The Pulmonary Neuroepithelial Body Microenvironment: A Multifunctional Unit in the Airway Epithelium 3030658163, 9783030658168

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Advances in Anatomy, Embryology and Cell Biology

Inge Brouns · Line Verckist  Isabel Pintelon  Jean-Pierre Timmermans  Dirk Adriaensen

The Pulmonary Neuroepithelial Body Microenvironment A Multifunctional Unit in the Airway Epithelium

Advances in Anatomy, Embryology and Cell Biology publishes critical reviews and state-ofthe-art research in the areas of anatomy, developmental and cellular biology. Founded in 1891, this book series has a long standing tradition of publishing focused and condensed information on a given topic with a special emphasis on biomedical and translational aspects. The series is open to both contributed volumes (each collecting 7 to 15 focused reviews written by leading experts) and single-authored or multi-authored monographs (providing a comprehensive overview of their topic of research). Advances in Anatomy, Embryology and Cell Biology is indexed in BIOSIS, Journal Citation Reports/Science Edition, Medline, SCImago, SCOPUS, Science Citation Index Expanded (SciSearch), Zoological Record. Manuscripts should be addressed to Editor-in-Chief Prof. Dr. PETER SUTOVSKY, Division of Animal Sciences and Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO, USA e-mail: [email protected] Series Editors Prof. Dr. FRANCISCO CLASCÁ, Department of Anatomy, Histology and Neurobiology, Universidad Autónoma de Madrid, Madrid, Spain e-mail: [email protected] Prof. Dr. Z. KMIEC, Department of Histology and Immunology, Medical University of Gdansk, Gdansk, Poland e-mail: [email protected] Prof. Dr. HORST-WERNER KORF, Anatomy and Brain Research Center, Department for Anatomy 1, Heinrich Heine University Düsseldorf, Düsseldorf, Germany e-mail: [email protected] Prof. Dr. MICHAEL J. SCHMEISSER, Institute of Microscopic Anatomy and Neurobiology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany e-mail: [email protected] Prof. Dr. BALJIT SINGH, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada e-mail: [email protected] Prof. Dr. JEAN-PIERRE TIMMERMANS, Laboratory of Cell Biology and Histology/Core Facility Biomedical Microscopic Imaging, Department of Veterinary Sciences, University of Antwerp, Wilrijk, Belgium e-mail: [email protected] Prof. Dr. SVEN SCHUMANN, Inst, f. Mikroskop. Anatomie u. Neurobio, Johannes Gutenberg University of Mainz, Mainz, Rheinland-Pfalz, Germany e-mail: [email protected]

233 Advances in Anatomy, Embryology and Cell Biology

Editor-in-Chief P. Sutovsky

Series Editors F. Clascá • Z. Kmiec • H.-W. Korf • M.J. Schmeisser • B. Singh • J.-P. Timmermans • S. Schumann More information about this series at http://www.springer.com/series/102

Inge Brouns • Line Verckist • Isabel Pintelon • Jean-Pierre Timmermans • Dirk Adriaensen

The Pulmonary Neuroepithelial Body Microenvironment A Multifunctional Unit in the Airway Epithelium

Inge Brouns Laboratory of Cell Biology and Histology, Department of Veterinary Sciences University of Antwerp Antwerpen (Wilrijk), Belgium

Line Verckist Laboratory of Cell Biology and Histology, Department of Veterinary Sciences University of Antwerp Antwerpen (Wilrijk), Belgium

Isabel Pintelon Laboratory of Cell Biology and Histology, Department of Veterinary Sciences University of Antwerp Antwerpen (Wilrijk), Belgium

Jean-Pierre Timmermans Laboratory of Cell Biology and Histology, Department of Veterinary Sciences University of Antwerp Antwerpen (Wilrijk), Belgium

Dirk Adriaensen Laboratory of Cell Biology and Histology, Department of Veterinary Sciences University of Antwerp Antwerpen (Wilrijk), Belgium

ISSN 0301-5556 ISSN 2192-7065 (electronic) Advances in Anatomy, Embryology and Cell Biology ISBN 978-3-030-65816-8 ISBN 978-3-030-65817-5 (eBook) https://doi.org/10.1007/978-3-030-65817-5 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

We would like to dedicate this issue to Geoffrey Burnstock (May 10th, 1929–June 3rd, 2020), whose contribution to the scientific world does not even fit in an entire book. His pioneering work on purinergic signalling and his discovery more than 50 years ago that ATP can act as a neurotransmitter have paved the way for an entirely new scientific domain which even today is still an exciting area of research. We had the privilege of working and publishing with him in our quest to unravel the purinergic signalling pathways in intrapulmonary receptors. We are most thankful to have had the opportunity to known him personally for many years, not only as a top scientist and great personality, but also as a warm, charming family man.

Acknowledgements

Special thanks to past and present colleagues of the research group studying airway sensory receptors, who have been involved in part of the reported research: Dr. I. De Proost, Dr. J. Van Genechten, Dr. R. Lembrechts, and Dr. K. Schnorbusch. The skilful technical assistance of S. Thys, E. Theuns, and K. Sterck is highly appreciated. Thanks to D. De Rijck for help with imaging and illustrations, D. Vindevogel for aid with the manuscript, and S. Kockelberg for administrative help.

