuppressive drugs in an immunocompromised population is controversial.

Bacterial Infections

Poststreptococcal Glomerulonephritis Poststreptococcal glomerulonephritis is the best-studied and classic immune complex–mediated glomerulonephritis. It is the result of skin or throat infection with nephritogenic strains of group A streptococci. The latent period between upper respiratory infection and nephritis is 7–10 days, and 2–4 weeks after skin infection. Antistreptococcal antibody titers are usually measured to demonstrate the existence of a preceding streptococcal infection. Antistreptolysin O titers and anti-DNase B titers are the most frequently elevated in upper respiratory and skin infections, respectively. Poststreptococcal glomerulonephritis is characterized by a nephritic syndrome consisting of smoky or rust-colored urine, generalized edema, hypertension, and nephritic urine sediment. Proteinuria is typically mild. Patients have rising titers of antistreptolysin and depressed C3 levels early in nephritis but normal

925

KEY CONCEPTS Infection-Related Nephropathies • Viral: Hepatitis B—membranous nephropathy; hepatitis C—cryoglobulinemic membranoproliferative glomerulonephritis; human immunodeficiency virus (HIV)—focal segmental glomerulosclerosis (HIV nephropathy) • Bacterial (mainly gram-positive): Nephritogenic streptococcal infections, prosthetic device (shunt) infections, subacute bacterial endocarditis, chronic deep tissue abscesses—mainly diffuse or focal proliferative glomerulonephritis

or minimally depressed C4 levels, indicating activation of the alternative complement pathway. Proliferative glomerulonephritis with polymorphonuclear leukocyte and monocyte infiltration, granular immune deposits of IgG and C3, and dome-shaped electron-dense subepithelial deposits (humps) are characteristic. The prognosis is excellent in children; with supportive care, almost all will recover. Progressive renal failure accompanied by severe hypertension appears to be more common in adults. Kidney biopsy is rarely needed in the child but may be warranted if there is an atypical presentation or evolution. . The classic childhood form is still seen in developing countries but is now rare in developed countries. However, there has been an increase in the incidence of nonstreptococcal postinfectious glomerulonephritis or “infection-related” glomerulonephritis, which tends to be seen in older patients with multiple comorbidities, especially diabetes, HIV infection, and malignancy. These clinical variants are usually related to other infective agents, such as Staphylococcus aureus, both methicillin-resistant (MRSA) and methicillin-sensitive S. aureus, and may be characterized by an IgA-dominant glomerular immune complex deposition. Etiology and pathogenesis.  Glomerular injury results from passive glomerular trapping of circulating immune complexes composed of nephritogenic bacterial antigens and IgG antibody or by the in situ formation of immune complexes. This is followed by immune cell recruitment, production of chemical mediators and cytokines, and local activation of the complement and coagulation cascades that drive an inflammatory response.1 Current therapeutic strategies rely on culture-guided systemic antibiotics, especially in older patients, in whom MRSA may be the causative agent. Steroids may be used in selected cases in which crescents and severe interstitial inflammation are present.

IgA Nephropathy KEY CONCEPTS Immunoglobulin A Nephritis (IgAN) • Common cause of asymptomatic microscopic hematuria, recurrent macroscopic hematuria, and/or low-grade proteinuria • Spectrum of disease, including idiopathic IgA nephritis and HenochSchönlein purpura nephritis; IgA in skin and renal biopsy samples • Mostly benign prognosis, especially in children • Patients with progressive renal insufficiency and/or crescentic glomerulonephritis warrant trial of glucocorticoids and/or cytotoxic drug therapy

926

Part Seven  Organ-Specific Inflammatory Disease

$

%

&

'

FIG 68.10  Immunoglobulin A Nephropathy (IgAN). (A) hematoxylin and eosin (H&E) stain and (B) periodic acid–Schiff (PAS) stain: Glomeruli manifest modestly increase mesangial hypercellularity and expanded mesangial matrix. C, Immunofluorescence showing predominantly IgA deposition within the mesangium. D, Electron microscopy showing deposits mainly within the mesangium but not along peripheral capillary loops.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide.32 IgAN can affect patients of all ages, especially children and young adults, with a male preponderance.34 There are geographical and ethnic differences in the prevalence of IgAN. The highest frequency is found among East Asians; IgAN is very uncommon in individuals of African ancestry. This observation and examples of familial clustering of IgA nephropathy favor an important element of genetic susceptibility. IgAN may be discovered during evaluation of asymptomatic microscopic hematuria.34 Alternatively, patients (especially children) can present with recurrent episodes of macroscopic hematuria that occur within 24–48 hours after an intercurrent infection, usually an upper respiratory or gastrointestinal (GI) tract infection. A transient elevation in serum creatinine has been associated with macroscopic hematuria in about one-third of cases. This has been attributed to tubular injury and obstruction caused by intraluminal RBC casts. A small percentage of patients present with either nephrotic syndrome or an acute RPGN picture characterized by edema, hypertension, renal insufficiency, and hematuria.

Pathology The characteristic findings on light microscopy are mesangial cell proliferation and mesangial matrix expansion.32 Electron microscopy typically reveals electron-dense deposits that are primarily limited to the mesangium, but a few deposits may also be present in subendothelial and subepithelial locations. The pathognomonic finding on immunofluorescence microscopy is globular deposits of IgA (often accompanied by C3 and IgG) in the mesangium and, to a lesser degree, along the glomerular capillary wall (Fig. 68.10). Etiology and Pathogenesis Aberrant glycosylation of O-linked glycans in the hinge region of IgA1 resulting in increased serum levels of galactose-deficient IgA1 (Gd-IgA1) plays a pivotal role in pathogenesis of IgAN.34 The aberrantly glycosylated IgA1 is recognized by antiglycan antibodies and leads to circulating IgA immune complexes that preferentially deposit in the mesangium, provoking local injury.

927

CHAPTER 68  Immunological Renal Diseases A genetic predisposition to IgAN has been linked with polymorphisms involving innate and adaptive immunities and the alternative complement pathway. A “second hit” may be needed in predisposed individuals. Infections may play a role because episodes of macroscopic bleeding often coincide with mucosal infections, including upper respiratory tract (synpharyngitic) or GI infections.

TABLE 68.2  Prevalence of Antineutrophil

Natural History Patients with IgAN who have low-grade proteinuria (1 g/day), and persistent azotemia.

Polyarteritis nodosa Microscopic polyangiitis Granulomatosis with polyangiitis (Wegener granulomatosis) Necrotizing and crescentic GN

Treatment The most effective treatment of progressive IgAN remains undefined and controversial.34 Angiotensin antagonists are recommended to achieve blood pressure control, to reduce proteinuria, and to slow the rate of deterioration of renal function. There are conflicting data about the value of fish oil dietary supplements (eicosanoids) in preventing renal progression in patients with IgAN. The practice of tonsillectomy in IgAN has not been confirmed. A large trial (STOP-IgA nephropathy) showed that immunosuppression (glucocorticoid monotherapy or a regimen that included prednisolone, cyclophosphamide, and azathioprine) did not have a significant beneficial effect on preservation of kidney function.33 However, a course of oral steroids may be considered in patients with high-grade proteinuria, and cytotoxic drugs are indicated in a small subset of patients with crescentic rapidly progressive IgAN. The lack of effective therapy have provided the impetus for several new phase II/III clinical trials testing novel therapies, such as B-cell activating factor (BAFF) inhibition, proteasome inhibition, and B-cell inhibition.34,35

Renal Vasculitis Associated With Antineutrophil Cytoplasmic Antibodies KEY CONCEPTS Antineutrophil Cytoplasmic Antibodies (ANCAs)– Associated Renal Vasculitis • Renal vasculitis with glomerular involvement includes microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and necrotizing crescentic glomerulonephritis (renal-limited vasculitis) • Associated with ANCAs • Rapidly progressive glomerulonephritis is common; early treatment includes pulse methylprednisolone, cyclophosphamide, rituximab, or possibly plasma exchange • Maintenance therapy: azathioprine, rituximab

ANCAs are associated with a distinct form of vasculitis that can affect many different types of vessels and any organ in the body. ANCA-associated vasculitis (AAV; Chapter 58) is categorized into four main types: microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, formerly called Wegener

Cytoplasmic Antibodies (ANCAs) in Renal Vasculitis

ANCAS TEST POSITIVITY (%)

Type of Renal Vasculitis

P-ANCAs or Anti-Mpo

C-ANCAs or Anti-Pr3

10–20 50–80 10–20

10–20 10–20 80–90

50–80

10–20

MPO, myeloperoxidase; PR3, proteinase 3.

granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA, formerly Churg-Strauss syndrome), and renal limited vasculitis. As shown in Table 68.2, the patterns of ANCAs differ, depending on the type of vasculitis. Renal involvement is common in AAV but varies in type and severity. It occurs more frequently in MPA (90%) and in GPA (80%) and less frequently in EGPA (45%). Clinically RPGN is a common manifestation of these renal vasculitides characterized by the presence of hematuria, proteinuria, active urinary sediments, and renal failure. Pathology The glomerular abnormalities are similar among the subtypes of ANCA-associated glomerulonephritides (Fig. 68.11). The glomerular lesions are characteristically focal and segmental in distribution, with fibrinoid necrosis and crescent formation. Breaks in the GBM may be seen. There may be accompanying necrotizing arteritis. In contrast to immune complex–mediated vasculitis, ANCA-associated vasculitis has little or no Ig deposition in injured glomerular vessels, with minimal or negative staining by immunofluorescence, a so-called pauci-immune pattern. Patients with large percentage of cellular crescents (>50%) typically present with severely reduced renal function but have a good chance for recovery of renal function with treatment, whereas those with a greater percentage of globally sclerotic glomeruli are less likely to recover renal function.36 Treatment and Prognosis ANCA-associated renal vasculitis tends to be severe and fulminant, making early detection critically important in management. Even with early diagnosis, approximately one-third of patients will progress to renal failure within 5 years. Relapsing courses are common in patients with microscopic polyangiitis, particularly GPA. This underscores the importance of prompt induction treatment followed by maintenance therapy. Glucocorticoids are important in the early treatment of renal vasculitis. Cyclophosphamide-based regimens are very effective in inducing remission in patients with AAVs. Most advocate daily oral cyclophosphamide regimens, but intermittent pulse cyclophosphamide may be substituted to reduce the toxicity of extended therapy. In patients with severe pulmonary hemorrhage or rapidly progressive glomerulonephritis caused by renal vasculitis, pulse methylprednisolone, followed by prednisone and daily cyclophosphamide, is clearly indicated. Adjunctive plasma exchange is commonly used in cases of aggressive pulmonary–renal syndrome. Rituximab has become a valuable alternative to

928

Part Seven  Organ-Specific Inflammatory Disease

$

%

&

'

FIG 68.11  Antineutrophil Cytoplasmic Antibody (ANCA)–Associated Systemic Vasculitis. A, Skin biopsy showing leukocytoclastic vasculitis of a small dermal artery (hematoxylin and eosin [H&E] stain). B, Glomerulus illustrating a characteristic segmental proliferative lesion forming an adhesion to the Bowman capsule in an early and mild case (H&E stain). C, Glomerulus with classical fibrinoid necrosis and an associated cellular crescent of a more severe and rapidly progressive case (H&E stain). D, Necrotizing vasculitis in a small renal artery in an extreme case of ANCA-associated renal vasculitis; large numbers of eosinophils are present in the perivascular inflammatory infiltrate (H&E stain).

cyclophosphamide for induction treatment based on the results of two pivotal prospective randomized controlled trials.37,38 Maintenance therapy is usually given for 12–18 months after achieving remission. Azathioprine has been the mainstay of maintenance therapy in AAVs after remission induction, but rituximab is also likely to have a prominent role in maintenance therapy, as indicated by favorable results from preliminary trials.39

Anti-GBM Antibody-Mediated Nephritis: Goodpasture Disease KEY CONCEPTS Goodpasture Disease • Circulating anti–glomerular basement membrane (GBM) antibody • Associated with pulmonary hemorrhage • Rapidly progressive glomerulonephritis with cellular crescents and linear deposits of immunoglobulin G (IgG) • Treated with high-dose steroids, cyclophosphamide, plasma exchange

Goodpasture disease is a rare but classic immune-mediated cause of severe pulmonary–renal syndrome. The cardinal pathogenic factor is an autoantibody to a component of type IV collagen (noncollagenous domain 1 of the α3 chain subunit) within the GBM or the alveolar basal membrane.40,41 These autoantibodies lead to GBM rupture with development of crescentic glomerulonephritis (Fig. 68.12) and account for the pathognomonic finding of linear deposition of IgG along the glomerular capillaries as assessed by immunofluorescence microscopy. The genesis of anti-GBM antibodies in sporadic cases is unknown. It is postulated that an inciting event(s) (i.e., viral infection or exposure to environmental factor, such as hydrocarbons, tobacco) perturbs the normal conformation of collagen in the basement membrane, exposing previously cryptic epitopes on the α3 subunit, eliciting the pathogenic autoantibody response. Iatrogenic cases have occurred when normal kidneys have been transplanted into patients with hereditary Alport nephropathy (which is caused by mutations in the α5 chain of type IV collagen). The absence of expression of α5 (and associated lack of α3 and

CHAPTER 68  Immunological Renal Diseases

929

Caucasians. Nephritis is a major cause of morbidity and mortality and accounts for a large portion of all hospital admissions in patients with SLE.

FIG 68.12  Goodpasture Disease. Circumferential cellular crescent fills the Bowman capsule and compresses the glomerular tuft (Silver stain).

α4) means that these molecules within the renal allograft are regarded as foreign antigens. Thus recipients mount an anti-GBM antibody response to the new donor antigens. Clinically malaise, weight loss, fever, or arthralgia may be the initial features of anti-GBM disease. Some patients present with isolated renal involvement, but more typically, pulmonary hemorrhage with hemoptysis accompanies acute renal failure. The devastating nature of the disease warrants aggressive treatment. Pulse methylprednisolone, cyclophosphamide, and plasma exchange are indicated early in the course of Goodpasture disease.42 The role of rituximab in this disease requires further study. Reversibility of renal disease is unlikely if renal function is substantially impaired or oliguria ensues before treatment is started. Immunosuppressive treatment is normally continued until the patient has been in sustained clinical remission and anti-GBM titers are minimal or absent for at least 3 months.

Lupus Nephritis KEY CONCEPTS Lupus Nephritis • Class I: normal glomeruli by light microscopy in patients with SLE. • Class II, mesangial: Immunosuppressive treatment is usually not indicated unless patient has proteinuria >1 g/day despite renin–angiotensin–aldosterone (RAA) system blockade, especially if urine sediment is nephritic • Class III, focal nephritis and class IV, diffuse nephritis: Mycophenolate mofetil (MMF) or intravenous cyclophosphamide (IVCY) plus glucocorticoids for induction followed by MMF or azathioprine plus low-dose corticosteroids as maintenance therapy • Class V, Membranous nephropathy: Alternate-day prednisone with MMF, or alternatively bimonthly pulse cyclophosphamide or low-dose daily cyclosporine

Glomerular disease affects the majority of patients with SLE (Chapter 51), but lupus nephritis has a wide spectrum of disease expression and outcomes among different patient populations. Lupus nephritis occurs more often and is associated with less favorable outcomes among Hispanic, Asian, Native American, and especially African American populations compared with

Pathogenesis Several different mechanisms appear to be involved in the pathogenesis of lupus nephritis, resulting in a wide spectrum of renal lesions. Deposition of immune complexes from the circulation into the kidney appears to be the initiating event in proliferative lupus nephritis; however, only a subset of immune complexes appears to be nephritogenic. DNA and anti-DNA antibodies are known to be concentrated in glomerular deposits in the subendothelial location and are likely to play a central role in the pathogenesis of proliferative lupus nephritis. Unfortunately, there are fewer insights into the pathogenesis of lupus MN with its characteristic epimembranous immune deposits. Although T cells are almost certainly involved in autoantibody production, it is unknown whether they have a direct role in the pathogenesis of lupus nephritis. Clinical Features Asymptomatic hematuria or proteinuria may be the presenting features, but they often progress to nephritic and/or nephrotic syndrome. Hypertension, azotemia, nephritic urine sediment (with hematuria and cellular casts), hypocomplementemia and high anti–double-stranded DNA (dsDNA) titers are more commonly found in patients with proliferative lupus nephritis. RPGN is usually associated with the appearance of cellular crescents and may be superimposed on severe proliferative or membranous forms of lupus nephritis. Pathology The former classification of renal biopsy in lupus nephritis by the World Health Organization (WHO) was revised by an international committee in 2004 (Figs. 68.13 to 68.15). A summary of the histological features in each class of lupus nephritis can be found in Table 68.3. Treatment In 2012, the American College of Rheumatology (ACR), the European League Against Rheumatism (EULAR), and the European Renal Association–European Dialysis and Transplantation Association (ERA-EDTA) published their recommendations for the management and treatment of patients with lupus nephritis.43,44 Immunosuppressive treatment of mesangial classes of lupus nephritis (classes I and II) is usually not indicated (ACR). However, the distinction between early mesangial lesions that are in transition to more ominous classes from those that reflect mild and stable nephropathy is difficult. Consequently, treatment with prednisone alone or in combination with azathioprine has been recommended for patients with proteinuria that exceeds 1 g/day despite blockade of the renin–angiotensin–aldosterone (RAA) system, especially if the patient also has a nephritic urinary sediment (EULAR/ERA-EDTA). For patients with focal or diffuse proliferative glomerulonephritis (classes III and IV), ACR and EULAR/ERA-EDTA recommended mycophenolate mofetil (MMF) or intravenous cyclophosphamide (IVCY) plus glucocorticoids. Low-dose IVCY (500 mg IV every 2 weeks in six doses) offers a favorable balance of efficacy and relatively low toxicity for Caucasian patients with Western or Southern European ancestry. Higher dose, monthly IVCY for 6 months, plus 3 daily IV infusions of methylprednisolone initially, followed by

930

Part Seven  Organ-Specific Inflammatory Disease

$

%

FIG 68.13  Classes of the Pathology of Lupus Nephritis (1). A, Class II, mesangial proliferative lupus nephritis; mesangial areas are expanded by cells and matrix but the peripheral capillary loops remain widely patent (periodic acid–Schiff [PAS] stain). B, Class III, focal lupus nephritis; solid lesion at the lower right portion of this glomerulus demonstrates segmental fibrinoid necrosis. Note the nuclear fragments (karyorrhexis) in the fibrinous exudate (hematoxylin and eosin [H&E] stain.)