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Abstract

Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance. Complexly organised pulmonary NEBs are an integral part of the intrapulmonary airway epithelium of all air-breathing vertebrates. For decades, a quest has been going on to unravel the functional significance of these intriguing structures that appear to be modified in the course of many pulmonary diseases. The pulmonary NEB microenvironment (ME) is composed of organoid clusters of pulmonary neuroendocrine cells (PNECs) that are able to store and release neurotransmitters and are closely contacted by extensive (mainly afferent) nerve terminals, emphasising a potential receptor/effector role and probable signalling to the central nervous system. PNECs are largely shielded from the airway lumen by a special type of Clara cells, the Clara-like cells, with potential stem cell characteristics. Since pulmonary NEBs are widely dispersed in the airway epithelium, and represent less than one percent of the cells in the epithelial lining, investigating the NEB ME largely depends on its unequivocal microscopic identification. Nowadays, multidisciplinary approaches allow to combine functional morphological investigations in cryosections and cleared whole lungs, live cell imaging in lung vibratome slices, and selective gene expression analysis after laser microdissection of genetically tagged NEBs. So far, functional studies of the pulmonary NEB ME revealed that PNECs can be activated by various mechanical and chemical stimuli, resulting in a calciummediated release of neurotransmitters. A number of publications in the past decades have exposed NEBs as potential hypoxia sensors. In the past few years, combination of in vivo experiments and gene expression analysis unveiled the pulmonary NEB ME as a quiescent stem cell niche in healthy postnatal mouse lungs. The stem cell population was shown to be activated by

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transient mild inflammation, and silencing appears to involve bone morphogenetic protein signalling that may be mediated by vagal afferents. Today, it is clear that only an integrated approach that takes all current information into account will be able to explain the full role of the pulmonary NEB ME in health and disease.

Contents

Pulmonary Sensory Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Sensory Nerves in the Airways . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Intrapulmonary Myelinated Vagal Sensory Airway Receptors . . . . 1.3 Why Focussing on the Pulmonary Neuroepithelial Body (NEB) Microenvironment (ME)? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .

1 1 3

.

5

2

The Pulmonary NEB ME Is a Complex Intraepithelial Unit . . . . . . . 2.1 Components of the NEB ME . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Pulmonary Neuroendocrine Cells (PNECs) . . . . . . . . . . . . 2.1.2 Clara-Like Cells (CLCs) . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3 Innervation of the NEB ME . . . . . . . . . . . . . . . . . . . . . . . 2.2 Receptor–Effector Properties of the Pulmonary NEB ME . . . . . . .

. 7 . 7 . 7 . 9 . 12 . 13

3

Studying the Pulmonary NEB ME: A Multidisciplinary Approach . . . . 3.1 Functional Morphological Characteristics of the NEB ME . . . . . . . . 3.2 Genetically Engineered Mouse Models to Study the NEB ME . . . . . 3.3 3D-Imaging and -Analysis of NEBs in Airway Whole Mount and in Cleared Whole Lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Models and Techniques for Functional Live Cell Imaging (LCI) of the NEB ME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Laser Microdissection and Selective Gene Expression Analysis of the NEB ME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19 19 20

Functional Exploration of the Pulmonary NEB ME . . . . . . . . . . . . . 4.1 Mechanosensing in the NEB ME . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Chemosensing in the NEB ME . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Oxygen Sensing in the NEB ME . . . . . . . . . . . . . . . . . . . 4.2.2 Activation of the NEB ME by Cigarette Smoke and Nicotine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Sensing Extracellular Ca2+ in the NEB ME . . . . . . . . . . . . 4.2.4 Hypercapnia and H+ Sensing . . . . . . . . . . . . . . . . . . . . . .

31 31 37 38

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. . . .

21 23 26

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Contents

4.3

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The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 The NEB ME During Lung Development and After Airway Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Modification of the NEB ME Related to Perinatal and Postnatal Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3 Selective Gene Expression in the Postnatal NEB ME: Special Focus on Stem Cell Characteristics . . . . . . . . . . . . 4.3.4 Selective Activation and Proliferation of a Quiescent Stem Cell Population in the NEB ME by Transient Acute Lung Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.5 Silencing Stem Cells in the Healthy Pulmonary NEB ME: Involvement of BMP Signalling . . . . . . . . . . . . . . . . . . . .

. 45 . 45 . 50 . 52

. 55 . 61

Concluding Remarks and Future Perspectives . . . . . . . . . . . . . . . . . . 69

Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

About the Authors

Dr. Inge Brouns obtained her Ph.D. in 2002 under the supervision of Profs. Adriaensen and Timmermans exploring the neurochemical coding and complex innervation pattern of pulmonary neuroepithelial bodies. She published >40 full papers mainly focusing on pulmonary sensory receptors and was the first author of a previous edition published in 2012. She is currently working as a coordinator for educational affairs within the Laboratory of Cell Biology and Histology. Dr. Line Verckist obtained her Ph.D. in 2018 under the supervision of Dr. Brouns and Prof. Adriaensen. She focused her research on the microenvironment of pulmonary neuroepithelial bodies being a potential stem cell niche in the airway epithelium. Dr. Isabel Pintelon obtained her Ph.D. in 2007 under the supervision of Prof. Adriaensen unravelling the neurochemical characteristics of sensory pulmonary and pleura receptors and in situ functional imaging in ex vivo lung models. She is currently a research manager and a core facility manager within the Laboratory of Cell Biology and Histology. Prof. Jean-Pierre Timmermans obtained his Ph.D. in zoological sciences in 1987 and completed his habilitation in 1994. Since 2003, he is a full professor at the Department of Veterinary Sciences of UAntwerp, where he is chair of the Laboratory of Cell Biology and Histology and director of the Antwerp Centre for Advanced Microscopy (ACAM). His main research interests relate to the autonomic nervous system, with specific attention to neuroimmune interactions in the gastrointestinal and respiratory tracts. His recent research activities are focused on the brain–gut– microbiome axis. He is (associated) editor of several international journals. He is a foreign member of the Deutsche Akademie der Naturforscher Leopoldina. He is a former president of the Royal Belgian Society for Microscopy, the International Society of Autonomic Neuroscience, and the Anatomische Gesellschaft. He is a fellow of the American Association of Anatomists and an honorary fellow of the xiii

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About the Authors

Anatomische Gesellschaft. He is (co-)author of over 330 peer-reviewed articles having received more than 9,700 citations (WoS) so far. Prof. Dirk Adriaensen obtained his Ph.D. in biological sciences in 1994 with an extensive study on the comparative functional histology of the pulmonary neuroepithelial endocrine system. In 2001, he obtained a research professorship, is currently a full professor at the Laboratory of Cell Biology and Histology, and leads a research team that focusses on experimental lung research and the development of models for functional molecular microscopic imaging of the pulmonary neuroepithelial body (NEB) microenvironment (ME). His main interest has been to unravel the complex interactions between neuroendocrine cells, a multitude of central nerve terminals, and a supposed population of stem cells within the NEB ME, and its potential involvement in repair after injury and in the pathogenesis of small cell lung carcinoma. He is currently President of the Royal Belgian Society for Microscopy. He is (co-)author of over 130 peer-reviewed papers that have been cited more than 3500 times.

List of Abbreviations

α7 nAChR αSMA 4-Di-2-ASP 5-HT [Ca2+]i [Ca2+]o [K+]o ACh ALI Ascl1 ATP A.U. BAL BALF BB bHLH Bl6 BMP BMPR BOM BrdU CA CALC CAE CAEctrl CAELPS CaSR CaV CB CC

α7 nicotinic acetylcholine receptor α-smooth muscle actin 4-(4-dimethylaminostyryl)-N-methylpryidinium iodide 5-Hydroxytryptamine, serotonin intracellular free ionised Ca2+ concentration extracellular free ionised Ca2+ concentration extracellular K+ concentration acetylcholine acute lung injury achaete–scute family bHLH transcription factor 1 adenosine 5’-triphosphate arbitrary units broncho-alveolar lavage broncho-alveolar lavage fluid bombesin receptors basic helix-loop-helix Black-6 bone morphogenetic protein BMP receptor bombesin bromo deoxyuridine carbonic anhydrase calcitonin control airway epithelium CAE in healthy control animals CAE in LPS-treated animals Ca2+-sensing receptor voltage-gated Ca2+ channels calbindin-D28k Clara cell xv

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CCK CDH CLC CCSP CGRP CNS CreERT CRLR Ctrl CYP2F2 DAB DCVs DEG/ENaC DIPNECH Dll DNES DRG ED eEF-2 ELISA Flt-1 GABA GAD GAD67-GFP GD GFP GPCRs GRP Hash1 Hes1 HRP ILC2 ir IR K2P Kv KI LCI LMD loxP LPS Mash1 MBP mOsmol

List of Abbreviations

cholecystokinin congenital diaphragmatic hernia Clara-like cell Clara cell secretory protein calcitonin gene-related peptide central nervous system tamoxifen-inducible Cre system calcitonin receptor-like receptor postnatal healthy control cytochrome P450, family 2, subfamily f, polypeptide 2 3,3’-diaminobenzidine dense-cored vesicles degenerin/epithelial sodium channel Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia Delta-like ligand diffuse neuroendocrine system dorsal root ganglion embryonic day(s) eukaryotic elongation factor-2 enzyme-linked immuno sorbent assay fms-like tyrosine kinase γ-aminobutyric acid glutamic acid decarboxylase expression of GFP in GAD67-expressing cells gestational day(s) green fluorescent protein G protein coupled receptors gastrin-releasing peptide human achaete–scute complex homologue-1 hairy and enhancer of Split-1 horse-radish peroxidase type 2 innate lymphoid cells immunoreactive (adjective) immunoreactivity (noun) ‘two-pore domain’ potassium channels voltage-gated potassium channels knock-in live cell imaging laser microdissection locus of X-over P1 lipopolysaccharide mammalian achaete–scute complex homologue-1 myelin basic protein milli-osmoles/kg H2O