%

$

FIG 68.14  Classes of the Pathology of Lupus Nephritis (2). A, Class IV, diffuse lupus nephritis; glomerulus with irregular but nearly global changes, including obliteration of many capillary loops resulting from endocapillary hypercellularity, “wire loop” thickening and hyaline thrombi (periodic acid–Schiff [PAS] stain). B, Class V, membranous lupus nephritis; glomerulus shows minimally increased mesangial cellularity with thickened but widely patent capillary loops (PAS stain).

prednisone has also been recommended for patients with ominous clinical and histological prognostic indicators, including cellular crescents and fibrinoid necrosis. ACR and EULAR/ERA-EDTA recommended either azathioprine (AZA) or MMF as maintenance therapy for patients showing a favorable response after initial immunosuppressive therapy. Although AZA and MMF appeared to be equally effective maintenance therapies in a European study, patients randomized to maintenance therapy with MMF had more favorable outcomes than those randomized to AZA in a larger study conducted worldwide. The duration of maintenance immunosuppressive therapy involves careful consideration of the risks of another renal flare-up versus the risks of drug toxicity.

EULAR/ERA-EDTA recommended at least 3 years of therapy in patients showing improvement after initial therapy. In general, we have offered a comparable recommendation that treatment should continue for at least 1 year after remission of renal disease to prevent exacerbations. Neither IVCY nor MMF is universally effective in the management of lupus nephritis, hence the search for more efficacious treatment regimens, including rituximab, belimumab (binds to BAFF), immunomodulators (e.g., laquinimod), cytokine inhibitors, immunoablation without or with stem-cell reconstitution, and immunological costimulation inhibitors (e.g., CTLA-4-Ig), continues.35,45 The importance of these ongoing efforts to

CHAPTER 68  Immunological Renal Diseases

931

For patients with “pure” class V lupus MN and nephrotic-range proteinuria, both the ACR and the EULAR/ERA-EDTA have recommended oral prednisone and MMF. A prospective controlled trial showed that both IVCY and CSA were more effective than steroids alone in inducing remission of proteinuria in lupus MN but that relapse of nephrotic syndrome occurred significantly more often in the CSA group than in the IVCY treatment group.

Scleroderma (Systemic Sclerosis) Renal Disease KEY CONCEPTS Nephropathies of Selected Connective Tissue Diseases

FIG 68.15  Ultrastructure of Proliferative Lupus Nephritis. Electron micrography demonstrates the characteristic mesangial deposits (dark materials interspersed within the centrally located amorphous, gray mesangial matrix) and subendothelial deposits (dark materials extending along the peripheral capillary loops).

TABLE 68.3  International Society of

Nephrology/Renal Pathology Society 2004 Classification of Lupus Nephritis Class

Histologic Features/Comments

I. Minimal mesangial

Normal light microscopy (LM); mesangial deposits by immunofluorescence (IF) and electron microscopy (EM) Pure mesangial hypercellularity and matrix expansion IF and EM: mesangial immune deposits Glomerular capillary obliteration in 50% of nephrons. Subsets defined as primarily global (class IV-G) or primarily segmental (class IV-S) involvement LM: Regular thickening of the peripheral capillary loops of the glomerulus. Mesangial expansion EM: Subepithelial, intramembranous, mesangial (but no or very rare subendothelial) immune complex deposits >90% global sclerosis without residual active lesions

II. Mesangial proliferative III. Focal

IV. Diffuse

V. Membranous

VI. Advanced

investigate novel approaches to treatment is underscored by observations that although the risk of renal failure caused by lupus nephritis decreased from the 1970s to the mid-1990s (coincident with the increased use of cyclophosphamide), that risk has shown a reverse trend and increased slightly in the last two decades.46

• Scleroderma renal crisis: Predominantly renal vasculopathy; moderate to severe (high renin) hypertension with progressive renal failure— treated with angiotensin-converting enzyme inhibitors (ACEIs); additional antihypertensive agents may be needed • Sjögren syndrome: Distal renal tubular acidosis, nephrogenic diabetes insipidus, interstitial nephritis, hypokalemia, and/or renal calculi; glomerulonephritis rare

The most common and potentially devastating renal manifestation of systemic sclerosis (Chapter 55) is scleroderma renal crisis (SRC).47 Most cases of SRC occur within 4 years of the onset of systemic sclerosis in patients with diffuse cutaneous scleroderma affecting the proximal extremities and the trunk. Several features, including rapid progression of skin thickening, palpable tendon friction rubs, anti-RNA polymerase III antibody, recent-onset cardiac events (e.g., pericardial effusion or heart failure), new-onset anemia (especially if associated with microangiopathic hemolysis and thrombocytopenia), and recent treatment with high-dose corticosteroids, help identify patients at increased risk for developing SRC. A classic clinical presentation may obviate the need for renal biopsy. However, renal biopsy may be necessary in atypical cases. For example, about 20% of SRC cases occur before the diagnosis of scleroderma has been established. Furthermore, patients with scleroderma have rarely developed other renal diseases, such as ANCA-associated vasculitis, which are important to recognize because they require treatments different from those usually recommended for SRC. Scleroderma renal crisis is characterized by abrupt onset of renin-mediated moderate to severe hypertension, rapid deterioration of renal function, and proteinuria (usually nonnephrotic). Associated findings may include microangiopathic hemolysis, hypertensive encephalopathy (including seizures), and hypertensive retinopathy, acute left ventricular failure, and pulmonary edema. Normotensive renal crisis occurs infrequently and may be recognized by the presence of microangiopathic hemolysis and/or unexplained azotemia. The primary pathogenic process appears to be a renal vasculopathy involving predominantly the interlobular arteries and arterioles. Marked intimal thickening with an attendant “mucoid” appearance, and fibrinoid necrosis in the absence of vasculitis, are common and characteristic of the disease (Fig. 68.16). Immune deposits are rarely observed by fluorescence or electron microscopy studies. Although a variety of treatments have been proposed for patients with scleroderma, none has been proven to be consistently efficacious. The most significant therapeutic advance in the treatment of SRC is the use of angiotensin-converting enzyme (ACE) inhibitors (ACEIs), which have dramatically

932

Part Seven  Organ-Specific Inflammatory Disease ON THE HORIZON • The advent of new genetic and molecular techniques and new disease models has led to exciting progress in our understanding of the biology of the glomerulus, the pathogenesis of many glomerular diseases, and the influence of genetic variants on disease predisposition and progression. • It is anticipated that these insights should lead to better noninvasive diagnostic techniques, biomarkers, and predictors of prognosis and relapse and facilitate a more personalized approach to therapy rather than a one-size-fits-all approach. • Novel targeted therapies are on the horizon that will interrupt or modulate the underlying pathophysiology of the individual diseases and also halt the downstream pathways of injury and fibrosis common to all of the glomerular diseases.

FIG 68.16  Scleroderma Renal Crisis. Renal arteriole demonstrates extensive fibrin deposition (dark material) within multiple layers of its wall. The lumen is further compromised by severe swelling and intimal hyperplasia (Masson trichrome stain).

Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES increased the 1-year survival of patients with SCR. Prompt treatment with ACEIs is recommended for hypertensive and normotensive SRC because these agents may reverse the process by interfering with angiotensin II–mediated vasoconstriction and by inhibiting the degradation of the potent vasodilator bradykinin by ACE. Despite this impressive impact of ACEIs on clinical outcomes, approximately one-third to one-half of patients still progress to early death or renal failure. This has prompted continued investigation of additional treatment strategies, including blockade of the RAA system at multiple sites (using, for example, a direct renin inhibitor) as well as addition of potent vasodilators (e.g., prostacyclin or an endothelin receptor antagonist).

Renal Disease of Sjögren Syndrome A variety of renal manifestations occur in approximately one-third of patients with primary Sjögren syndrome (Chapter 54), including tubular dysfunction (proximal or, more often, distal tubular acidosis, Fanconi syndrome, hypokalemia that may be profound and associated with paralysis, Bartter syndrome, Gitelman syndrome, nephrogenic diabetes insipidus), as well as renal calculi, nephrocalcinosis, interstitial nephritis, pseudolymphoma, necrotizing vasculitis, and glomerulopathy.48 Studies of renal pathology among patients with primary Sjögren syndrome have shown that acute and/or chronic tubulointerstitial nephritis is the predominant lesion. A diverse range of glomerular diseases, including MN and proliferative glomerulonephritis (focal or diffuse, and membranoproliferative), have been reported in primary Sjögren syndrome. In such cases, the possibility of overlap with SLE should be considered. Immunosuppressive therapy for renal manifestations of primary Sjögren syndrome should be individualized on the basis of activity and severity of the glomerular and/or tubulointerstitial nephritis. Patients with serious complications of tubular dysfunction may warrant immunosuppressive therapies based on preliminary evidence that autoantibodies (against, for example, carbonic-anhydrase-II) as well as cell-mediated injury likely contribute to at least some of those disorders.

1. Couser WG, Johnson RJ. The etiology of glomerulonephritis: roles of infection and autoimmunity. Kidney Int 2014;86:905–14. 2. Mastroianni-Kirsztajn G, Hornig N, Schlumberger W. Autoantibodies in renal diseases - clinical significance and recent developments in serological detection. Front Immunol 2015;6:221. 3. Floege J, Amann K. Primary glomerulonephritides. Lancet 2016;387:2036–48. 4. Waldman M, Crew RJ, Valeri A, et al. Adult minimal-change disease: clinical characteristics, treatment, and outcomes. Clin J Am Soc Nephrol 2007;2:445–53. 5. Maas RJ, Deegens JK, Wetzels JF. Permeability factors in idiopathic nephrotic syndrome: historical perspectives and lessons for the future. Nephrol Dial Transplant 2014;29:2207–16. 6. Fornoni A, Sageshima J, Wei C, et al. Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis. Sci Transl Med 2011;3:85ra46. 7. Yu CC, Fornoni A, Weins A, et al. Abatacept in B7-1-positive proteinuric kidney disease. New Engl J Med 2013;369:2416–23. 8. D’Agati VD, Alster JM, Jennette JC, et al. Association of histologic variants in FSGS clinical trial with presenting features and outcomes. Clin J Am Soc Nephrol 2013;8:399–406. 9. Fernandez-Fresnedo G, Segarra A, Gonzalez E, et al. Rituximab treatment of adult patients with steroid-resistant focal segmental glomerulosclerosis. Clin J Am Soc Nephrol 2009;4:1317–23. 10. Iijima K, Sako M, Nozu K, et al. Rituximab for childhood-onset, complicated, frequently relapsing nephrotic syndrome or steroid-dependent nephrotic syndrome: a multicentre, double-blind, randomised, placebo-controlled trial. Lancet 2014;384:1273–81. 11. Malaga-Dieguez L, Bouhassira D, Gipson D, et al. Novel therapies for FSGS: preclinical and clinical studies. Adv Chronic Kidney Dis 2015;22:e1–6. 12. Coppo R. Different targets for treating focal segmental glomerular sclerosis. Contrib Nephrol 2013;181:84–90. 13. Beck LH Jr, Bonegio RG, Lambeau G, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 2009;361:11–21. 14. Tomas NM, Beck LH Jr, Meyer-Schwesinger C, et al. Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy. New Engl J Med 2014;371:2277–87. 15. Hoxha E, Thiele I, Zahner G, et al. Phospholipase A2 Receptor Autoantibodies and Clinical Outcome in Patients with Primary Membranous Nephropathy. J Am Soc Nephrol 2014;25:1357–66. 16. Waldman M, Austin HA 3rd. Treatment of idiopathic membranous nephropathy. J Am Soc Nephrol 2012;23:1617–30.

CHAPTER 68  Immunological Renal Diseases 17. Fervenza FC, Cosio FG, Erickson SB, et al. Rituximab treatment of idiopathic membranous nephropathy. Kidney Int 2008;73:117–25. 18. Fervenza FC, Abraham RS, Erickson SB, et al. Rituximab therapy in idiopathic membranous nephropathy: a 2-year study. Clin J Am Soc Nephrol 2010;5:2188–98. 19. Remuzzi G, Chiurchiu C, Abbate M, et al. Rituximab for idiopathic membranous nephropathy. Lancet 2002;360:923–4. 20. Pickering MC, D’Agati VD, Nester CM, et al. C3 glomerulopathy: consensus report. Kidney Int 2013;84:1079–89. 21. Fakhouri F, Fremeaux-Bacchi V, Noel LH, et al. C3 glomerulopathy: a new classification. Nature Rev Nephrol 2010;6:494–9. 22. De Vriese AS, Sethi S, Van Praet J, et al. Kidney Disease Caused by Dysregulation of the Complement Alternative Pathway: An Etiologic Approach. J Am Soc Nephrol 2015;26:2917–29. 23. Bertino G, Ardiri A, Proiti M, et al. Chronic hepatitis C: This and the new era of treatment. World J Hepatol 2016;8:92–106. 24. Bruchfeld A, Lindahl K, Reichard O, et al. Pegylated interferon and ribavirin in haemodialysis patients. Nephrol Dial Transplant 2006;21:1444–5. 25. Feng B, Eknoyan G, Guo ZS, et al. Effect of interferon-α-based antiviral therapy on hepatitis C virus-associated glomerulonephritis: a meta-analysis. Nephrol Dial Transplant 2012;27:640–6. 26. Sise ME, Bloom AK, Wisocky J, et al. Treatment of hepatitis C virus-associated mixed cryoglobulinemia with direct-acting antiviral agents. Hepatology 2016;63:408–17. 27. Sneller MC, Hu Z, Langford CA. A randomized controlled trial of rituximab following failure of antiviral therapy for hepatitis C virus-associated cryoglobulinemic vasculitis. Arthritis Rheum 2012;64:835–42. 28. Saadoun D, Resche-Rigon M, Sene D, et al. Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinaemia vasculitis. Ann Rheum Dis 2008;67:1431–6. 29. Lan X, Rao TK, Chander PN, et al. Apolipoprotein L1 (APOL1) Variants (Vs) a possible link between Heroin-associated Nephropathy (HAN) and HIV-associated Nephropathy (HIVAN). Front Microbiol 2015;6:571. 30. Booth JW, Hamzah L, Jose S, et al. Clinical characteristics and outcomes of HIV-associated immune complex kidney disease. Nephrol Dial Transplant 2016;31:2099–107. 31. Nobakht E, Cohen SD, Rosenberg AZ, et al. HIV-associated immune complex kidney disease. Nature Rev Nephrol 2016;12:291–300. 32. Cattran DC, Coppo R, Cook HT, et al. The Oxford classification of IgA nephropathy: rationale, clinicopathological correlations, and classification. Kidney Int 2009;76:534–45.

933

33. Rauen T, Eitner F, Fitzner C, et al. Intensive Supportive Care plus Immunosuppression in IgA Nephropathy. N Engl J Med 2015;373:2225–36. 34. Wyatt RJ, Julian BA. IgA Nephropathy. N Engl J Med 2013;368:2402–14. 35. Karras A, Jayne D. New biologics for glomerular disease on the horizon. Nephron 2014;128:283–91. 36. Berden AE, Ferrario F, Hagen EC, et al. Histopathologic classification of ANCA-associated glomerulonephritis. J Am Soc Nephrol 2010;21:1628–36. 37. Stone JH, Merkel PA, Spiera R, et al. Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med 2010;363:221–32. 38. Jones RB, Tervaert JW, Hauser T, et al. Rituximab versus cyclophosphamide in ANCA-associated renal vasculitis. N Engl J Med 2010;363:211–20. 39. Guillevin L, Pagnoux C, Karras A, et al. Rituximab versus azathioprine for maintenance in ANCA-associated vasculitis. N Engl J Med 2014;371:1771–80. 40. Pedchenko V, Bondar O, Fogo AB, et al. Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis. N Engl J Med 2010;363:343–54. 41. Greco A, Rizzo MI, De Virgilio A, et al. Goodpasture’s syndrome: a clinical update. Autoimmun Rev 2015;14:246–53. 42. Pusey CD. Anti-glomerular basement membrane disease. Kidney Int 2003;64:1535–50. 43. Hahn BH, McMahon MA, Wilkinson A, et al. American College of Rheumatology guidelines for screening, treatment, and management of lupus nephritis. Arthritis care & research 2012;64:797–808. 44. Bertsias GK, Tektonidou M, Amoura Z, et al. Joint European League Against Rheumatism and European Renal Association-European Dialysis and Transplant Association (EULAR/ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis. Ann Rheum Dis 2012;71:1771–82. 45. Mok CC. Towards new avenues in the management of lupus glomerulonephritis. Nature Rev Rheumatol 2016;12:221–34. 46. Tektonidou MG, Dasgupta A, Ward MM. Risk of End-stage Renal Disease in Patients with Lupus Nephritis, 1970 to 2015 A systematic review and Bayesian meta-analysis. Arthritis Rheumatol 2016;68:1432–41. 47. Mouthon L, Bussone G, Berezne A, et al. Scleroderma renal crisis. J Rheumatol 2014;41:1040–8. 48. Francois H, Mariette X. Renal involvement in primary Sjogren syndrome. Nature Rev Nephrol 2016;12:82–93.