List of Abbreviations

Na+/K+-ATPase α3 NADPH oxidase NCAM NEstem NEB ME NEB MEctrl NEB MELPS NEBs NEHI NICD NNK nNOS NPY NRQ NSE PACAP pCO2 PD PGP9.5 PNECs pO2 qRT-PCR; qPCR Ramp1 RARs Rb1 RIN ROI ROS RT-PCR SARs SCLC SCGB Shh SMARs SSEA-1 SP SP1 SPNC SV2 SYN TASK TEM

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sodium/potassium-adenosine 5’-triphosphatase α3 heme-linked nicotinamide adenine dinucleotide phosphate oxidase neural cell adhesion molecules minor population of PNECs in NEBs with reserve stem cell activity neuroepithelial body microenvironment NEB ME in healthy control animals NEB ME in LPS-treated animals neuroepithelial bodies neuroendocrine cell hyperplasia of infancy Notch intracellular domain 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone neuronal nitric oxide synthase neuropeptide Y normalised relative quantity neuron-specific enolase Pituitary adenylate cyclase-activating polypeptide partial CO2 pressure postnatal day(s) protein gene product 9.5 pulmonary neuroendocrine cells partial O2 pressure quantitative (real-time) RT-PCR receptor activity-modifying protein 1 rapidly adapting (stretch) receptors Retinoblastoma1 RNA integrity number region of interest reactive oxygen species reverse transcription polymerase chain reaction slowly adapting (stretch) receptors small cell lung carcinoma secretoglobin sonic hedgehog smooth muscle-associated airway receptors stage-specific embryonic antigen-1 substance P specificity protein 1 SSEA-1-positive, peri-NEB, Notch-active, CC10-negative synaptic vesicle protein 2 synaptophysin TWIK-related acid-sensitive K2P channel transmission electron microscopy

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TH TRAAK TREK TrkB TRP TRP53 TRPC5 TRPV1 TRPV4 U-CCs UCHL1 Upk3A VAChT v-CC v-CE vClub VGAT VGLUT VIP vSC WT

List of Abbreviations

tyrosine hydroxylase TWIK-related arachidonic-acid-stimulated K+ channel TWIK-related K+ channel tyrosine receptor kinase B transient receptor potential transformation-related protein 53 transient receptor potential canonical-5 transient receptor potential vanilloid-1 transient receptor potential vanilloid-4 Upk3A-expressing cells ubiquitin carboxyl-terminal hydrolase L1 uroplakin3A vesicular acetylcholine transporter variant Clara cells variant CCSP-expressing cells variant Club cells vesicular GABA transporter vesicular glutamate transporter vasoactive intestinal polypeptide variant secretory cells wild-type

Chapter 1

Pulmonary Sensory Receptors

1.1

Sensory Nerves in the Airways

Airways and lungs comprise a large-surfaced area that is in extensive contact with environmental air, ensuring the principal physiological function of the lung in any situation, i.e. the uptake of oxygen and elimination of carbon dioxide. In order to detect potential physical and chemical changes, the pulmonary system is equipped with an extensive panel of sensory nerve terminals, enabling transduction of information of the local environment to the central nervous system (CNS). The diverse populations of ‘airway sensory nerves’ provide input to the respiratory centres, which may lead to changes in breathing pattern or may trigger defensive responses. Activation of sensory nerve endings in the airways elicits reflexes that are responsible for two primary functions essential to the overall respiratory function. One is the regulation of breathing and bronchomotor tone, including the maintenance of homeostasis. The other concerns the defence reflexes that protect the lung and the rest of the body from potential health hazards caused by inhaled irritants, toxic substances, cold or heat, and many other environmental factors (for review, see Lee and Yu 2014; Mazzone and Undem 2016). Respiratory reflexes controlled by the input from pulmonary and airway afferents are known to cause a wide range of physiological effects, such as the Hering–Breuer reflex, cough, airway mucus secretion, vaso- and bronchodilation (for review, see Kubin et al. 2006; Mazzone and Undem 2016). Dysfunction of the communication between airway afferents and the brain, or inappropriate activation of airway receptors, may contribute to symptoms of pulmonary disease, including dyspnoea, bronchospasm, excessive cough, and mucus secretion (Coleridge and Coleridge 1994, 1997; Sant’Ambrogio and Sant’Ambrogio 1997; Lee and Pisarri 2001; Canning 2006; Widdicombe 2006, 2009; Adriaensen and Timmermans 2011; Mazzone and Undem 2016). Afferent activity arising from sensory terminals located in the airways and lungs is believed to be mainly conducted via fibres travelling in the vagal nerve, with cell © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 I. Brouns et al., The Pulmonary Neuroepithelial Body Microenvironment, Advances in Anatomy, Embryology and Cell Biology 233, https://doi.org/10.1007/978-3-030-65817-5_1

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1 Pulmonary Sensory Receptors

bodies in nodose and jugular ganglia. These vagal sensory neurons innervate the entire respiratory tract and project to the nucleus of the solitary tract in the medulla (Agostoni et al. 1957; Jammes et al. 1982). Some sensory terminals in the airways originate from spinal afferents, with cell bodies in the thoracic dorsal root ganglia, but are believed to be subsidiary to the vagal pathways (see recent review by Undem and Sun 2020). Spinal afferents have been investigated less extensively, since most studies found that the majority of reflexes evoked by the activation of airway sensory nerves are abolished by section or conduction block of the vagal nerve (Widdicombe 1986; Kummer et al. 1992; Carr and Undem 2003; Oh et al. 2006). Depolarisation of afferent nerve terminals induces discharges that are directed towards the CNS. Therefore, the activity of airway sensory nerves has mainly been examined by extracellular recordings of action potentials travelling the cervical vagal nerve from the lungs towards the CNS. Based on their sensitivity to physical and chemical stimuli, adaptation to mechanical stimulation, myelinisation, conduction velocity, activity during tidal breathing, reflexes associated with their activity, ganglionic origin, and presumable sites of termination in the airways and the lungs, these so-called single-fibre recordings have resulted in the electrophysiological characterisation and classification of various subtypes of airway-related vagal afferent nerves (for reviews, see Carr and Undem 2003; Mazzone 2005). From a physiological point of view, bronchopulmonary afferent nerve fibres fall broadly into two types: those that are the mainly mechanosensitive slowly and rapidly adapting receptors (SARs and RARs) (Sant’Ambrogio and Widdicombe 2001; Schelegle and Green 2001; Schelegle 2003; Widdicombe 2003; Lee and Yu 2014), corresponding with myelinated fast-conducting A-fibres, and those that are relatively insensitive to mechanical stimuli, namely chemosensitive/nociceptive unmyelinated C-fibre receptors (Lee and Pisarri 2001; Carr and Undem 2003; Lee et al. 2003; Lee and Undem 2005; Undem and Sun 2020). Given that some airway receptors have been classified as ‘subtypes’ or not even fit in the (simplified) electrophysiological classification (Lee and Yu 2014), recent reviews additionally define subsets of airway vagal afferents by their embryonic origin, molecular profile, neurochemistry, functionality, or anatomical organisation (Mazzone and Undem 2016). In view of the current review, special attention should be given to the anatomical and morphological classification of ‘airway receptors’ (recent review see Mazzone and Undem 2016). Intrapulmonary sensory nerve terminals can be found widely distributed throughout all levels of the airway tree. Generally, the mucosal lining is supplied with a dense plexus of ‘varicose’ non-myelinated C-fibre-like nerve terminals, which is typically in intimate association with the epithelium. Although present in the entire airway tree, the density of the fibres is highest in the large conducting extrapulmonary and intrapulmonary airways. The majority of these fibres can be recognised at the light microscopy level by their varicose (beaded) appearance and their relatively simple terminal structure/profile. These nerve fibres may express one or more neuropeptides [notably substance P (SP), calcitonin gene-related peptide (CGRP), and/or vasoactive intestinal polypeptide (VIP)] and often co-express the capsaicin receptor, transient receptor potential

1.2 Intrapulmonary Myelinated Vagal Sensory Airway Receptors

3

vanilloid 1 (TRPV1) (Dey et al. 1990; Lee et al. 1995; Lamb and Sparrow 2002; Dinh et al. 2003; Watanabe et al. 2005; Undem and Sun 2020). Although most of these terminals have a vagal jugular origin, species differences exist and some populations may also derive from dorsal root ganglia. Myelinated nerve fibres show a rather restricted distribution in the airways and an often complex terminal anatomy (see Sect. 1.2).

1.2

Intrapulmonary Myelinated Vagal Sensory Airway Receptors

The myelinated vagal sensory nerve fibres described in most detail are those innervating the laryngeal mucosa, tracheobronchial airways, and/or lung parenchyma. Typically, the myelin sheath of these fibres is lost immediately prior to ramifying into an often complex terminal structure, the nerve terminals of which can be found beneath or within the airway epithelium, or in the lung parenchyma. So far, there have been no reports of myelinated sensory fibres innervating intrinsic airway autonomic ganglia (for review, see Mazzone and Undem 2016). While the main axon and the first-order branches can be easily detected by their immunoreactivity for myelin markers or large molecular weight neurofilaments, it is hard to discriminate these fibres based on their neuropeptide content or by their surface receptors (for reviews, see Brouns et al. 2000; Brouns et al. 2012; Mazzone and Undem 2016). Terminal structures, on the other hand, can be visualised by immunohistochemistry, anterograde axonal dye filling, staining with fluorescent styryl pyridinium dyes (for review, see Brouns et al. 2012; Mazzone and Undem 2016), viral transfection (Hennel et al. 2018) or using genetic tools (Chang et al. 2015), combined with chemical or mechanical denervation and confocal microscopy. From a functional (mainly mechanosensitive) point of view, antibody markers for potential intrapulmonary mechanosensory nerve terminals have been used in a wide range of studies exploring the location, morphology, and neurochemical coding of both subepithelial (Yu et al. 2003; Yu and Zhang 2004; Adriaensen et al. 2006; Brouns et al. 2006a, 2012) and intraepithelial (Brouns et al. 2000; Adriaensen et al. 2003; Brouns et al. 2003, 2004; Adriaensen et al. 2006; Brouns et al. 2006a, 2009b, 2012) sensory receptor-like structures in rat and mouse airways. From these studies, it became clear that a single large-diameter myelinated axon gives rise to a number of ramifying terminal branches with terminal boutons. Nerve terminals seem to share a nearly identical neurochemical coding and a typical ‘laminar’ appearance. In rodents, subepithelial laminar terminals originating from myelinated vagal afferents have been shown to be consistently associated with the airway smooth muscle layer (Yu 2002, 2005, 2009), and were named ‘smooth muscle-associated airway receptors’ (SMARs, Fig. 1.1A) (Brouns et al. 2006a, b; De Proost et al.