CHAPTER 68  Immunological Renal Diseases

933.e1

MULTIPLE-CHOICE QUESTIONS 1. A 45-year-old woman presented to the emergency department (ED) with swelling in both ankles, 15-lb weight gain over the past 3 months and shortness of breath. During a physical examination 1 year prior, 1+ protein was noted on dipstick urinalysis, but no further workup was done. There was no family history of renal disease. The patient has blood pressure of 120/80 mm Hg, elevated respiratory rate of 25, and oxygen saturation of 90%. Physical examination was notable for pitting edema in her legs up to the mid-thighs. Lungs were clear. Laboratory testing revealed a hemoglobin level of 14 g/dL, hematocrit of 42%, serum creatinine level of 1.0 mg/dL, blood urea nitrogen (BUN) of 28 mg/dL, albumin level of 1.6 g/ dL, serum total cholesterol level of 350 mg/dL (normal, 8 degrees in uniocular ocular excursion in any one direction 10. Decrease of acuity equivalent to 1 Snellen line

support. The general health benefits of smoking cessation are numerous; in addition, smokers are more likely to relapse after a course of medical therapy compared with nonsmokers. In parallel to restoring and maintaining euthyroidism, the successful treatment of GO depends on staging the activity and severity of the disease. In patients with mild, active GO, an observational policy can be employed with symptomatic measures, such as artificial tears and dark glasses. Selenium supplements, at a dose of 100 µg twice daily for 6 months, have been shown to significantly improve quality of life, slow disease progression, and reduce ocular involvement in these patients.21 In those with moderate to severe, active, and progressive GO, a course of oral or intravenous (IV) glucocorticoids (steroids) is indicated. Orbital radiotherapy is also efficacious in people with active inflammation and diplopia, but since it has a delayed onset of action, it often needs to be used in conjunction with other therapies, such as steroids or orbital decompression surgery. In patients with sightthreatening optic neuropathy, high-dose IV steroids are used,

CHAPTER 70  Autoimmune Thyroid Diseases and orbital pressure is relieved by urgent orbital decompression surgery. In patients whose eyelids do not close completely, eye ointments and protective eye pads are essential to protect the eyes against corneal damage and ulceration. Once disease activity has burned out, rehabilitative surgery can greatly improve the function and cosmetic appearance of the eyes. Orbital decompres­ sion, strabismus correction, and eyelid surgery are commonly used procedures.22

FUTURE DEVELOPMENTS FOR GRAVES HYPERTHYROIDISM AND OPHTHALMOPATHY Novel approaches to modulating the immune response in GD and GO as a therapeutic strategy are under investigation. In light of the significant side effects associated with steroid therapy for GO, steroid-sparing agents, including methotrexate and mycophenolate mofetil, are of particular interest. Novel biological agents have been evaluated to a limited extent in these condi­ tions and are subject to further ongoing studies.23 Rituximab, a CD20 monoclonal antibody (mAb) that depletes circulating B cells, appeared to be potentially efficacious in early studies; however, in two randomized controlled trials in individuals with moderate to severe active GO, results have been conflicting, and therefore further studies are now needed. A longer-term goal is the development of an anti–TSH-R antibody or small molecule antagonist that could block binding of stimulatory TSH-R anti­ bodies or inhibit TSH-R signaling, thus ameliorating the cause of hyperthyroidism.

AUTOIMMUNE HYPOTHYROIDISM The most common cause of autoimmune hypothyroidism (AH) is chronic (or lymphocytic) autoimmune thyroiditis. There are two variants, atrophic and goitrous (Hashimoto thyroiditis).

Epidemiology In populations living in iodine sufficient areas, AH is common, affecting between 1 and 10% of the population. The prevalence increases with age, with 3–20% of individuals over 75 years of age being hypothyroid. Like GD, AH is more common in women than in men. In a UK community survey, the incidence of hypothyroidism in women was 3.5/1000/year, which increased to 13.7/1000/year in women between 75 and 80 years of age. In men, the incidence was just 0.6/1000/year.24

Etiology AH, like GD, is a complex genetic condition. Familial clustering provides evidence for a genetic etiology, which, in several studies, appears stronger than that for GD. The λs for AH is estimated to be between 10 and 45, suggesting that AH is more heritable compared with GD. In families with autoimmunity, frequently a mixture of individuals affected by AH and GD are seen, sug­ gesting some shared genetic factors. The differing prevalence of AH in different ethnic groups, with AH being more common in Caucasian than in black populations, also supports a genetic background. Knowledge about the genetics of AH is limited. The MHC class II HLA-DR3, -DR4, and -DR5 alleles have been associated with AH in Caucasians only. Conflicting results have been reported for HLA-DQ alleles. One study reported that the HLA-DQ alleles DQA1*0301 and DQB1*0201 confer sus­ ceptibility to AH in Caucasians, with certain HLA-DQ alleles (DQA1*0102 and DQB1*0602) reported to confer a protective

953

effect. These data highlight the need to further investigate the MHC region to clarify its role in AH susceptibility. In common with GD, the CTLA-4 gene also appears to influ­ ence AH susceptibility. Three CTLA-4 polymorphisms have been associated with AH in a number of populations. An A/G SNP located downstream of the 3’UTR (designated CT60), an A/G polymorphism at codon 17, and a 106-bp microsatellite repeat in the 3’UTR of exon 3. A locus on chromosome 8q24 containing the thyroglobulin gene was linked to AH, and a number of SNPs were subsequently studied in AH individuals with modest reported ORs for association of between 1.32 and 1.56. Other loci impli­ cated in AH susceptibility include the tumor necrosis factor (TNF-α) gene, PTPN22, CYP27B1, T-cell receptor (TCR) genes, and several immunoglobulin genes and cytokine regulatory genes. In contrast to GD, environmental factors in AH susceptibility have been challenging to identify. However, the role of iodine is widely accepted, since population studies have reported an increase in the prevalence of thyroid lymphocytic infiltration and auto­ antibodies following public health salt iodization programs. Infectious agents have also been implicated in susceptibility to AH. Several studies have identified an increased prevalence of IgG and/or IgA antibodies to virulence-associated outer mem­ brane proteins of Yersinia enterocolitica in AH patients and in relatives of individuals with AH, suggesting that susceptibility genes for Yersinia infection may also confer risk for AH. The effect of radiation, either “internal” (nuclear “fallout” or from RAI treatment) or “external” (radiotherapy or direct exposure during a nuclear accident), on AH susceptibility has been extensively studied. Following the nuclear reactor accident at Chernobyl, a rise in thyroid autoantibodies was noted 15 years following exposure; however, this was not accompanied by thyroid dysfunction.25 Long-term follow-up studies of thyroid function in Japanese survivors of the atomic bombing of Nagasaki and Hiroshima have demonstrated a clear link between radiation exposure and thyroid cancer; however, the association with AH remains disputed. One study, at 40 years follow-up, demonstrated a significant relationship between dose of radiation exposure at Nagasaki and AH. However, a further study, at more than 50 years of follow-up, showed that radiation exposure did not correlate with either the occurrence of thyroid autoantibodies or AH.26

Immunopathogenesis The mechanisms by which tolerance to thyroid antigens is lost in the first instance remain obscure. It appears that both a susceptible genetic background and a permissive environ­ ment are required before AH develops. Notably, AH is much more frequent in the autoimmune polyendocrinopathy type 1 (APECED) syndrome than GD, suggesting that central thymic T-cell selection, and therefore central tolerance, may be more important in AH than in GD. Histologically, lymphocytic infiltrates can be seen in the thyroid, consisting of both T and B cells (Fig. 70.7). These infiltrates can be diffuse or focal. Scarring and fibrosis may also be seen, with destruction of the normal thyroid architecture and an absence of colloid in thyroid follicles. Both cell-mediated and humoral immune mechanisms are important in the continuing thyroid damage seen in AH. T cells are known to play a pivotal role in both the initiation and perpetu­ ation of AH. Studies in which researchers induced hypothyroidism in Rag1-deficient transgenic mice that were unable to produce autoantibodies confirm this.27 T cells respond to antigen-presenting

954

Part seven  Organ-Specific Inflammatory Disease TABLE 70.3  Common and Rare Clinical

Manifestations of Hypothyroidism Common

Rare

Neuropsychiatric Lethargy Impaired cognitive function Slow speech Depression

Cerebellar ataxia Deafness Psychosis

Gastrointestinal Anorexia Weight gain Constipation and bloating Abnormal liver function tests

FIG 70.7  Lymphocytic infiltration of the thyroid in a patient with autoimmune hypothyroidism.

cells (APCs) and release cytotoxic and lytic factors, which result in thyrocyte death. Thyroid follicular cells have themselves been demonstrated to express HLA class II molecules, suggesting that they may also have a direct role in antigen presentation. The humoral immune response is also important in AH. More than 90% of individuals with AH have detectable TPO antibodies. Autoantibodies directed against Tg and, to a lesser degree, the TSH-R are also commonly detected. In vitro, TPO antibodies can fix complement and directly induce cell damage.17 Their presence within thyroid follicles in AH patients suggests they may have the same effect in vivo, although the thyrocyte destruc­ tion found in the Rag1-deficient mouse suggests TPO antibodies are not necessary for AH. Interestingly, the epitopes toward which TPO antibodies are directed in both GD and AH overlap, and there is no disease specificity for the targeted TPO domain.17 TSH-R autoantibodies found in rare patients with AH are likely to exert a blocking or antagonist effect, thus inducing hypothyroid­ ism. As thyroid hormone secretion falls, increasing thyrocyte stimulation by elevation of serum TSH may induce or augment thyroid autoantigen expression (e.g., TPO, Tg), thereby perpetuat­ ing the autoimmune response.

Clinical Presentation Hypothyroidism can result in changes in almost every organ system in the body (Table 70.3). Initially, the signs and symptoms may be subtle and nonspecific, including tiredness, cold sensitivity, and constipation. Hypothyroidism is frequently diagnosed incidentally following blood tests for another problem. The typical goiter palpable in Hashimoto thyroiditis is moderate in size and firm with a finely granular surface. Individuals often report a gradual increase in size over a number of years. However, rapid growth is unusual. In atrophic AH, the size of the thyroid gland is reduced.

Investigation and Diagnosis Hypothyroidism is detected biochemically by a raised serum TSH with reduced fT4. AH is differentiated from other forms of hypothyroidism by the presence of circulating autoantibodies, including TPO and Tg. On ultrasound scanning, the thyroid gland appears finely heterogeneous and hypoechoic, a change that predates serum autoantibody positivity in some. Fine-needle aspiration (FNA) demonstrates lymphocytes and Hürthle cells,

Ascites

Cardiorespiratory Shortness of breath on exertion Reduced exercise tolerance Bradycardia Diastolic hypertension Cardiomegaly Low-voltage electrocardiogram Peripheral edema (nonpitting)

Pericardial effusion

Genitourinary Oligomenorrhea, amenorrhea, menorrhagia Reduced libido Early fetal loss Impotence

Cutaneous Cold intolerance Skin dryness and thickening Malar flush Edema of the face, hands, and feet Change in face shape Pallor Nail abnormalities Alopecia

Musculoskeletal Bradykinesia Joint and muscular pains Delayed relaxation of tendon reflexes

Miscellaneous Goiter (in Hashimoto thyroiditis) Reduced basal metabolic rate Increased sensitivity to exogenous insulin Abnormal lipid metabolism

but this investigation is generally only indicated if a discrete nodule is present. Reflecting the effects of hypothyroidism on multiple organ systems, many biochemical and hematological abnormalities, such as mild anemia, hyponatremia, or elevated serum creatine kinase, transaminases, lactate dehydrogenase, and low-density lipoprotein (LDL) cholesterol, are also commonly detected in patients with AH.

Management AH requires lifelong treatment with thyroid hormone replacement therapy. The most commonly prescribed is synthetic thyroxine, levothyroxine (L-T4), which is widely available and inexpensive.

CHAPTER 70  Autoimmune Thyroid Diseases Except for individuals with known heart disease or the very old, a full, weight-related replacement dose (≈1.6 µg/kg/day) should be started. Once the patient is on a stable dose, thyroid function should be assessed annually to ensure that the patient continues to receive the appropriate dose. Some commonly prescribed medications, such as calcium and iron supplements, and proton pump inhibitors interfere with the absorption of L-T4, and patients should be advised to take these at least 4 hours before or after their L-T4 to ensure maximum absorption.

Subclinical Hypothyroidism Although the need to treat individuals with overt AH is universally accepted, it is unclear whether thyroid hormone replacement is beneficial in individuals with persistent subclinical hypothyroidism (serum TSH raised, but fT4 and fT3 within the normal reference range on at least two separate occasions). Progression to overt hypothyroidism from this state occurs in about 2% of individuals per year who are TPO antibody negative, rising to 5% per year if antibodies are present. Persistent subclinical hypothyroidism has been associated with a number of markers of cardiac and vascular dysfunction in observational studies, including left ventricular diastolic dysfunction, increased vascular resistance, and atherosclerosis. A randomized controlled study is now underway to determine whether thyroid hormone replacement can be used to improve cardiovascular outcomes in patients with persistent subclinical hypothyroidism.28

CLINICAL PEARLS Management of Persistent Subclinical Hypothyroidism • If thyroid-stimulating hormone (TSH) is elevated, immediate treatment should be offered: • In pregnancy • Preconception, if planning a pregnancy • If TSH is elevated but 10 mIU/L, treatment should be offered: • To individuals under the age of 70 years • To individuals over the age of 70 years if there is a clear history of hypothyroid symptoms or there are significant risk factors for cardiovascular disease.

955

1–4 months. During the thyrotoxic phase, preformed thyroid hormone stores are released from the thyroid follicles, leading to thyrotoxicosis, which may be severe. The hypothyroid phase follows when these preformed stores are exhausted and the thyroid has become depleted of hormones. In about 90% of cases this hypothyroidism is transient, but in some cases, it never resolves.

POSTPARTUM THYROIDITIS Postpartum thyroiditis (PPT) is a common endocrine condition that manifests within 1 year following pregnancy.29 It affects between 5% and 10% of women in the general population. PPT is classically a biphasic disorder, consisting of a period of transient thyrotoxicosis (median onset 12–14 weeks post partum) followed by a period of transient hypothyroidism (median onset 18–20 weeks post partum); however, a monophasic (thyrotoxicosis or hypothyroidism alone) or reversed biphasic (hypothyroidism followed by thyrotoxicosis) pattern can also occur. During pregnancy, there is a state of relative immune tolerance, followed by a rebound in immune function following delivery, coinciding with the occurrence of PPT. The presence of thyroid autoantibod­ ies and a lymphocytic infiltrate on thyroid biopsy supports an autoimmune basis for this condition.29 Clinically, the thyrotoxic phase of PPT is often mild, resulting in symptoms of fatigue and irritability, which can be misdiagnosed as postnatal depression. If the thyrotoxic episode is short, it may even go unnoticed. Neck pain is not a feature. Women with PPT who are thyrotoxic may benefit from a beta-blocker, such as propranolol, for symptom relief. Antithyroid drugs are not effective, as the thyrotoxicosis is caused by release of preformed thyroid hormones. Following the episode of thyroid dysfunction, 10–20% of women remain permanently hypothyroid.29 In women who have had PPT and then recovered, an annual assessment of thyroid function is recommended, as their risk of long-term hypothyroidism is considerable.29 In those women who return to being euthyroid, there is a 75% risk of PPT in subsequent pregnancies and a 50% risk of permanent hypothyroidism at 7 years.30

TRANSLATIONAL RESEARCH ON THE HORIZON

FUTURE DEVELOPMENTS

New Approaches to Therapy of Graves Disease

The genetics of AH remain understudied considering its frequency as the commonest autoimmune disease in humans. Considerable work remains to be done on whether treatment of subclinical hypothyroidism is beneficial. Given the insidious nature of the development of AH, it remains a good target for a preventative immunotherapeutic intervention, if a safe and economic treatment can be found.