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1 Pulmonary Sensory Receptors

Fig. 1.1 (A) A myelinated nerve fibre (open arrowhead), characterised by its myelin basic protein (MBP)-immunoreactive (ir) myelin sheath (blue fluorescence) gives rise to a smooth muscleassociated airway receptor (SMAR) upon termination of its myelin sheath. The Na+/K+-ATPase α3-ir (red fluorescence) nerve fibre (arrow) branches (open arrow) and forms laminar nerve endings (arrowheads) between the subepithelial alpha smooth muscle actin-ir (αSMA; green fluorescence) and smooth muscle cell bundles in the wall of a rat intrapulmonary airway. E: epithelium; L: airway lumen. (B) A myelinated P2X3-ir (green FITC fluorescence) nerve fibre (open arrowhead) approaches a pulmonary neuroepithelial body (NEB) in a rat bronchiole. The corpuscle is visualised by its calbindin D28k (CB)-ir (blue fluorescence) composing NEB cells. Upon termination of the MBP-ir (red fluorescence) myelin sheath, the approaching P2X3-ir nerve fibre gives rise to an intraepithelial arborisation (arrowheads). E: epithelium; L: airway lumen (Adapted with permission from Adriaensen and Timmermans 2004)

2007b; Lembrechts et al. 2011; Brouns et al. 2012) in order to point out the difference with the intraepithelial subgroup, the terminals of which are invariably associated with NEBs (see further) (Brouns et al. 2006a, b; Brouns et al. 2012). Staining of airway whole mounts (De Proost et al. 2007b) revealed that nerve bundles traverse the airways, give off smaller bundles and eventually single fibres that can be followed over long distances. These fibres give rise to terminals that are aligned parallel with the arrangement of the bundles of smooth muscle cells underlying the airway epithelium. Although general pan-neuronal markers, such as PGP9.5, NSE, SV2, or SYN, are able to stain SMARs, the inability of these markers to discriminate between motor and sensory terminals prompted researchers to use more selective ‘mechanosensory’ markers, such as the ‘two-pore domain’ potassium (K2P) channel TRAAK (Lembrechts et al. 2011) or Na+/K+-ATPase α3 (Yu et al. 2004; Brouns et al. 2006b). An overview of the neurochemical features of these structures can be found in our previous review (Brouns et al. 2012). A few years later, the existence of SMARs was confirmed in human airway biopsies (West et al. 2015). Myelinated vagal sensory nerve fibres that give rise to intraepithelial terminals invariably coincide with corpuscles termed pulmonary ‘neuroepithelial bodies’ (NEBs; Fig. 1.1B), an integral part of the normal epithelium at all levels of the intrapulmonary airways in man, mammals, and in all other air-breathing vertebrate groups studied so far (for review, see Hoyt et al. 1982; Scheuermann 1987; Sorokin and Hoyt 1990; Adriaensen and Scheuermann 1993; Linnoila 2006).

1.3 Why Focussing on the Pulmonary Neuroepithelial Body (NEB). . .

1.3

5

Why Focussing on the Pulmonary Neuroepithelial Body (NEB) Microenvironment (ME)?

The term ‘neuroepithelial bodies’ (NEBs) first appeared in the seventies (Lauweryns et al. 1972; Lauweryns and Peuskens 1972) to indicate the innervated clusters of ‘pulmonary neuroendocrine cells’ (PNECs) found in the airway epithelium of humans and other mammalian species. For many years the PNEC clusters (see Sect. 2.1.1) and their innervation (see Sect. 2.1.3) were considered as most important structures, and were hence the subject of many studies. General interest in the involvement of the Clara-like cells (CLCs), which typically cover the apical surface of NEBs (see Sect. 2.1.2), has grown over the years, and they are now regarded as essential components for at least some of the functions of this complex organoid. Close association between all the components prompted several investigators in the field to use the term ‘NEB microenvironment’ (NEB ME) to describe the complete shielded and highly innervated intraepithelial corpuscles (Reynolds et al. 2000a; Linnoila 2006; De Proost et al. 2008). This term will also be adopted throughout the current review, and ‘NEB’ will be used when only PNEC clusters are considered. We will provide insight into the different components of the NEB ME (Chap. 2) and highlight both their intracorpuscular relationship in healthy physiological conditions, and changes in view of pathological conditions (Chap. 4). Starting with an overview of the different techniques that are nowadays used (Chap. 3), and showing their application in different functional investigations, will finally help to clarify the intriguing picture of an intrapulmonary structure with multifunctional role(s). These functions may change over the life span of an organism and may be critically involved in normal physiology as well as in the pathobiology of many paediatric and adult lung diseases. The review mainly focuses on the NEB ME in rodents, and particularly in mice, which possess a set of airway sensory receptors and pulmonary reflexes that is similar to that typically reported for larger animals (Zhang et al. 2006). Studying these structures in mice has the additional advantage that it allows the use of transgenic and knock-out models. The evolutionary conservation of complexly innervated PNECs implies that data and interpretations obtained via mouse studies may be extrapolated to other mammals, including humans. Given that the NEB ME comprises different components that will turn out to be involved in various disease processes, it is likely that understanding the mechanisms intrinsic to the NEB ME may reveal novel concepts and potential therapeutic targets.