• Novel immunotherapeutic agents for Graves orbitopathy • Anti–thyroid-stimulating hormone receptor (TSH-R) blocking antibodies for control of hyperthyroid Graves disease • Small molecule TSH receptor antagonists for management of hyperthyroidism in Graves disease

OTHER FORMS OF THYROIDITIS The term thyroiditis relates to conditions resulting in inflammation of the thyroid gland. A number of etiologies have been described, including infection, radiation exposure, drugs, and autoimmune factors. A common pattern to the natural history of several thyroiditides is frequently seen, involving an initial thyrotoxic phase of 1–3 months, followed by a rapid drop in serum thyroid hormones and a transient hypothyroid phase, often lasting another

The major challenge of the next 5–10 years is to take novel immunotherapeutic agents, including “biologicals” that have been developed for rheumatic disorders into the clinical arena for autoimmune thyroid diseases. The primary target of these efforts should be GO, which remains a disfiguring condition, often with substantial functional impairment of vision and associated low quality of life. Two randomized controlled trials of B-lymphocyte–depleting agents have yielded conflicting results, and therefore other powerful agents need to be investigated as this remains a disorder with no wholly satisfactory treatment. Early diagnosis of GO and development of markers that predict

956

Part seven  Organ-Specific Inflammatory Disease

progressive or severe disease will also be helpful in identifying patients for early intervention. The development of anti–TSH-R– blocking antibodies as a therapeutic agent for hyperthyroid GD seems likely. These agents might have a role in patients unlikely to gain medical remission from thionamide antithyroid drugs or in those for whom rapid control of hyperthyroidism is desir­ able. Small-molecule TSH-R antagonists may also come to the clinic, with a similar role. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Tunbridge WM, Evered DC, Hall R, et al. The spectrum of thyroid disease in a community: the Whickham survey. Clin Endocrinol (Oxf) 1977;7:481–93. 2. Laurberg P, Pedersen KM, Vestergaard H, et al. High incidence of multinodular toxic goitre in the elderly population in a low iodine intake area vs. high incidence of Graves’ disease in the young in a high iodine intake area: comparative surveys of thyrotoxicosis epidemiology in East-Jutland Denmark and Iceland. J Intern Med 1991;229:415–20. 3. Brix TH, Kyvik KO, Christensen K, et al. Evidence for a major role of heredity in Graves’ disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001;86:930–4. 4. Vaidya B, Kendall-Taylor P, Pearce SH. The genetics of autoimmune thyroid disease. J Clin Endocrinol Metab 2002;87:5385–97. 5. Eschler DC, Hasham A, Tomer Y. Cutting edge: the etiology of autoimmune thyroid diseases. Clin Rev Allergy Immunol 2011;41:190–7. 6. Ueda H, Howson JM, Esposito L, et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003;423(6939):506–11. 7. Velaga MR, Wilson V, Jennings CE, et al. The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves’ disease. J Clin Endocrinol Metab 2004;89:5862–5. 8. Brent GA. Environmental exposures and autoimmune thyroid disease. Thyroid 2010;20:755–61. 9. Vestergaard P. Smoking and thyroid disorders—a meta-analysis. Eur J Endocrinol 2002;146:153–61. 10. Cawood TJ, Moriarty P, O’Farrelly C, et al. Smoking and thyroid-associated ophthalmopathy: a novel explanation of the biological link. J Clin Endocrinol Metab 2007;92:59–64. 11. Winsa B, Adami HO, Bergström R, et al. Stressful life events and Graves’ disease. Lancet 1991;338:1475–9. 12. Matos-Santos A, Nobre EL, Costa JG, et al. Relationship between the number and impact of stressful life events and the onset of Graves’ disease and toxic nodular goitre. Clin Endocrinol (Oxf) 2001;55:15–19. 13. Chen F, Day SL, Metcalfe RA, et al. Characteristics of autoimmune thyroid disease occurring as a late complication of immune

reconstitution in patients with advanced human immunodeficiency virus (HIV) disease. Medicine (Baltimore) 2005;84:98–106. 14. Smith BR, Bolton J, Young S, et al. A new assay for thyrotropin receptor autoantibodies. Thyroid 2004;14:830–5. 15. Czarnocka B. Thyroperoxidase, thyroglobulin, Na(+)/I(−) symporter, pendrin in thyroid autoimmunity. Front Biosci 2011;16:783–802. 16. Costagliola S, Bonomi M, Morgenthaler NG, et al. Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity. Mol Endocrinol 2004;18:3020–34. 17. McLachlan SM, Rapoport B. Thyroid peroxidase as an autoantigen. Thyroid 2007;17:939–48. 18. Hegedus L. Treatment of Graves’ hyperthyroidism: evidence-based and emerging modalities. Endocrinol Metab Clin North Am 2009;38:355–71, ix. 19. Perros P, Dayan CM, Dickinson AJ, et al. Management of patients with Graves’ orbitopathy: initial assessment, management outside specialised centres and referral pathways. Clin Med (Lond) 2015;15:173–8. 20. Bahn RS. Graves’ ophthalmopathy. N Engl J Med 2010;362:726–38. 21. Marcocci C, Kahaly GJ, Krassas GE, et al. Selenium and the course of mild Graves’ orbitopathy. NEJM 2011;364:1920–31. 22. Bartalena L, Baldeschi L, Dickinson A, et al. Consensus statement of the European Group on Graves’ orbitopathy (EUGOGO) on management of GO. Eur J Endocrinol 2008;158:273–85. 23. Salvi M, Campi I. Medical treatment of Graves’ orbitopathy. Horm Metab Res 2015;47:779–88. 24. Vanderpump MP, Tunbridge WM, French JM, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 1995;43:55–68. 25. Agate L, Mariotti S, Elisei R, et al. Thyroid autoantibodies and thyroid function in subjects exposed to Chernobyl fallout during childhood: evidence for a transient radiation-induced elevation of serum thyroid antibodies without an increase in thyroid autoimmune disease. J Clin Endocrinol Metab 2008;93:2729–36. 26. Imaizumi M, Usa T, Tominaga T, et al. Radiation dose-response relationships for thyroid nodules and autoimmune thyroid diseases in Hiroshima and Nagasaki atomic bomb survivors 55-58 years after radiation exposure. JAMA 2006;295:1011–22. 27. Quaratino S, Badami E, Pang YY, et al. Degenerate self-reactive human T-cell receptor causes spontaneous autoimmune disease in mice. Nat Med 2004;10:920–6. 28. Pearce SHS, Brabant G, Duntas LH, et al. 2013 ETA guideline: management of subclinical hypothyroidism. Eur Thyroid J 2013;2:215–28. 29. Landek-Salgado MA, Gutenberg A, Lupi I, et al. Pregnancy, postpartum autoimmune thyroiditis, and autoimmune hypophysitis: intimate relationships. Autoimmun Rev 2010;9:153–7. 30. De Groot L, Abalovich M, Alexander EK, et al. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2012;97:2543–65. 31. Davies TF, Ando T, Lin RY, et al. Thyrotropin receptor-associated diseases: from adenomata to Graves’ disease. J Clin Invest 2005;115:1972–83.

CHAPTER 70  Autoimmune Thyroid Diseases

956.e1

MULTIPLE-CHOICE QUESTIONS 1. When assessing an individual with thyrotoxicosis, which feature is NOT consistent with a diagnosis of Graves disease? A. Increasing severity of symptoms over weeks to months B. Grittiness of the eyes and blurred vision on waking in the morning C. Neck pain D. A family history of autoimmune hypothyroidism and pernicious anemia E. Relief of symptoms with beta-blocker medication 2. Which of the listed clinical features is in keeping with a diagnosis of postpartum thyroiditis (PPT)?

A. Proptosis of the eyes B. Pretibial myxedema C. Previous history of Graves disease D. Goiter with a bruit E. Onset of symptoms 4 months after delivery 3. In moderate to severe Graves ophthalmopathy (GO), the following treatments are evidence based and effective except: A. Oral selenium supplements B. Topical lubricants C. Systemic glucocorticoids D. Orbital radiotherapy E. Orbital decompression surgery

71  Type 1 Diabetes Leonard C. Harrison

Diabetes is not a single disease but a metabolic syndrome in which different mechanisms lead to deficiency of insulin and/ or impaired insulin action and persistent hyperglycemia. The American Diabetes Association classified diabetes into four categories based on etiology rather than age of onset (juvenile-onset versus adult-onset) or requirement for insulin therapy (insulindependent versus noninsulin-dependent).1 The vast majority of cases, approximately 10% and 90%, respectively, are attributed to type 1 and type 2 diabetes. This chapter focuses mainly on type 1 diabetes, which results from an absolute deficiency of insulin secondary to the loss of pancreatic β cells. Type 1 diabetes is classified as 1A (immune-mediated) or 1B (idiopathic), primarily depending on the presence or absence, respectively, of pancreatic islet autoantibodies. However, as discussed below, type 1A diabetes (T1DA) and type 1B diabetes (T1DB) also differ in their natural history and clinical features. Diabetes is diagnosed on the basis of the following criteria1: symptoms in association with a casual plasma glucose ≥200 mg/dL (11.1 mmol/L) OR fasting plasma glucose ≥126 mg/dL (7.0 mmol/L) OR 2-hour plasma glucose ≥200 mg/dL (11.1 mmol/L) in an oral glucose tolerance test (OGTT; 75 g glucose in water). Without symptoms, the diagnosis of diabetes must rest on confirmation of a raised plasma glucose concentration. A further diagnostic criterion introduced by the World Health Organization (WHO) in 2011, in particular for the diagnosis of type 2 diabetes, is a confirmed blood glycated hemoglobin (HbA1c) ≥48 mmol/mol (6.5%). Because HbA1c is an integrated measure of glycemia over many weeks, it is not suitable for diagnosing children or in the following circumstances: suspected type 1 diabetes; symptoms of diabetes for 24

959

No data

FIG 71.1  Global Incidence of type 1A diabetes (T1DA) in 100 000 children ages 0–14 years per year. (From IDF diabetes atlas, 7th ed. Brussels, Belgium: International Diabetes Federation; 2015.)

Type 1A diabetes lifetime risk General population

3% Mother

5%

4%

0.25%

First-degree relative (FDR)

HLA high risk genes

No HLA risk genes

5% Father

8% Sibling

0.01% Protective HLA allele

10–20% FDR and HLA high risk genes

95% of caucasians cases have at least one high risk HLA allele

FIG 71.2  Lifetime risk of type 1A diabetes.

interactions between the environment and β cells. In addition, evidence suggests that immune activation and β-cell destruction may be accelerated in the late preclinical stage. The appearance of predictive islet autoantibodies in the first years of life5 means that the stage for developing T1DA is set very early, even before birth, on a background of genetic susceptibility. These early years must provide clues to environment–gene interactions that lead to immune dysfunction and disease. It is generally agreed that β-cell destruction in T1DA is mediated by autoreactive T cells, the ultimate effectors being CD8 cytotoxic T cells (Fig. 71.4). The evidence for this is unequivocal in the inbred nonobese diabetic (NOD) mouse model of T1DA,

which shares several key features with T1DA in outbred humans (Table 71.1) despite 65 million years of evolutionary distance. The molecular mechanisms of β-cell death, gleaned mostly from the NOD mouse, encompass a combination of both apoptosis induced by activation of extrinsic (e.g., tumor necrosis factor [TNF] receptor or Fas ligation) or intrinsic (e.g., endoplasmic reticulum [ER] stress) pathways and necroptosis induced by cytotoxic CD8 T-cell granule components (granzymes and perforin), reactive oxygen species (ROS), or ischemia. The evidence from human studies is obviously less compelling, as access to human pathology is limited. Studies of pancreas biopsies and organ-donor pancreas, more recently from the

960

Part seven  Organ-Specific Inflammatory Disease Environmental modifiers 100% Beta-cell function

Autoantibodies to insulin, GAD65, IA2, ZnT8 Loss of first phase insulin response (iv glucose) Genetic susceptibility

Immune cells invade the islets beta-cell injury

Loss of glucose tolerance (oral glucose)

Prediabetes

Diabetes

Birth Months Autoimmune disorder

Years Metabolic disorder

FIG 71.3  Natural history of type 1A diabetes.

Activated cytotoxic CD8 T cells

Pancreatic islet

β cell β cell CD8 T cells Activated CD4 helper T cell β cell antigen

TCR

CD40L CD40

MHC II

Pancreatic lymph node

β cell antigen mimic

TCR MCHI

Activated dendritic cell

Super-activated dendritic cell

FIG 71.4  Immune mechanisms of β-cell destruction in type 1A diabetes.

Network for Pancreatic Organ Donors with Diabetes (nPOD; www.jdrfnpod.org), have not revealed the florid immune cell infiltration of islets (insulitis) seen in NOD mice but rather patchy insulitis predominantly caused by CD8 T cells and macrophages. A further observation, confirming earlier observations,17 is hyperexpression of HLA class I by islets, in association with immunoreactive interferon-α (IFN-α), even in atrophic islets lacking insulin, which can be taken as presumptive evidence for persisting virus. A key question is: why are β cells specifically targeted? The answer may lie in insulin itself. Central to the concept of autoimmune disease is the notion that the pathology

is driven by loss of immune tolerance to self-antigens. In the case of T1DA, considerable evidence, direct in the NOD mouse, identifies (pro)insulin as a key disease-initiating self-antigen (Table 71.2).

GENES The concordance for T1D in monozygotic twins, who are almost totally genetically identical, approaches 50%, indicating that both genetic and environmental–epigenetic mechanisms contribute to disease. Large case/control studies and more recently genome-wide

CHAPTER 71  Type 1 Diabetes TABLE 71.1 The Nonobese Diabetic

(NOD) Mouse as a Model of Human Type 1A Diabetes Feature

NOD Mouse

Human

Preclinical stage Gender Genetic susceptibility   MHC class II fi57 non-Asp   Polygenic non-MHC Environmental influence on gene penetrance Disease transmission via bone marrow Mononuclear cell infiltration of islets (insulitis) Other organs Impaired immune regulation Autoantigens:  (Pro)insulin   Glutamic acid decarboxylase (GAD) Clinical response to autoantigen-specific therapy

Yes F>M

Yes M > F after puberty

Yes (I-Ag7) Yes Yes

Yes (HLA-DQ8) Yes Yes

Yes

Yes

Marked

Moderate

Yes Yes

Sometimes Yes

Yes Yes

Yes Yes

Yes

Not yet shown

TABLE 71.2 Evidence for the Key Role of

Proinsulin as Primary Autoantigen in Type 1A Diabetes (T1DA)

• β cell–specific (except for thymus) • Second strongest genetic locus in humans (IDDM2) = VNTR 5’ of INS allelism correlates with proinsulin transcription in the thymus • Early target of autoimmunity in humans and nonobese diabetic (NOD) mice • Main target of T cells isolated from islets of NOD mice and humans with T1DA Genetic manipulation in the NOD mouse: • Transgenic expression of proinsulin (but not glutamic acid decarboxylase [GAD]) in antigen-presenting cells (APCs) prevents insulitis/diabetes • Transfer of hematopoietic stem cells (HSCs) or myeloid progenitors encoding proinsulin in APC progeny prevents diabetes • Knockout of proinsulin II (expressed in thymus) accelerates diabetes • Induction of mucosal tolerance to (pro)insulin prevents diabetes

association (GWA) studies have identified over 40 chromosomal loci associated with risk for T1DA18,19 (Fig. 71.5). Many are weak associations defined only by single nucleotide polymorphisms (SNPs), and the genes with which the SNPs are in linkage and/or their functional contribution to pathogenesis are not known. It is likely that different combinations of genes/susceptibility loci in different environmental contexts lead by different routes to the final outcome of β-cell destruction and account for phenotypic– clinical heterogeneity in T1D. Nevertheless, what is clear across populations globally is the dominance of the HLA locus (IDDM1) as the single most important genetic contributor to T1D risk (see Table 71.1), accounting for about half the lifetime risk. The highest risk HLA haplotypes in Europeans are DR3 (DRB1*03:01DQA1*05:01-DQB1*02:01) and DR4 (usually DRB1*04:01 or *04:04 with DQA1*03:01-DQB1*03:02). It should also be noted that the HLA DQ6 haplotype DQA1*01:02-DQB1*06:02, which is in linkage with DR15 (DRB1*15:01), is dominantly protective for

961

T1DA but denotes susceptibility for a subtype of multiple sclerosis and for narcolepsy. The second most important contribution is from the insulin gene (INS) locus (IDDM2), which maps to a variable number of tandem repeats (VNTR) 5’ of the coding sequence. Apart from the HLA locus, IDDM2 is still the only locus for which genome-wide association is reflected by linkage, which might be explained by disease heterogeneity. Long (class III) and short (class I) variable number tandem repeat (VNTR) alleles are associated, respectively, with higher and lower transcription of proinsulin messenger RNA (mRNA) in medullary thymic epithelial cells (mTECs) under the control of the autoimmune regulator gene (AIRE)20 and with lower and higher T1DA risk.21 This leads to the inference that IDDM2 controls the extent of deletion of proinsulin-specific T cells during their intrathymic development and predicts that the long VNTR would be associated with less proinsulin-specific T cells in the periphery; there is evidence for and against this, perhaps because of the difficulty of measuring islet antigen-specific T cells in human blood. The INS polymorphism is unique to humans. Mice, instead, have two insulin genes; INSI is expressed in the β cell and INSII in the thymus, and expression of the latter is impaired in the NOD mouse. In summary, IDDM2 provides a genetic mechanism for autoimmunity targeting proinsulin and the β cell. Many of the other candidate genes are involved in immune function and are associated with other autoimmune diseases. For example, a nonsynonymous gain-of-function (GOF) polymorphism in PTPN22 (lymphoid tyrosine phosphatase) results in enhanced T-cell suppression, which may impair negative selection of autoreactive T cells in the thymus and decrease the number and function of natural regulatory T cells (nTregs). Polymorphisms around IL2RA (interleukin-2 [IL-2] receptor α chain; CD25) and IL2 are associated with impaired IL-2 signaling, which in both humans and NOD mice impairs the generation and maintenance of nTregs. Polymorphisms involving the VDR (vitamin D receptor) and CYP27B1 (25-hydroxyvitamin vitamin D3 1-α-hydroxylase) and IFIH1 (IFN-induced with helicase C domain 1; which enhances the type I IFN response to virus infection) are clues to gene–environment interactions in T1DA.