Chapter 2

The Pulmonary NEB ME Is a Complex Intraepithelial Unit

2.1 2.1.1

Components of the NEB ME Pulmonary Neuroendocrine Cells (PNECs)

The cellular components of the NEB ME that have been explored most extensively are definitely the pulmonary neuroendocrine cells (PNECs) (Fig. 2.1 A–C). For many years, PNECs have been considered as the most important players in the NEB ME. PNECs have always been hard to study due to their widespread and dispersed distribution, since they account for less than 1% of the cells that build up the airway epithelium (Boers et al. 1996; Gosney 1997). Being part of the diffuse neuroendocrine system (Feyrter 1938; Fröhlich 1949; Feyrter 1953) (for review, see Montuenga et al. 2003), PNECs can be easily recognised in the transmission electron microscope (TEM) by their dense-cored vesicles (DCVs) content (Fig. 2.1A) (for review, see Scheuermann 1987; Adriaensen and Scheuermann 1993; Scheuermann 1997). These vesicles typically consist of an electron-dense central part, surrounded by a limiting membrane that is separated from this central part by a clear space, hence their name (Stahlman and Gray 1984). Storage of different bioactive substances in these DCVs led researchers to assume that the mechanism of action for PNECs involves paracrine secretion of bioactive neuropeptides, amines, purines, and growth factors (Uddman et al. 1985; Dey and Hoffpauir 1986; Adriaensen et al. 2001; Fu et al. 2002; Adriaensen et al. 2003; Linnoila 2006). The amine, peptide, purine, and amino acid most frequently reported in DCVs of PNECs are serotonin (5-hydroxytryptamine; 5-HT) (Cutz et al. 1982; Lauweryns et al. 1982; Adriaensen et al. 1993; Pan et al. 2004), CGRP (Uddman et al. 1985; Cadieux et al. 1986; Lauweryns and Van Ranst 1987; Scheuermann et al. 1987; Keith et al. 1991; Luts et al. 1991), ATP (Brouns et al. 2000; Linnoila 2006; Burnstock 2009; De Proost et al. 2009), and γ-aminobutyric acid (GABA) © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 I. Brouns et al., The Pulmonary Neuroepithelial Body Microenvironment, Advances in Anatomy, Embryology and Cell Biology 233, https://doi.org/10.1007/978-3-030-65817-5_2

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L

STIMULUS

PNECs - CCSP L

E

C

A

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C

Fig. 2.1 (A) Conceptual scheme of the NEB ME. Pulmonary neuroendocrine cells (PNECs) (yellow) are crowned by Clara-like cells (CLCs) (dark grey), leaving only thin apical processes of PNECs in contact with the airway lumen (L ). When stimulated (red dashed arrows), PNECs may differentially discharge the contents of part of their DCVs, and these substances may exert local paracrine effects, act on nearby blood vessels (red arrows) and smooth muscle bundles (blue arrows), or synaptically interact with intracorpuscular sensory nerve endings (white arrowheads) (Adapted from Adriaensen and Scheuermann 1993; Adriaensen et al. 2003). (B) Confocal optical section showing a NEB corpuscle built up of PNECs (green GFP fluorescence) and overlying CLCs (blue fluorescence, visualising CCSP (¼SCGB1A1) immunoreactivity (IR)). CCSP IR is less pronounced in CLCs (arrowheads) than in Clara cells (open arrowheads), which are intermingled with unlabelled ciliated cells in the rest of the airway epithelium. L: airway lumen; E: epithelium. (C) Schematic summary of the innervation of a pulmonary NEB (in yellow), as found in mouse intrapulmonary airways. Known characteristics of the represented cells and nerve fibre populations are included in the same colour as the respective structures (adapted from Brouns et al. 2009a)

(Yabumoto et al. 2008; Schnorbusch et al. 2013; Barrios et al. 2017), respectively, although postnatal changes and interspecies differences do occur. Molecules involved in vesicle packaging, exocytosis, or other biological roles have also been identified, as such providing evidence that PNECs are capable of releasing neurotransmitters (for review, see Brouns et al. 2012; Cutz 2015). The presence of background potassium channels (Kv) and voltage-gated calcium channels (Cav) on the surface membrane of PNECs (Fu et al. 2000; De Proost et al.