β CELLS Why are β cells selectively destroyed by immune inflammation that involves the whole islet, which also contains a minority of glucagon-secreting α cells and other endocrine cells? First, cytotoxic CD8 T cells recognize autoepitope peptides (e.g., from insulin) presented by hyperexpressed HLA class I proteins on β cells. Second, because of their unique metabolic wiring, β cells may contribute to their own death at the hands of the immune system. Evidence for the hypothesis of “assisted suicide” is compelling.22 Studies of rodent islets, mainly in vitro, have indicated that β cells lack efficient antioxidant and free radical scavenging mechanisms and are especially sensitive to mitochondrial oxidative and ER stress in response to cytokines and granzymes,22 although more recent studies of human islets do not support this view.23 Third, many of the candidate T1D genes identified in GWA studies are transcribed in β cells (see Fig. 71.4) and encode proteins that interact with the immune system.23 Finally, in response to inflammation, human islets generate hundreds of RNA splice variants for proteins, which, translated as neoantigens, may not be subject to immune tolerance. Although it is still unclear whether human β cells are more sensitive than other autoimmune target cells to immune

962

Part seven  Organ-Specific Inflammatory Disease 7.0

6.5

Odds ratio

6.0

5.5 2.5

2.0

1.5

HLA *INS PTPN22 IL2RA C10orf59 *SH2B3 *ERBB3 *COBL *PTPN2 *CTRB1/2 *CLEC16A CTLA4 IL18RAP PTPN2 IL10 CCR5 *C6orf173 *C14orf181 *PRKD2 *IFIH1 *CTSH CD226 IL27 GAB3 IL2RA *SKAP2 *GLIS3 *ORMDL3 *PRKCQ IL2 BACH2 UBASH3A *RGS1 IL7R CIQTNF6 SIRPG *TNFAIP3 4p15 CD69 14q22 TAGAP *SMARCE1

1.0

Locus

FIG 71.5  Candidate genes in type 1A diabetes (T1DA). The y-axis shows the odds ratio for risk alleles at each of the loci on the x-axis.18 The majority of the known genes are involved in immune responses, and many (marked *) are expressed in islets. Colors indicate era of identification: blue 1970–2000; green 2001–2006; red 2007–2008; yellow 2009–2010.

effectors, what is clear is that having undergone apoptotic or necrotic death, β cells are not restorable in the face of autoimmune memory.

KEY CONCEPTS Type 1A Diabetes Is an Autoimmune Disease • Genetic and familial clustering, as well as association with other autoimmune diseases, including autoimmune polyendocrinopathy syndromes (e.g., APS-1 due to mutations in AIRE) • Presence of autoantibodies and T cells reactive with islet cell antigens • Strong association with specific human leukocyte antigen (HLA) alleles and haplotypes • Transfer of disease by bone marrow or development of disease in normal pancreas transplanted from identical twin without diabetes to the twin with diabetes • Neonatal onset with loss of natural regulatory T cells with mutations in FOXP3 (immune dysfunction/polyendocrinopathy/enteropathy/X-linked [IPEX] syndrome)

ENVIRONMENT—OUTSIDE AND INSIDE The environment in its various manifestations may impact on β-cell function in several possible ways: by activating innate immune cells (macrophages) in the islets to release cytokines (e.g., IL-1β, TNF) that elicit mitochondrial oxidative ER stress; by driving adaptive immunity to β cells in people with T1D risk genes, either generally or specifically by inducing posttranslational modifications in β-cell proteins that render them immunogenic;

and by promoting obesity and insulin resistance and thereby increasing the workload of the β cell. The environment in Western societies has changed dramatically in many ways during the last century (Fig. 71.6), and some of these changes have been associated epidemiologically or in animal studies with development of T1DA.6 An index of the modern “exposome” is the ongoing rise in the prevalence of obesity. When children at increased genetic risk for T1DA (with an affected first-degree relative) were monitored from birth, weight gain in the first 2–3 years of life was shown to be a risk factor for islet autoimmunity.24 When such at-risk children developed islet autoantibodies and were then followed up over time, those with insulin resistance progressed most rapidly to clinical diabetes.15 Whether insulin resistance is a risk factor for the development of islet autoimmunity is an open question that should be answered by ongoing pregnancy–birth cohort studies.25 If insulin resistance synergizes with impaired insulin secretion to promote T1DA, then lifestyle factors must be considered in the efforts to forestall or prevent T1DA. Excessive energy consumption leading to obesity is part of a complex interplay of related factors that promote low-grade inflammation and insulin resistance. These include a poor-quality diet and lack of physical exercise, sunlight (vitamin D), and sleep. The highly processed fast-food “Western” diet lacks diversity of components, lacks plant-derived prebiotics and complex carbohydrates (starches and fiber), is high in saturated fats and in sucrose and fructose sugars, and has added artificial preservatives, emulsifiers, and sweeteners. All of these alter the composition of the gut microbiome and reduce its diversity, which is a feature

CHAPTER 71  Type 1 Diabetes

963

Th The T he E Environment has h as Changed Excess calories * Different, less diverse food Less physical activity * Less sunlight-vitamin D * More ‘hygiene’ * Fewer infections More C-sections * Less breast milk * More antibiotics * Less thermoregulation Less sleep More pollution

Modifiers: culture, education, wealth, access to technology, family size, maternal age...

FIG 71.6  Changes in the modern environment. Those that have been associated with an increased risk for type 1A diabetes (T1DA) in humans or nonobese diabetic (NOD) mice are indicated (marked *).

found in children at risk for T1DA.26 Diets containing a diverse range of plant products (cereals, fresh fruits, and vegetables) provide complex carbohydrates for fermentation by colonic bacteria to short-chain fatty acids (SCFAs), which are antiinflammatory.27 The dramatic influence of the environment is seen in the comparison between the highly developed Finland and the nearby undeveloped Russian Karelia. Finland has the highest incidence of T1DA in the world, currently 57.6 cases/100 000 population ≤14 years of age (www.diabetesatlas.org). In 2005, there was a sixfold difference in the incidence of T1DA between Finland and Karelia despite overlap in ethnic background and a similar distribution of HLA-DQ risk genotypes.28 This difference could reflect a relatively lower rate of infections (in Finland), in keeping with the “hygiene hypothesis.”29 However, viral infections have long been considered triggers of T1DA, but the evidence remains inconclusive.30,31 Viral infection is circumstantially associated with the onset of T1DA, but given the long presymptomatic stage of the disease, this may simply represent a nonspecific inflammatory insult that precipitates clinical presentation. Viral mechanisms in T1DA could be direct or indirect (e.g., infection of β cells, infection of the exocrine pancreas with bystander death of β cells, mimicry between T-cell epitopes in a viral protein and β-cell autoantigens, or activation of endogenous retroviruses in β cells by environmental agents). If an exogenous virus was clearly identified, then protective vaccination early in life would be an approach to primary prevention. Interest in viral triggers of T1DA was initiated by the observation that a minority of children with congenital rubella developed insulin-dependent diabetes32 and were found to have a higher frequency of what was later recognized as the T1D susceptibility haplotype HLA-A1 B8-(DR3-DQ2). Clearly, this may have been an anomaly because congenital rubella has been virtually eliminated by vaccination and yet the incidence of T1D has continued to climb. Much circumstantial evidence exists for

enteroviruses in T1DA31 but has not been consolidated in followup studies. Even if a particular enterovirus strain were shown directly to be diabetogenic, vaccination would be elusive because among the many thousands of strain variants, the only one for which a vaccine exists is poliovirus. Mumps virus epidemics have been associated with T1DA onset after 2–4 years, and introduction of a mumps vaccine was associated with a plateau in the rising incidence of T1D in Finland, but this was temporary, and mumps vaccination has clearly not prevented T1D. It must be emphasized that there is no scientific evidence that any form of vaccination itself triggers T1DA. Rotavirus is the commonest cause of gastroenteritis in young children. The discovery of strong sequence similarities between T-cell epitopes in the VP7 protein of rotavirus and the IA2 and GAD islet antigens in autoantibody-positive children led to speculation that mimicry of rotavirus might contribute to islet autoimmunity. Subsequently, in the Australian BabyDiab Study, rotavirus infection was associated over time with the first appearance of or an increase in islet autoantibodies in children before they developed diabetes.33 Moreover, rotavirus has been shown to infect β cells in islets from mice, pigs, and monkeys and cause transient involution of the pancreas and hyperglycemia in a Toll-like receptor 3 (TLR3)–dependent manner in mice.34 Ubiquitous rotavirus infections that drive cross-reactive immunity to islet autoantigens are unlikely to be diabetogenic but could complement and sustain the immune response to direct infection of β cells. As with rubella and mumps, it will be interesting to observe if recently introduced rotavirus vaccines have any effect on the incidence of T1DA. The microbiome—the trillions of microorganisms (bacteria, fungi, archaea, protozoa, and viruses; Chapter 14) and their millions of genes and proteins that reside within the human mucosae, skin, and secretions—has gained increasing attention as a bellwether of health and disease (“dysbiosis”). Most

964

Part seven  Organ-Specific Inflammatory Disease

microbiome analyses are based on DNA extraction and polymerase chain reaction (PCR) amplification of regions of the 16S ribosomal RNA (rRNA) gene that are conserved among bacteria. Although other internal regions of the 16S gene are variable and enable taxonomic classification from the phylum to genus level, 16S sequencing does not have the sensitivity to distinguish individual species and their strains. Direct metagenomic sequencing of all species, as well as analysis of their transcriptomes, epigenomes, and functions, is required to fully comprehend the role of the microbiome in T1DA and other chronic immune– inflammatory diseases. The marked difference in the incidence of T1DA between Finland and neighboring Karelia was found to be associated in Finnish children having decreased bacterial diversity overall, a dominance of the phylum Bacteroidetes, and lack of butyrate-producing bacteria.26 These changes were seen after the appearance of autoantibodies, suggesting that they followed, rather than preceded, the disease process. However, in a further small study in Finnish children, metagenomic sequencing identified a relative abundance of Bacteroides dorei, which peaked around 7–8 months of age with the introduction of solids and preceded the appearance of islet autoantibodies. Gut Bacteroides species are abundant in Finnish children, including B. dorei, which produces a lipopolysaccharide (LPS) endotoxin that inhibits the immunostimulatory activity of Escherichia coli LPS, known to protect against development of diabetes in NOD mice. These findings suggest that an approach to lowering the environmental contribution to T1DA will be intervention with prebiotics, probiotics, or other means to favorably alter the composition of the gut microbiome.

KEY CONCEPTS Type 1A Diabetes: Epidemiology, Environment, and Genes • Incidence is increasing as a result of the impact of environment. • Environment factors enable increasing penetrance of lower-risk human leukocyte antigen (HLA) alleles. • Environmental factors are multifactorial but generally “pro-inflammatory” and impact via microbiome dysbiosis and epigenetic modifications on polygene expression to cause immune dysregulation in early life.

TREATMENT AND PREVENTION Treatment of the metabolic syndrome of T1D is focused on optimizing blood glucose control with various modes of insulin delivery to prevent the short- and long-term complications of hyperglycemia. Refinements and advances over the past 30 years in insulin delivery and improved control of blood glucose and blood pressure have greatly improved the lives and prognosis of individuals with T1DA. Nevertheless, despite the introduction of pure, recombinant forms of human insulin, which have varied kinetics of action and can be injected via a syringe or continuously infused via a pump, and blood glucose self-monitoring, which can now be done continuously in real time, those with T1DA have not been rescued from a life sentence, and their blood glucose control remains less than physiological. The “cure” of T1DA requires the transplantation of insulin-secreting cells or their progenitors or the regeneration of β cells in situ, in conjunction with approaches to prevent allograft rejection and/or recurrence of autoimmunity (there will be no cure for T1DA without prevention). Currently, in some economically developed societies, allografts of whole pancreas or isolated islets are offered

on a limited scale and at considerable cost to individuals with T1DA and life-threatening complications, in particular lack of awareness of hypoglycemia that is unresponsive to conventional treatment. However, shortage of donor tissue, cost, and the need for life-long immune suppressive agents militate against this solution. Longer-term, genetically engineered pig islet xenografts may overcome the tissue supply barrier. Stem cells, in particular autologous induced pluripotent stem cells (iPSCs), remain the great hope, but early proof-of-concept results in rodents await scale-up and translation to humans. The incidence of spontaneous autoimmune diabetes in inbred NOD mice is decreased by many immune and other (including environmental) interventions introduced early in the disease course, although various interventions are often only effective in a proportion of mice, sometimes only delay disease onset, and sometimes have no effect (and are therefore not reported). Nevertheless, these findings in the NOD mouse suggest that T1DA is potentially preventable in outbred humans. On the basis of the key role of proinsulin in driving β-cell autoimmunity, the NOD mouse has provided “proof-of-concept” for the efficacy of antigen-specific vaccination strategies.35 In humans, it has been convenient to classify prevention into primary (before the onset of islet autoimmunity), secondary (after the onset of islet autoimmunity), and tertiary (after the onset of clinical disease) prevention. Of course, real prevention is primary prevention—identifying those at genetic risk and intervening to avert islet autoimmunity. Neonatal screening based on HLA genotyping to identify at-risk individuals would be a basis for primary prevention, but even in countries with a high incidence of T1DA, this would have only modest predictive value, and intervention could be justified if it was practical and safe, if not efficacious. The mandate Primum non nocere (“first do no harm”) dictates that many immune modulating agents are off limits for either primary or secondary prevention in T1DA. Options (e.g., to modification of the diet or microbiome or of forms of vaccination) are greatly restricted. Agents that are effective in other autoimmune diseases are more likely to be effective in the early asymptomatic stage of T1DA, not after clinical presentation when most β cells have been destroyed. Indeed, clinical trials of tertiary prevention with more than 70 different agents since the early 1980s have failed to unequivocally demonstrate sustained preservation of residual β-cell function.36,37 Interestingly, the number of trials reporting a positive outcome decreased from the 1980s, together with a shift in the primary outcome measure away from clinical remission to the more rigorous measure of residual insulin (stimulated C-peptide) secretion. Is there a potential solution to the “off limits” dilemma? Classically, T1DA is regarded as a metabolic disorder diagnosed at clinical presentation with symptoms and signs of hyperglycemia. However, based on pathology, T1DA is an immune disease—autoimmune β-cell disorder (ABCD)—and only becomes a metabolic disorder at the end stage of its pathology. Accepting this reality would amount to a paradigm shift that could profoundly change the approach to and outcome of secondary prevention. Consider the situation in other autoimmune diseases. In rheumatoid arthritis, the pathology and clinical features (painful swollen joints) appear concomitantly when effective disease-sparing treatment with newer biological agents is initiated, not at the end stage of pathology when the joints are irreversibly deformed. A further benefit of diagnosing of T1DA early in the asymptomatic stage, based on evidence of underlying pathology, is that it would markedly

CHAPTER 71  Type 1 Diabetes reduce the risk of life-threatening ketoacidosis associated with the classic symptomatic presentation. In health, immune responses to autoantigens are regulated to prevent development of autoimmune diseases. Autoantigenspecific immunotherapy aims to boost or restore autoantigenspecific immunoregulatory mechanisms. Its parallel, allergen-specific immunotherapy, has been shown in randomized trials to be effective in allergic asthma and rhinitis. Such “negative vaccination” can be achieved in several ways: by administering antigen via a “tolerogenic” route (mucosal, dermal), cell type (resting dendritic cell), mode (with blockade of costimulation molecules), or form (as an “altered peptide ligand”). Mechanisms of antigen-induced tolerance include deletion and/or anergy of effector T cells and induction of regulatory T cells (iTregs). Of clinical importance is the ability of iTregs to exert antigen nonspecific “bystander” suppression. Thus by direct cell contact and/or the release of soluble immunosuppressive factors, such as IL-10, iTregs impair the function of antigen-presenting DCs to elicit effector T-cell responses to the same or other antigens presented locally in the lesion or draining lymph nodes. Bystander suppression does not require that the “tolerizing” antigen is necessarily the primary driver of pathology. Its clinical importance is that it obviates specificity restrictions imposed by polymorphisms in the HLA and human T-cell receptors. In NOD mice, administration of insulin, proinsulin peptides, or proinsulin DNA via oral or nasorespiratory routes, acting locally on the mucosal immune system, induces Tregs and decreases the incidence of diabetes. Results of randomized secondary prevention trials of insulin or GAD in relatives at risk for T1DA (ClinicalTrials.gov) have been summarized elsewhere.35-37 In the DPT-1 oral insulin trial, relatives with a 5-year diabetes risk of 25–50% received 7.5 mg human insulin or placebo daily for a median of 4.3 years. There was no effect overall, but posttrial hypothesis testing revealed a significant delay in diabetes onset in participants with significant IAA at entry.38 This outcome has led to an ongoing follow-up international trial by NIH-TrialNet using the same dose of 7.5 mg daily, which is not optimal because on a body-weight basis this dose is very small compared with that required to induce protective iTregs in the NOD mouse. Apart from the dose, other variables have not been systematically tested in humans, in part because of the expense and duration of prevention trials. These variables include route of administration, form of the antigen, combinations of antigen with antigennonspecific agents, and the nature of induced immune responses. Unfortunately, surrogate markers of a potential therapeutic response have not been included in most trials and, in the case of antigen-specific T cells in blood, are not universally accepted as being sufficiently robust and reproducible. Oral delivery might not be optimal because proteins are degraded after ingestion, and the concentration or form of peptide reaching the upper small intestine may be variable and unpredictable. T-cell responses that were observed after nasorespiratory administration of a peptide have not been observed after oral administration. In a randomized trial of nasal insulin in individuals with recent-onset T1D not initially requiring insulin treatment, participants in the nasal insulin arm had markedly blunted insulin antibody responses after subsequent subcutaneous insulin.39 This was the first demonstration, in humans, of immune regulation induced by mucosally administered exogenous autoantigen. Although this result provides a rationale for further disease endpoint trials, a Finnish study40 suggested that this result cannot be extrapolated to endogenous “autoantigenic” insulin. Nasal insulin (1 unit/kg

965

daily) did not alter the rate of progression to diabetes in islet autoantibody-positive children Th1) IL-4, IL-6, IL-8

TGF-β, IGF, PDGF Angiogenesis Endothelin-1 Fibroproliferation

Fibroblast Myofibroblast Foci

KEY CONCEPTS

Environment?

Failure of apoptosis

Extracellular matrix deposition

Lung remodeling

Pathogenesis of the Idiopathic Interstitial Pneumonias (IIPs) • Although the inciting event(s) is unknown in the different diseases, a common result is a dysregulated fibroproliferative response (similar to wound healing), which leads to excessive extracellular matrix (ECM) production and lung remodeling. • A genetically determined inability to repair and reepithelialize the denuded basement membranes adequately may be a contributing factor and may relate to the familial occurrence of some cases of idiopathic pulmonary fibrosis (IPF). • The presence of a chronic stimulus (autoantigen), as is seen in the pneumoconioses, may result in a persistent inflammatory and immune response and lead to a failure in the normal healing process. • The release of transforming growth factor-β (TGF-β) following epithelial injury stimulates collagen synthesis and the prevention of apoptosis of proliferating fibroblasts in the lung and may impair collagen degradation by inhibiting the production of metalloproteases. • A predominant T-helper type 2 (Th2) response in the lung and the absence of interferon-γ (IFN-γ) favor the development of a fibrotic response.

Fibrosis

FIG 72.4  Events Hypothesized to Be Involved in the Pathogenesis of Idiopathic Pulmonary Fibrosis. The initiating event(s) leading to persistent lung injury remains poorly understood. The interaction between genetic factors, environmental exposures, and infectious agents leads to epithelial and endothelial injury, resulting in the secretion of macrophage-derived growth factors, including transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), and platelet-derived growth factor (PDGF). This cytokine milieu stimulates fibroblast proliferation and collagen deposition. In addition, the resulting T-helper type 2 (Th2) immune response stimulates extracellular matrix production and fibroblast proliferation, resulting in lung remodeling and, eventually, lung fibrosis.

972

Part Seven  Organ-Specific Inflammatory Disease

The history and physical findings in IPF are nonspecific. However, extrapulmonary involvement does not occur; the presence of fever, arthralgias, myalgias, or pleuritis should suggest a connective tissue disorder. Antinuclear antibodies (ANAs) and rheumatoid factor are present in 10–20% of patients with IPF, but titers greater than 1 : 320 should suggest an alternative diagnosis. The majority of patients with IPF have abnormal chest radiography results at the time of presentation. Basal peripheral reticular opacities are the characteristic radiographic findings. A confident diagnosis of IPF from HRCT of the lung requires the presence of patchy, peripheral bibasal reticular abnormalities with honeycombing.23 The presence of extensive ground-glass opacities on HRCT should suggest an alternative diagnosis, such as DIP, hypersensitivity pneumonitis, bronchiolitis obliterans organizing pneumonia (BOOP), or NSIP. A surgical lung biopsy is recommended in patients with suspected IPF without a definitive HRCT appearance and who do not have contraindications to the procedure. This is especially important in patients with atypical clinical or radiographic findings, which could suggest the possibility of one of the other histological patterns of the IIPs and an improved prognosis. Biopsy may be omitted in older patients with cardiovascular disease, or those with evidence of extensive honeycomb change. Biopsy through video-assisted thoracoscopy (VATS) is the preferred surgical technique and has been associated with less morbidity and a decreased length of hospital stay compared with open lung biopsy. Treatment and Outcome The usual course of IPF is relentless progression without spontaneous remission, commonly with a fatal outcome. The most common cause of death in patients with IPF is progression of the underlying disease with two-thirds of deaths caused by respiratory failure or cardiovascular complications. Other causes of death in IPF include bronchogenic carcinoma, infection, and pulmonary embolism. Recent studies in patients with biopsy-proven IPF indicate a poor prognosis (30–50% 5-year survival).24 Previously, there was no evidence to support the use of any specific therapy in the management of IPF. However, recent clinical trials have shown decreased decline in forced vital capacity (FVC) after use of either pirfenidone25 or nintedanib.26 Thus for the first time, there are two drugs approved by the US Food and Drug Administration (FDA) for the treatment of IPF, with multiple other phase II and III clinical trials scheduled for completion in the near future. Finally, lung transplantation should be considered in patients who have progressive clinical and physiological deterioration and who meet established criteria.

Acute Interstitial Pneumonia AIP is a fulminant form of IIP. Although it was previously thought to represent an acute phase of UIP, some studies have suggested that it is a distinct entity.18 However, patients with documented UIP/IPF experiencing acute exacerbations can have the pathology of AIP superimposed on UIP.27 Clinical Manifestations AIP presents with the abrupt onset of dyspnea followed by rapid progression to respiratory failure. The clinical, radiographic, physiological, and histological features are identical to those of the acute respiratory distress syndrome (ARDS) but without any identifiable cause. Most patients are previously healthy individuals over 40 years of age. Men and women are equally

FIG 72.5  Histopathology of Acute Interstitial Pneumonitis. Diffuse thickening of the alveolar septum with an infiltration of mononuclear cells is the characteristic abnormality. The temporal uniformity of this process is also apparent.

affected. A viral prodrome is common, with symptoms that include fever, nonproductive cough, and dyspnea. Laboratory studies are nonspecific. Chest radiography and HRCT show diffuse airspace opacities and ground-glass attenuation, respectively. A similar presentation may occur as the initial manifestation of a CTD. Histopathology AIP is characterized by diffuse interstitial fibrosis that is temporally uniform (Fig. 72.5).28 The changes are identical to the organizing phases of diffuse alveolar damage, as seen in ARDS. Within the thickened interstitial space, there is active, diffuse fibroblast proliferation similar to the focal fibroblast foci seen in UIP. If this is progressive, honeycomb change occurs. Other features of acute lung injury, which are frequently seen in AIP, are intra­ alveolar hyaline membranes. Diagnosis The diagnosis of AIP is based on a clinical syndrome of idiopathic ARDS and the presence of organizing diffuse alveolar damage on lung biopsy. Lung biopsy is occasionally performed to establish the diagnosis and exclude other causes of acute ILD. Treatment and Outcome No effective therapy exists for patients with AIP. Glucocorticoids are utilized in most cases, but no survival benefit has been shown. Overall, the prognosis of patients with AIP is poor, with mortality rates in the range of 50–88%. Half the patients die within 6 months of disease onset. However, those who survive may have complete recovery of lung function, and AIP rarely recurs in survivors.

Desquamative Interstitial Pneumonitis DIP represents fewer than 3% of all cases of interstitial lung disease.29 However, it is a distinct clinicopathological entity that differs substantially from UIP. Clinical Manifestations DIP affects individuals in their fourth to fifth decades of life with a male predominance. It predominantly occurs in cigarette

CHAPTER 72  Immunological Lung Diseases

FIG 72.6  Radiographic Manifestations in Desquamative Interstitial Pneumonitis. High-resolution computed tomography scan in a patient with desquamative interstitial pneumonitis shows ground-glass attenuation in the periphery of the upper and lower lung fields.

smokers. Clinically, most individuals present with subacute onset of a dry, nonproductive cough and dyspnea. Clubbing is present in approximately 50% of patients with DIP. Laboratory evaluation is usually nonspecific. Although the chest radiography results can be normal in up to 20% of symptomatic individuals, it typically shows nonspecific bibasilar ground-glass opacities. Reticulonodular interstitial infiltrates have also been reported. HRCT confirms the presence of ground-glass attenuation in the periphery of the lower lung zones (Fig. 72.6). Pulmonary function testing shows a restrictive defect with hypoxemia and a decrease in diffusion capacity. Histopathology DIP is a misnomer. It was initially thought that the intraalveolar cells represented sloughed or desquamated alveolar epithelial cells. However, DIP is pathologically characterized by uniform, diffuse accumulation of macrophages in the alveolar space (Fig. 72.7). At low magnification, the overall appearance is one of uniformity from one field of view to the next as opposed to the variegated appearance of UIP. In addition, there is scant interstitial inflammation with varying degrees of fibrosis of the alveolar septum. Diagnosis The diagnosis of DIP requires tissue confirmation of the pathological lesion. This is important since DIP has a better prognosis and response to therapeutic intervention compared with IPF. A DIP-like pattern is frequently seen in other IIPs as well as in pulmonary Langerhans cell histiocytosis, CTDs, and drug reactions. Thus the diagnosis of DIP requires careful correlation of pathological findings with clinical and radiological findings. Treatment and Outcome The primary intervention in DIP is smoking cessation. Since this is a rare condition with relatively few published cases, it is unclear whether glucocorticoids alter the natural history of this

973

FIG 72.7  Histopathology of Desquamative Interstitial Pneumonitis. A high-magnification photomicrograph of desquamative interstitial pneumonitis shows the uniform, diffuse accumulation of macrophages within the alveolar space with associated thickening of the alveolar septum. These aggregates of macrophages almost completely fill the alveolar spaces.

disease. A mortality rate of 28% with a mean survival of 12 years has been reported compared with a 30–50% 5-year survival in UIP.24 Of note, 22% patients improved spontaneously, and 60% responded to glucocorticoid therapy. This picture is dramatically different from that of IPF, in which spontaneous improvement rarely, if ever, occurs. However, a significant minority of patients with DIP fail to respond to treatment and progress to respiratory failure secondary to advanced fibrosis.

Respiratory Bronchiolitis–Associated Interstitial Lung Disease RB-ILD is a distinct clinical entity that occurs in current or former cigarette smokers. It is unclear whether RB-ILD and DIP represent different diseases or different ends of the spectrum of the same disease process.29 DIP occurs predominantly and RB-ILD occurs exclusively in cigarette smokers, suggesting a common pathogenesis related to cigarette smoke exposure. Clinical Manifestations The mean age at presentation with RB-ILD is 36 years. Males are more often affected, and all individuals with RB-ILD are cigarette smokers. Symptoms include a dry, nonproductive cough and dyspnea. Clubbing is absent in RB-ILD, whereas it is frequently present in DIP. Laboratory evaluation is nonspecific. Chest radiography typically shows diffuse, fine reticular or nodular interstitial opacities with normal lung volumes. Additional findings include bronchial wall thickening and a prominent peribronchovascular interstitium. HRCT may reveal ground-glass opacification and emphysema. Pulmonary function tests most commonly reveal a mixed restrictive–obstructive pattern with a reduced diffusing capacity and mild hypoxemia. The residual volume may be increased, with no change in other spirometric parameters. Histopathology The pathology of RB-ILD is similar to that of DIP. However, in RB-ILD, the intraalveolar macrophages accumulate primarily

974

Part Seven  Organ-Specific Inflammatory Disease

FIG 72.8  Histopathology of Respiratory Bronchiolitis–Interstitial Lung Disease. An ectatic bronchiole with a thickened wall is shown, with a mononuclear infiltrate extending into the immediately surrounding alveoli.

within the peribronchiolar airspaces and are associated with thickening of the alveolar septum in these areas (Fig. 72.8). The differentiation of this lesion from DIP requires sparing of distal airspaces with the lesion confined to the peribronchiolar airspaces in RB-ILD. Diagnosis RB-ILD should be suspected in young individuals who have a history of cigarette smoking and complain of cough and dyspnea with chest radiography or HRCT showing nodular and/or reticular interstitial opacities. The diagnosis requires tissue confirmation of the pathological findings noted above. Treatment and Outcome The key therapeutic intervention in RB-ILD is cessation of smoking. The use of glucocorticoids has been associated with favorable results. At present, the clinical course and prognosis of patients with RB-ILD are unknown. In most clinical series, patients either improved or stabilized, and mortality is uncommon.29,30

Nonspecific Interstitial Pneumonitis The term NSIP was first used to describe cases of interstitial pneumonia that did not demonstrate a pattern of UIP, AIP, or DIP. Currently, the term NSIP is applied to an IIP or to a similar histological pattern that occurs in CTD, hypersensitivity pneumonitis, infection, or drug-induced lung disease. Thus the diagnosis of NSIP should prompt investigation for a causative agent.18 In fact, 16% of patients in the original description of NSIP had one of the CTDs.31 Clinical Manifestations Idiopathic NSIP occurs in middle-aged individuals, with a slight female predominance. A dry, nonproductive cough and exertional dyspnea are the most common symptoms, although fever is present in 25% of patients. Symptoms are usually present for 6–10 months before diagnosis. As in other IIPs, the laboratory evaluation is nonspecific. Chest radiography usually shows bilateral interstitial infiltrates, and sometimes the result can be normal in a symptomatic patient.

FIG 72.9  Histopathology of Nonspecific Interstitial Pneumonitis. Low-magnification photomicrograph of cellular nonspecific interstitial pneumonitis shows diffuse uniform thickening of the alveolar septum as a result of the presence of a lymphoplasmacytic infiltrate.

HRCT characteristically shows bilateral, patchy ground-glass attenuation indistinguishable from DIP or RB-ILD.23 Histopathology NSIP is characterized by varying, but temporally uniform, degrees of fibrosis and inflammation of the alveolar septum, without the histopathological features indicative of UIP, AIP, or DIP (Fig. 72.9). NSIP has been divided into three groups, depending on the presence or absence of interstitial fibrosis: interstitial lympho­ plasmacytic inflammation (48% of cases); inflammation and fibrosis (38%); and fibrosis (14%). Although the changes are temporally uniform, they may be patchy with intervening areas of normal lung. This temporal uniformity is in contrast to the variegated pattern seen in UIP. Fibroblast foci, the earliest lesions seen in UIP, are found in 20% of patients with NSIP, making it difficult to differente fibrotic NSIP from UIP. The key feature in this circumstance is the temporal uniformity of the lesions in NSIP. Treatment and Outcome Unlike patients with UIP, individuals with NSIP have a favorable prognosis. In the original description of the disease, 45% of subjects completely recovered, and the condition of another 42% remained stable or improved.31 Only 11% of patients died, with a mean survival of 16 months. All of the individuals with an aggressive course were in the fibrotic group. Ten-year survival in the cellular group was 90%, compared with 35% in patients with the fibrotic pattern. Despite the worse prognosis of NSIP with a fibrosing pattern, this is still significantly better than the 15% 10-year survival rate for patients with UIP.32

Cryptogenic Organizing Pneumonia COP is a specific clinicopathologic disorder of unknown etiology characterized by excessive proliferation of granulation tissue within the lumen of distal airspaces.33 The term COP is reserved for cases demonstrating bronchiolitis obliterans organizing pneumonia (BOOP) without an obvious cause, since this histological appearance occurs in a variety of inflammatory lung

CHAPTER 72  Immunological Lung Diseases disorders, including CTDs, malignancy, infections, and those caused by medications. Clinical Manifestations The onset of disease is usually in the fifth to sixth decades of life; men and women are equally affected. Most individuals have symptoms for less than 2 months before diagnosis. The initial presentation is usually with a dry, nonproductive cough and flu-like symptoms, including fever, sore throat, and malaise. This is followed by progressive dyspnea, and routine laboratory evaluation is nonspecific. Chest radiography shows diffuse, often patchy alveolar opacities in the setting of normal lung volumes (Fig. 72.10A). These

975

opacities can be migratory and usually have a peripheral distribution similar to those seen in chronic eosinophilic pneumonia. Rarer radiographic manifestations include linear or nodular interstitial opacities and honeycombing. The presence of a pleural effusion or pleural thickening should suggest an associated CTD. HRCT shows patchy airspace consolidation, especially in the lung periphery with a lower-lung zone predominance (see Fig. 72.10B). Other findings include ground-glass attenuation, small nodular opacities, and bronchial wall thickening. As in other ILDs, a restrictive ventilatory defect is the most common pulmonary function abnormality. Gas exchange abnormalities are common and are accompanied by decreased diffusing capacity, widening of the alveolar–arterial gradient, and exercise-induced hypoxemia. Histopathology The histopathology of COP is characterized by excessive proliferation of granulation tissue in the small airways and alveolar ducts with associated chronic inflammation in the alveolar walls (Fig. 72.11).33 The intraluminal fibrotic buds (Masson bodies) consist of loose collagen-embedding fibroblasts and myofibroblasts and have a tendency to extend from one alveolus to the next, giving a characteristic “butterfly” pattern. The lesions are patchy in nature and have a uniform temporal appearance at low magnification, with preservation of the underlying lung parenchyma. This pattern has been described as the prototypical healing response of the lung to a variety of insults.

A

Diagnosis The presence of BOOP in a lung biopsy does not necessarily represent COP, since COP is a diagnosis of exclusion. Organizing pneumonia is a nonspecific response to many lung injuries and can occur in conjunction with another pathological process or as a component of other primary pulmonary disorders, such as infections, irradiation, CTD, hypersensitivity pneumonitis, granulomatosis with polyangiitis, or chronic eosinophilic pneumonia (Table 72.3).

B

FIG 72.10  Radiographic Findings in Cryptogenic Organizing Pneumonia. (A) Chest radiograph in a patient with cryptogenic organizing pneumonia shows bilateral patchy alveolar opacities with a peripheral distribution in the setting of normal lung volumes. (B) Chest computed tomography shows a dense right lower lung consolidation with the presence of air bronchograms.

FIG 72.11  Histopathology of Cryptogenic Organizing Pneumonia. A photomicrograph of cryptogenic organizing pneumonia shows intraalveolar fibroblast proliferation (arrows) and early collagen production. In addition, thickening of the alveolar septa with a lymphoplasmacytic infiltrate consistent with cellular nonspecific interstitial pneumonitis is present.

976

Part Seven  Organ-Specific Inflammatory Disease

TABLE 72.3  Disorders Associated With a

Bronchiolitis Obliterans Organizing Pneumonia (BOOP) Pattern

Idiopathic BOOP (cryptogenic organizing pneumonia) Connective tissue diseases • Systemic lupus erythematosus • Rheumatoid arthritis • Polymyositis/dermatomyositis • Sjögren syndrome Hypersensitivity pneumonitis Chronic eosinophilic pneumonia Drug-induced • Gold • Penicillamine • Amiodarone • Bleomycin • Sulfa drugs Granulomatosis with polyangiitis (wegener granulomatosis) Bone marrow transplantation Lung transplantation/rejection Inhalational injury Neoplasms Lung irradiation Virus-associated • Human immunodeficiency virus (HIV) • Influenza virus • Adenovirus

Treatment and Outcome Treatment with glucocorticoids usually offers dramatic clinical and radiographic improvement within days to weeks.33 Complete clinical, physiological, and radiographic recovery occurs in twothirds of cases. In the remainder, persistent disease progresses to fibrosis. It is common for relapses to occur with glucocorticoid tapering, followed by improvement with reintroduction of treatment; consequently at least 6 months of therapy is recommended. The 5-year survival rate in COP is 73%, compared with 5-year survival rates of 44% in patients with BOOP resulting from other causes (e.g., CTD) or 30% for IPF.

LUNG INVOLVEMENT IN CONNECTIVE TISSUE DISEASES CTDs are a heterogeneous group of systemic autoimmune diseases that frequently involve the lungs. The pleuropulmonary manifestations of these diseases are diverse, affecting all parts of the respiratory tract (i.e., airways, alveoli, blood vessels, and pleura) (Table 72.4). Although pulmonary complications generally occur in patients with well-established disease, occasionally the lung involvement is the first manifestation of the autoimmune disorder. This section discusses the pleuropulmonary manifestations of systemic lupus erythematosus (SLE), RA, and systemic sclerosis (SSc) (for a discussion of other manifestations in these diseases, see Chapters 51, 52, and 55).

Systemic Lupus Erythematosus SLE is a disease of unknown etiology characterized by the presence of autoantibodies directed against various nuclear antigens. These autoantibodies and the resultant immune complexes mediate many of the manifestations of SLE (Chapter 51). This disease

TABLE 72.4 Pleuropulmonary

Manifestations of Connective Tissue Diseases Pulmonary hypertension Vasculitis Pleural disease Bronchiolitis obliterans Aspiration pneumonia Diaphragmatic dysfunction Lung nodules Diffuse alveolar damage BOOP UIP Capillaritis LIP NSIP

SLE

RA

SSc

+ + +++ ± − ++ − + ± + ++ + +

+ ± +++ ++ − − ++ ± + ++ + + ++

+++ ± + + ++ − − ± ± + ± + ++

BOOP, bronchiolitis obliterans organizing pneumonia; LIP, lymphocytic interstitial pneumonia; NSIP, nonspecific interstitial pneumonia; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SSc, systemic sclerosis; UIP, usual interstitial pneumonitis.

mainly affects young women (female-to-male ratio >8 : 1) and may involve virtually every organ system. Pleuropulmonary involvement occurs at some point in the disease course in 38–89% of cases.34 Thus the respiratory system is affected more commonly in SLE than in any other CTD. However, infectious pneumonia remains the commonest cause of pulmonary disease and death in these patients. Thus in patients with SLE presenting with a febrile illness and pulmonary infiltrates, a community-acquired or opportunistic infection must be promptly excluded. Acute Lupus Pneumonitis Acute lupus pneumonitis is an uncommon pulmonary manifestation of SLE, occurring in fewer than 5% of cases.34 The clinical presentation mimics that of an infectious pneumonia with the abrupt onset of fever, cough, and dyspnea. Serum complement levels are often low, and chest radiography typically shows diffuse alveolar opacities. Acute lupus pneumonitis can be accompanied by pericarditis and often pleuritis and pleural effusion. It can be difficult to distinguish acute lupus pneumonitis from an infectious pneumonia. BAL followed by thoracoscopic lung biopsy is recommended before instituting corticosteroid therapy. The histopathology varies and includes diffuse alveolar damage, BOOP, NSIP, or a combination of these. There are no controlled trials of therapy for acute lupus pneumonitis. Treatment includes high-dose glucocorticoids (1–2 mg/kg/day) with or without accompanying cytotoxic drugs, such as cyclophosphamide. Mortality rates as high as 50% have been reported. In those patients who fail to respond to treatment, respiratory failure is the usual cause of death. Diffuse Alveolar Hemorrhage DAH occurs in fewer than 5% of patients with SLE, and it represents the initial manifestation of disease in 11–20% of those cases.34 However, most cases develop in individuals with wellestablished diagnoses of SLE, usually with preexisting lupus nephritis. The symptoms of DAH mimic those of infectious pneumonia and acute lupus pneumonitis.34 Hemoptysis is present in 42–66%

CHAPTER 72  Immunological Lung Diseases of patients at presentation. Therefore the absence of hemoptysis does not exclude the diagnosis, particularly in the setting of a falling hematocrit, diffuse pulmonary infiltrates, and blood-stained BAL fluid. DAH in SLE most often results from pulmonary capillaritis, but it can also be caused by diffuse alveolar damage. Immunofluorescence studies show granular deposits of immunoglobulin G (IgG) and C3 along alveolar walls, interstitium, and capillary endothelial cells. There are no controlled trials for the treatment of alveolar hemorrhage in SLE. Glucocorticoids, cytotoxic drugs, and plasmapheresis have been used in various combinations. The mortality rate associated with DAH is approximately 50%. Poor prognostic factors include the need for mechanical ventilation, presence of infection, and prior treatment with cyclophosphamide. Lupus Pleuritis The pleura are the most common site of respiratory involvement in SLE, with pleurisy and pleural effusions occurring in 50–80% of patients. Lupus pleuritis can be the presenting manifestation of disease, but more commonly, it develops in patients with established SLE. It is often recurrent. The clinical manifestations include chest pain, fever, and dyspnea, and chest radiography typically shows bilateral pleural effusions. The pleural fluid is serous or serosanguineous and exudative in nature. Compared with effusions in RA, the glucose is higher, and the lactate dehydrogenase level is lower. The most helpful measurement is a pleural fluid ANA titer greater than 1 : 160. Examination of the pleura reveals infiltration with plasma cells and lymphocytes, accompanied by pleural thickening and fibrosis. Treatment with nonsteroidal antiinflammatory drugs (NSAIDs) and/or glucocorticoids is usually effective for relief of pleural discomfort. Interstitial Lung Disease The presence of ILD in SLE is uncommon, especially compared with SSc or RA. However, minor interstitial abnormalities can be found on HRCT in approximately one-third of patients with SLE who have normal results of chest radiography and physiological testing. The significance and natural history of these subclinical findings are uncertain. The presence of anti-SSA (Ro) has been noted in approximately 80% of patients who have lupus with interstitial changes. In addition, the prevalence of ILD is increased in a subset of patients with SLE who have sclerodermatous skin changes. The diagnosis of SLE is usually well established in patients who develop the insidious form of ILD. The disease course is characterized by progressive dyspnea and cough; chest radiography shows reduced lung volumes and reticular interstitial infiltrates. A restrictive lung function pattern with reduced diffusing capacity and exercise-induced hypoxemia are typical. The histopathology of chronic interstitial disease in SLE resembles NSIP, although cases of BOOP, LIP, and UIP have been described. Response to therapy depends on the underlying histopathology, with the UIP-like form being least responsive. Pulmonary Vascular Disease Although previously thought to be unusual, the development of pulmonary hypertension has been increasingly noted in SLE, with an incidence ranging from 0.5–14%. Pulmonary hypertension in SLE has been associated with the presence of Raynaud syndrome, serositis, digital vasculitis, and antiphospholipid antibodies.35 Dyspnea and fatigue, despite normal results on chest radiography,

977

is the most common presentation. Most of the patients with SLE who have pulmonary hypertension are female, with 3- and 5-year survival rates of 45% and 17%, respectively, representing a worse prognosis compared with that for patients with idiopathic pulmonary hypertension. The vascular changes of SLE-associated pulmonary hypertension are similar to those seen in idiopathic pulmonary hypertension with intimal hyperplasia, smooth muscle hypertrophy, and medial thickening. Several pathological mechanisms have been proposed for the development of pulmonary hypertension, including vasoconstriction, in addition to vasculitis and thrombosis associated with antiphospholipid and anticardiolipin antibodies. Serum endothelin levels are elevated in patients with SLE-associated pulmonary hypertension and correlate with pulmonary arterial pressures. As the pulmonary hypertension advances, the central pulmonary arteries enlarge. Pulmonary function testing shows an isolated decrease in the diffusing capacity for carbon monoxide. Patients with SLE-associated pulmonary hypertension may respond to immunosuppressive therapy. In a small study, five of 12 patients with SLE responded to monthly intravenous bolus doses of cyclophosphamide in addition to systemic glucocorticoids. A positive response was indicated by sustained hemodynamic improvement after at least 1 year of treatment without the need for additional pulmonary hypertension–specific therapies. Patients who responded to immunosuppression could be maintained on azathioprine or mycophenolate mofetil to avoid potential adverse effects of cyclophosphamide. Patients with SLE who were treated with bosentan did not have clinical worsening and showed an improvement in 6-minute walk distance.36 Overall, the long-term survival of patients with SLE-associated pulmonary hypertension is poor,37 and the optimal treatment regimen for SLE-associated pulmonary hypertension remains unknown. Respiratory Muscle Dysfunction The shrinking lung syndrome is caused by diaphragmatic weakness as well as weakness of other respiratory muscles. This entity accounts for the findings of dyspnea without evidence of interstitial infiltrates or pulmonary vascular disease. It occurs in 25% of patients with SLE. Chest radiography typically shows elevated diaphragms and basilar atelectasis. The pathogenesis of respiratory muscle weakness is unknown, but it is not associated with generalized muscle weakness. Glucocorticoids are frequently ineffective in the treatment of this syndrome. Improvement has been noted with inhaled β-agonist and theophylline therapy. Despite a variable response to therapy, it is unusual for this manifestation of SLE to be progressive.

Rheumatoid Arthritis RA is an autoimmune disease associated with autoantibodies directed against citrullinated antigens and characterized by the presence of a symmetrical, inflammatory polyarthritis (Chapter 52). It occurs more frequently in women, with a female-to-male ratio of 2 : 1. Disease onset is most commonly in the fourth to fifth decades of life. The pleuropulmonary complications of RA occur more commonly in individuals with subcutaneous nodules, high titers of rheumatoid factor, and more severe chronic articular involvement. Although RA itself is more common in women, the pleuropulmonary manifestations have a higher incidence in men. The pleuropulmonary complications of RA are numerous, but the treatment-related lung toxicity and pulmonary infections

978

Part Seven  Organ-Specific Inflammatory Disease

are difficult to differentiate from the primary pleuropulmonary manifestations of the disease.

occupational dust exposures have also been associated with this syndrome.

Pleuritis and Pleural Effusions As in SLE, pleural abnormalities are one of the most common pulmonary complications of RA. Pleural effusion is clinically evident in approximately 5% of patients, and this can occur before the development of arthritis. Pleural disease is often discovered as an incidental finding on routine chest radiography, but nonspecific chest pain, dyspnea, and fever are not unusual. The effusion can be unilateral or bilateral and can coexist with ILD. Typically, the effusion is an exudate, with a glucose level less than 30 mg/mL in 70–80% of cases. The mechanism underlying the low pleural fluid glucose is impaired membrane transport of glucose. A low pleural fluid pH is thought to occur secondary to impaired carbon dioxide exit from the pleural space. If the effusion is chronic, the cholesterol concentration can be increased, and the pleural fluid can have a milky appearance (pseudochylothorax). Cytological examination reveals multinucleated giant cells, spindle-shaped macrophages, and necrotic debris. Most rheumatoid effusions are small and asymptomatic, thus requiring no treatment. They resolve over several months without complications. The use of glucocorticoids for active articular disease hastens the resolution of the pleural process.

Airway Disease Airflow limitation is a common finding in patients with RA, being present in approximately one-third of patients. The mechanism(s) responsible for airway disease is poorly understood. The interplay of cigarette smoking and RA may play a role. A life-threatening complication of RA is upper airway obstruction, resulting from synovitis of the cricoarytenoid joint. Common presenting complaints include a sore throat, hoarseness, and fullness in the throat. It can progress to inspiratory stridor and upper airway obstruction. This complication occurs more commonly in women, particularly in those with advanced RA. Seventy-five percent of patients were found to have cricoarytenoid abnormalities when screening with direct or indirect laryngoscopy and computed tomography (CT) was utilized. The treatment of cricoarytenoid arthritis includes antiinflammatory medications. Bronchiolitis obliterans is a progressive form of obstructive lung disease that is being increasingly recognized as a complication of RA.38 This entity was thought to develop secondary to the use of penicillamine in the treatment of RA, but most cases occur in the absence of this therapy. The histopathological lesion of bronchiolitis obliterans is constrictive bronchiolitis, which is characterized by concentric submucosal and peribronchiolar fibrosis resulting in extrinsic compression and obliteration of the bronchiolar lumen. The typical clinical presentation is insidious onset of cough and dyspnea, with a normal or hyperinflated chest on radiography. This complication occurs more commonly in women than in men. Pulmonary function studies show airflow limitation with hyperinflation and a reduced diffusing capacity. Expiratory HRCT shows multiple areas of air trapping (mosaic pattern). Some individuals respond to high-dose glucocorticoids and cytotoxic drugs, but in most patients, bronchiolitis obliterans progresses to respiratory failure. Bronchiectasis occurs at an increased frequency in RA, usually in individuals with long-standing articular disease. Productive cough and dyspnea are the most common respiratory symptoms. In most patients, bronchiectasis is not clinically significant. Recurrent pneumonia and respiratory failure are potentially fatal complications of this problem.

CLINICAL PEARLS Lung Involvement in Rheumatoid Arthritis (RA) • RA is more common in women, but pleuropulmonary complications occur more frequently in men. • Factors associated with pleuropulmonary complications of RA include more severe articular involvement, subcutaneous nodules, and high levels of rheumatoid factor. • Pleural effusions are the most common complication, characterized by an exudate and a low glucose and low pH. • The differentiation of rheumatoid nodules from malignant lesions can be difficult. • The rapid growth of a nodule should prompt aggressive investigation for a malignant cause.

Rheumatoid Nodules Rheumatoid or necrobiotic nodules are the only pleuropulmonary manifestation specific for RA. These nodules are most commonly seen in men with active articular disease, high rheumatoid factor titers, and subcutaneous nodules. Most individuals are asymptomatic and are diagnosed on routine chest radiography. Radiographically, these nodules can be singular or multiple with an upper to midlung zone predominance. Cavitation occurs in approximately 50% of cases. HRCT indicates a higher frequency of nodules than previously thought. Rarely, subpleural necrobiotic nodules can erode into the pleural space, resulting in a pneumothorax with a complicating bronchopleural fistula. It can be difficult to differentiate these nodules from malignant lesions, and open-lung biopsy is frequently necessary. Evidence of rapid growth on chest radiography should prompt an aggressive diagnostic evaluation. Caplan syndrome refers to the rapid development of pulmonary nodules predominantly in the upper lung zone. This syndrome was originally described in Welsh coalminers with RA. Histologically, these nodules are identical to necrobiotic nodules. Other

Interstitial Lung Disease Although ILD is a common complication of RA, the incidence is difficult to determine, since different methods of detection have been employed and dissimilar populations of patients have been studied. However, clinically significant ILD occurs in approximately 14% of patients.39 The development of ILD in relation to the onset of arthritis is variable. Most often, the ILD develops subsequent to arthritis, but in approximately 20% of patients, the lung disease precedes the onset of arthritis and is associated with cigarette smoking, presence of the shared HLA-DR4 epitope, and RA-specific anticitrullinated protein antibodies. The most common histopathologies identified in this patient population are UIP, LIP, NSIP and BOOP. The clinical manifestations of ILD in RA resemble those seen in idiopathic disease and include a dry, nonproductive cough and dyspnea on exertion. Chest radiography and HRCT show increased reticular markings with a predilection for the peripheral lower lung zones. Often, pleural abnormalities accompany the interstitial changes. With advanced disease, progression to honeycomb lung occurs. LIP usually occurs in cases of RA complicated by Sjögren syndrome;

CHAPTER 72  Immunological Lung Diseases the presence of keratoconjunctivitis sicca and xerostomia in a patient with RA and ILD should suggest this histological type. In general, ILD in RA appears more indolent than in the IIPs. Thus because of uncertain treatment benefits and possible adverse effects, the decision to institute therapy should be based on clinical, radiographic, and physiological deterioration. Drug-Induced Lung Disease Methotrexate and gold are the two main anti-RA drugs capable of causing lung injury. Methotrexate administered weekly (10–20 mg/week) is associated with the development of interstitial changes in 1–5% of patients with RA. No correlation with age, sex, disease duration, or cumulative dose has been identified. The clinical presentation is subacute, with fever, cough, and dyspnea occurring 1–5 months after initiation of the drug. Chest radiography shows mixed interstitial–alveolar infiltrates. Nonspecific laboratory abnormalities include leukocytosis, sometimes with mild eosinophilia, and an elevated erythrocyte sedimentation rate (ESR). In most cases, BAL reveals a lymphocytosis. Histologically, cellular NSIP is seen with areas of organizing pneumonia. Noncaseating, granulomatous inflammation similar to that seen in hypersensitivity pneumonitis may also be present. The primary treatment of methotrexate-induced pneumonitis is withdrawal of methotrexate, as well as appropriate supportive care. Gold-induced pneumonitis occurs in fewer than 1% of patients with RA who are treated with gold. Dyspnea and cough usually begin after 4–6 weeks of therapy; eosinophilia occurs in a minority of patients. Chest radiography typically reveals mixed alveolar– interstitial opacities with a predilection for the upper lung zone. The histology is similar to that seen in patients with RA-associated ILD. Thus the differentiation of gold-induced pneumonitis from RA-associated ILD can only be established when discontinuation of the medication results in remission.

Systemic Sclerosis (Scleroderma) SSc is characterized by excessive deposition of ECM in the skin and internal organs, and vascular involvement (Chapter 55). The degree of visceral organ involvement determines morbidity and mortality. Pulmonary involvement occurs in 70–100% of patients with SSc. There is no correlation with the degree of extrapulmonary disease. ILD is the most common pulmonary manifestation of SSc. Of note, with the improved mortality associated with renal involvement in SSc, lung disease has become the most important cause of morbidity and mortality. Interstitial Lung Disease The incidence of ILD in SSc depends on the method of detection. Autopsy studies have reported an ILD incidence of 60–100% of cases, whereas studies based on chest radiography have noted interstitial changes in 14–66% of cases. Cough and dyspnea on exertion are the most common symptoms. Physical examination reveals bibasilar rales. Radiographic findings include basal reticulonodular infiltrates, enlargement of pulmonary arteries, and progressive volume loss. Pulmonary function testing reveals restrictive lung disease, preservation of flow rates, and decreased diffusing capacity. A disproportionate decrease in diffusing capacity compared with lung volume changes should suggest pulmonary hypertension, especially in individuals with limited scleroderma (calcinosis, Raynaud phenomenon [RP], esophageal dysmotility, sclerodactyly, telangiectasia [CREST] syndrome). The predominant histopathologic abnormality is NSIP. Rarely, LIP may complicate cases of SSc associated with Sjögren syndrome.

979

Although the 5-year survival for patients with SSc and ILD is 38–45%, it is better than that of patients with IPF. Pulmonary Vascular Disease Pulmonary hypertension is a frequent complication of SSc, occurring in approximately 30% of patients with diffuse scleroderma and in 10–50% of those with limited scleroderma (Chapter 55). It is a major cause of morbidity and mortality in systemic sclerosis and has also become part of the diagnostic criteria for the disease.40 Pulmonary hypertension can either be associated with interstitial fibrosis or result from involvement of small and medium-sized arteries and arterioles with smooth-muscle hyperplasia, medial hypertrophy, and intimal proliferation (plexogenic). Direct involvement of the pulmonary circulation is more common with limited scleroderma, whereas pulmonary hypertension in patients with diffuse scleroderma is more likely associated with ILD. The clinical presentation is characterized by the insidious onset of fatigue and dyspnea on exertion. Physical examination and chest radiography show signs typical of pulmonary hypertension, whereas a decreased diffusing capacity is seen on pulmonary function testing. Risk factors for developing SSc-associated pulmonary hypertension include limited skin involvement, duration of disease greater than 10 years, onset of SSc at older age, and severity and duration of RP. The pathogenesis of SSc-associated pulmonary hypertension is poorly understood. Vascular changes occur at an early stage in SSc and include apoptosis, endothelial cell activation with increased expression of cell adhesion molecules, inflammatory cell recruitment, intimal proliferation, and adventitial fibrosis leading to vessel obliteration. Endothelial injury is reflected by increased levels of soluble cell adhesion molecules, disturbances of angiogenesis with increased levels of circulating vascular endothelial growth factor (VEGF), and the presence of angiostatic factors. To what extent dysregulated angiogenesis in SSc-associated pulmonary hypertension is driven by an inflammatory process or other as yet unidentified mechanisms remains unclear. Treatment of SSc-associated pulmonary hypertension has been disappointing, with no therapy showing a significant survival benefit. Calcium channel blockers are not usually indicated for patients with SSc-associated pulmonary hypertension, although often used at lower doses for RP. Continuous intravenous epoprostenol improves exercise capacity and hemodynamics.41 Randomized clinical trials with phosphodiesterase inhibitors, including sildenafil, showed a modest effect on exercise capacity, hemodynamic parameters, and functional class after 12 weeks of treatment. Carefully selected patients may be considered for heart–lung transplantation but are often excluded because of the risk of postoperative complications arising from SSc-related gastroesophageal reflux disease (GERD) and renal dysfunction.

CONCLUSIONS ILDs comprise a diverse group of disorders ranging from idiopathic etiologies to those related to an underlying autoimmune condition. There is likely a complex interplay between the innate and adaptive arms of the immune system and the profibrotic pathways that lead to the development of these various disease states. Future work should focus on better understanding this relationship and, more importantly, translating these findings to the clinical setting. The role of standard immunosuppressive approaches with the ongoing discovery of novel approaches to

980

Part Seven  Organ-Specific Inflammatory Disease

ON THE HORIZON • Our understanding of the idiopathic interstitial pneumonias (IIPs) and autoimmune-related interstitial lung diseases (ILDs) has evolved over time. With increased emphasis being placed on diagnosis, because of the implications for treatment and prognosis, we are more accurately phenotyping the diseases. This has led to a better understanding of genetic associations, biological pathways, as well as more accurate identification of risk factors for disease development and progression. • However, there are still many areas in need of further understanding and research. In the future, determining whether risk factor identification and modification can lead to primary and/or secondary prevention strategies in the treatment of immunological lung diseases will be essential. • Testing if the novel antifibrotic therapies approved for idiopathic pulmonary fibrosis (IPF) are more broadly applicable to other forms of ILDs, including autoimmune-related ILD, will be important. • Other therapies that target the immune system also need to be more carefully studied in diseases outside of IPF.

targeting the immune system as well as novel antifibrotic therapies will need to be determined. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Luzina IG, Todd NW, Iacono AT, et al. Roles of T lymphocytes in pulmonary fibrosis. J Leukoc Biol 2008;83:237–44. 2. Ginhoux F, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 2010;330:841–5. 3. Ginhoux F, Jung S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat Rev Immunol 2014;14:392–404. 4. Hashimoto D, et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 2013;38:792–804. 5. Yona S, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 2013;38:79–91. 6. Schulz C, et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 2012;336:86–90. 7. Parra ER, Kairalla RA, Ribeiro de Carvalho CR, et al. Inflammatory cell phenotyping of the pulmonary interstitium in idiopathic interstitial pneumonia. Respiration 2007;74:159–69. 8. Wells AU, et al. Fibrosing alveolitis in systemic sclerosis: increase in memory T-cells in lung interstitium. Eur Respir J 1995;8:266–71. 9. Simonian PL, et al. Gammadelta T cells protect against lung fibrosis via IL-22. J Exp Med 2010;207:2239–53. 10. Wynn TA. Integrating mechanisms of pulmonary fibrosis. J Exp Med 2011;208:1339–50. 11. Sonnenberg GF, et al. Pathological versus protective functions of IL-22 in airway inflammation are regulated by IL-17A. J Exp Med 2010;207:1293–305. 12. Wilson MS, et al. Bleomycin and IL-1beta-mediated pulmonary fibrosis is IL-17A dependent. J Exp Med 2010;207:535–52. 13. Kotsianidis I, et al. Global impairment of CD4+CD25+FOXP3+ regulatory T cells in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2009;179:1121–30. 14. Travis WD, et al. An official American Thoracic Society/European Respiratory Society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2013;188:733–48. 15. Fischer A, et al. An official European Respiratory Society/American Thoracic Society research statement: interstitial pneumonia with autoimmune features. Eur Respir J 2015;46:976–87.

16. Ahluwalia N, Shea BS, Tager AM. New therapeutic targets in idiopathic pulmonary fibrosis. Aiming to rein in runaway wound-healing responses. Am J Respir Crit Care Med 2014;190:867–78. 17. Coultas DB, Zumwalt RE, Black WC, et al. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med 1994;150:967–72. 18. American Thoracic S, European Respiratory S. American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am J Respir Crit Care Med 2002;165:277–304. 19. Seibold MA, et al. A common MUC5B promoter polymorphism and pulmonary fibrosis. N Engl J Med 2011;364:1503–12. 20. Feghali-Bostwick CA, et al. Cellular and humoral autoreactivity in idiopathic pulmonary fibrosis. J Immunol 2007;179:2592–9. 21. Dreisin RB, Schwarz MI, Theofilopoulos AN, et al. Circulating immune complexes in the idiopathic interstitial pneumonias. N Engl J Med 1978;298:353–7. 22. Lee CG, et al. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 2001;194:809–21. 23. Lynch DA, et al. High-resolution computed tomography in idiopathic pulmonary fibrosis: diagnosis and prognosis. Am J Respir Crit Care Med 2005;172:488–93. 24. Ley B, Collard HR, King TE Jr. Clinical course and prediction of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2011;183:431–40. 25. King TE Jr, et al. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med 2014;370:2083–92. 26. Richeldi L, et al. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med 2014;370:2071–82. 27. Hyzy R, Huang S, Myers J, et al. Acute exacerbation of idiopathic pulmonary fibrosis. Chest 2007;132:1652–8. 28. Katzenstein AL, Myers JL, Mazur MT. Acute interstitial pneumonia. A clinicopathologic, ultrastructural, and cell kinetic study. Am J Surg Pathol 1986;10:256–67. 29. Ryu JH, et al. Desquamative interstitial pneumonia and respiratory bronchiolitis-associated interstitial lung disease. Chest 2005;127:178–84. 30. Portnoy J, et al. Respiratory bronchiolitis-interstitial lung disease: long-term outcome. Chest 2007;131:664–71. 31. Katzenstein AL, Fiorelli RF. Nonspecific interstitial pneumonia/fibrosis. Histologic features and clinical significance. Am J Surg Pathol 1994;18:136–47. 32. Travis WD, Matsui K, Moss J, et al. Idiopathic nonspecific interstitial pneumonia: prognostic significance of cellular and fibrosing patterns: survival comparison with usual interstitial pneumonia and desquamative interstitial pneumonia. Am J Surg Pathol 2000;24:19–33. 33. Cordier JF. Cryptogenic organising pneumonia. Eur Respir J 2006;28:422–46. 34. Kamen DL, Strange C. Pulmonary manifestations of systemic lupus erythematosus. Clin Chest Med 2010;31:479–88. 35. Hassoun PM. Pulmonary arterial hypertension complicating connective tissue diseases. Semin Respir Crit Care Med 2009;30:429–39. 36. Mok MY, et al. Bosentan use in systemic lupus erythematosus patients with pulmonary arterial hypertension. Lupus 2007;16:279–85. 37. Qian J, et al. Survival and prognostic factors of systemic lupus erythematosus-associated pulmonary arterial hypertension: a PRISMA-compliant systematic review and meta-analysis. Autoimmun Rev 2016;15:250–7. 38. Schwarz MI, Lynch DA, Tuder R. Bronchiolitis obliterans: the lone manifestation of rheumatoid arthritis? Eur Respir J 1994;7:817–20. 39. Gabbay E, et al. Interstitial lung disease in recent onset rheumatoid arthritis. Am J Respir Crit Care Med 1997;156:528–35. 40. van den Hoogen F, et al. 2013 classification criteria for systemic sclerosis: an American college of rheumatology/European league against rheumatism collaborative initiative. Ann Rheum Dis 2013;72:1747–55. 41. Highland KB. Recent advances in scleroderma-associated pulmonary hypertension. Curr Opin Rheumatol 2014;26:637–45.

CHAPTER 72  Immunological Lung Diseases

980.e1

MULTIPLE-CHOICE QUESTIONS 1. The most common pulmonary manifestations of systemic lupus erythematosus (SLE) is: A. Interstitial lung disease (ILD) B. Diffuse alveolar hemorrhage (DAH) C. Pleural effusion D. Pulmonary hypertension 2. Idiopathic pulmonary fibrosis (IPF) is characterized by: A. The pathological lesion of usual interstitial pneumonitis B. A relentless progression and a poor prognosis despite therapeutic intervention C. The presence of interstitial opacities in a basilar and peripheral distribution D. All of the above

3. Which of the following statement regarding systemic sclerosis (SSc) is correct? A. Renal involvement and renal crisis remain the predominant cause of mortality in SSc. B. Pulmonary hypertension is a newly added diagnostic criteria for SSc. C. The most common lung pathological lesion in SSc is lymphocytic interstitial pneumonitis (LIP). D. Calcium channel blockers are indicated for patients with SSc-associated pulmonary hypertension.

73  Sarcoidosis Edward S. Chen, David R. Moller

Sarcoidosis is a systemic inflammatory disorder characterized by the presence of noncaseating granulomas and lymphocyte inflammation.1,2 The disease most frequently involves lungs and thoracic lymph nodes, although any organ system can be involved (Table 73.1). The manifestations and clinical course of sarcoidosis vary greatly.3 An estimated 30–60% of patients with sarcoidosis are asymptomatic, usually with isolated bilateral hilar adenopathy, and clearly some of these patients remain unidentified. Symptomatic manifestations most frequently involve the respiratory system, with symptoms of cough, dyspnea, and chest discomfort. Löfgren syndrome is a distinct presentation of acute sarcoidosis, with polyarthritis, bilateral hilar adenopathy, erythema nodosum, and often uveitis. This form of sarcoidosis usually resolves completely. Other clinical manifestations depend on the range and extent of extrapulmonary involvement. More than one-third of patients will experience chronic active disease, which is associated with considerable risk of long-term morbidity resulting from progressive organ dysfunction and complications of immunosuppressive treatment. Given the lack of validated biomarkers of disease activity, the management of sarcoidosis involves serial assessment of symptoms, radiographic imaging, and objective measurement of organ function.

EPIDEMIOLOGY Sarcoidosis is found worldwide, although there are striking differences in the prevalence of the disease in different geographical areas and racial groups.4 In Europe and North America, the estimated prevalence ranges from 10 to 64 cases per 100 000, with higher rates in African-American populations. Although it can occur at any age, over 80% of cases are diagnosed between 20 and 50 years of age, with a further peak after age 50 years.4,5 Globally, there is a slight female preponderance. Sarcoidosis may present differently in men and women,6 and it presents differently in older people, complicating case identification.7 Recent studies have supported prior observations of worse outcomes in African American women compared with other demographic groups in the United States.8,9 The frequency of clinical manifestations and disease severity varies among different groups. Löfgren syndrome has a particularly high frequency in the Scandinavian countries and in Ireland but occurs in 50%

of Japanese patients with sarcoidosis, but in only 10–25% of European and North American patients. Retrospective studies suggest that sarcoidosis is the direct cause of death in 1–6% of cases, usually from pulmonary, cardiac, or neurological involvement.10

GENETICS There is substantial evidence for a genetic predisposition to sarcoidosis, including higher concordance among monozygotic twins than among dizygotic twins and familial clustering in 5–16% of patients. A US-based multicenter study compared 706 newly diagnosed cases of biopsy-proven sarcoidosis to age-, sex-, and race-matched relatives and found an approximate fivefold increased relative risk in siblings and parents of individuals with sarcoidosis.11 Major histocompatibility complex (MHC) class II alleles are major contributors to disease susceptibility across different ethnic populations.12 Human leukocyte antigen–D related 3 (HLA-DR3) is associated with increased sarcoidosis risk in Scandinavian and European populations, whereas HLA-DR1 and HLA-DR4 alleles are associated with disease protection. A significant association was found with HLA-DRB1*11:01 in African Americans and Caucasians, whereas HLA-DRB1*15:01 was a risk factor only in Caucasians. Residues forming pocket 4 of HLA-DR and pocket 9 of HLA-DQ seem to influence sarcoidosis risk, which suggests specific antigenic peptides are involved in causing sarcoidosis. HLA class I alleles (B*07, A*03) also confer risk for sarcoidosis, independently from HLA class II alleles. Together, these HLA associations with sarcoidosis may reflect the effects of an 8.1 ancestral haplotype (A1, B8, DR3, DQ2) that is linked to several other immune-related diseases; larger studies will be necessary to determine the independent risks associated with each allele. HLA class I and II alleles are associated with clinical course. HLA DRB1*03:01 and DQB1*02:01 are associated with favorable outcomes, whereas DR14 and DR15 are associated with severe, chronic disease in European and Japanese populations, as are the closely linked alleles DRB1*1501:01 and DQB1*06:02. Studies of non-HLA polymorphisms have yielded few clues to the genetic basis of sarcoidosis. A meta-analysis found polymorphism of the gene for tumor necrosis factor-α (TNF-α) associated with a 1.5-fold increased risk of developing sarcoidosis.13 Associations were reported between sarcoidosis and the CC chemokine receptors CCR2 and CCR5 and the receptor for

981

982

Part seven  Organ-Specific Inflammatory Disease

TABLE 73.1  Major Clinical Features of

analysis suggested that T-helper 1 (Th1) and Th17 signaling pathways are involved in sarcoid immunopathogenesis.19

Organ System (Approx. % Involvement)

ENVIRONMENTAL FACTORS

Systemic Sarcoidosis

Pulmonary (90)

Upper airway (5–10)

Ocular (25)

Skin (20–30)

Hepatic (10) Cardiac (10–15) Central nervous system (5–10)

Salivary and parotid gland (