2.1 Components of the NEB ME

9

2007a, 2009) (for review, see Brouns et al. 2012) demonstrates that PNECs exhibit characteristics of excitable cells. In vivo, activated PNECs (e.g. depolarisation with high potassium) show an intracellular calcium rise that initiates exocytosis of DCVs (De Proost et al. 2007a) and hence neurotransmitter release (De Proost et al. 2009; Lembrechts et al. 2012; Schnorbusch et al. 2013), which in turn may trigger other cells and mechanisms. The distribution of the DCVs, which are mainly situated at the basal PNEC pole, may point to secretion directed towards the basal lamina and hence to neighbouring cells, nerve endings, smooth muscle, or capillaries (for review, see Adriaensen and Scheuermann 1993; Adriaensen et al. 2003; Cutz et al. 2013; Cutz 2015). The so far explored signalling mechanisms in the NEB ME are outlined in Sect. 2.2. It is believed that PNEC secretory products are involved in organogenesis, local (immune) responses, and signalling to the CNS (Cutz et al. 2013; Cutz 2015). Table 2.1 gives a non-exhaustive overview of molecules identified in and on PNECs. Interspecies differences occur, but were neglected. More information on molecules that are specifically involved during development and in repair processes can be found in Sect. 4.3.

2.1.2

Clara-Like Cells (CLCs)

The second cellular component of the pulmonary NEB ME is the ‘modified’ Clara cells, henceforth referred to as Clara-like cells (CLCs). Multiple seminal studies using scanning and transmission electron microscopy that explored the ultrastructure of pulmonary NEBs in mice (Hung et al. 1973; Hung and Loosli 1974; Hung et al. 1979; Hung 1982; Haller 1994), rats (Cutz et al. 1974; Van Lommel and Lauweryns 1993; Haller 1994), hamster (Pearsall et al. 1985), and humans (Stahlman and Gray 1984) already reported that PNEC corpuscles are always largely covered by modified Clara cells. The latter cells exhibit a different morphology than the ciliated cells and Clara cells found in the ‘NEB boundary’, and leave only thin apical processes of PNECs directly exposed to the airway lumen (Fig. 2.1A and C). Although at the time PNECs were considered as the essential cells in the NEB ME, researchers felt that also these ‘modified’ Clara cells should be included as a constituent cell of the NEB corpuscle (Hung et al. 1979). Clara-like cells have also been reported in rabbit NEBs but, especially in prenatal and neonatal stages, appear to leave a larger surface area of PNECs in contact with the air space (Lauweryns et al. 1972; Cutz et al. 1978a, b). Meanwhile, many attempts have been undertaken to selectively visualise CLCs in light microscopic experiments. The resemblance to Clara cells prompted researchers to use Clara cell markers. Most often used was Clara-cell secretory protein (CCSP) (for review, see Linnoila 2006; Brouns et al. 2012), which was detected in Clara cells in rabbit lungs (Gupta et al. 1987). CCSP belongs to the group of cytokine-like secreted proteins of small molecular weight and is nowadays referred to as

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Table 2.1 Products and markers of mammalian PNECs in postnatal lungs Secretory products:

ϒ-aminobutyric acid (GABA) Acetylcholine (ACh) (indirect evidence) Adenosine triphosphate (ATP) Bombesin/gastrin releasing peptide (BOM/GRP) Calcitonin (CALC) Calcitonin-gene related peptide (CGRP) Cholecystokinin (CCK) Endothelin Enkephalin Helodermin Peptide YY Pituitary adenylate cyclase-activating polypeptide (PACAP) Serotonin (5-hydroxytryptamine, 5-HT) Somatostatin Substance P

Vesicle-associated proteins:

Chromograning A, B Secretogranin II Synaptic vesicle protein 2 (SV2) Synaptophysin (SYN) Vesicular ACh transporter (VAChT) Vesicular GABA transporter (VGAT)

Cytoplasmic proteins:

Acetylcholine esterase Aromatic amino acid decarboxylase Calbindin D28k (CB) Glutamic acid decarboxylase (GAD) Neu 7/NHK Neuron specific enolase Protein gene product 9.5 (PGP9.5) (=ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) Tryptophane hydroxylase

Receptors, channels and other surface membrane molecules: α7 nAChR Calcium-sensing receptor (CaSR) GABA receptors Go alpha NADPH oxidase (gp91 phox/NOX2, p22Phox, p47Phox, P67Phox, rac2) Neural cell adhesion molecules (NCAM) TRAAK (K2P) TRPC5

Modified from (Brouns et al. 2012) and (Cutz et al. 2013), supplemented with information of recent publications used throughout the current review

SCGB1A1, as a member of the secretoglobin gene superfamily (nomenclature committee report: Klug et al. 2000). It is the most widely studied member of the SCGB proteins, and is thought to be involved in modulation of inflammation, tissue repair, and tumour genesis. SGB1A1 is expressed in both Clara cells and CLCs, although CLCs show a lower SCGB1A1 expression (Fig. 2.1B). Recently, spatial

2.1 Components of the NEB ME

11

and/or temporal differences in the expression of SCGB1A1, SCGB3A1, and SCGB3A2 were demonstrated in Clara cells during embryonic development and in adult mice (Naizhen et al. 2019). Furthermore, SCGB3A2 was found to be enriched, but not exclusively present, in CLCs in the NEB ME during development (Guha et al. 2012; Naizhen et al. 2019). Global expression profile screening, combined with genetic tagging and in situ hybridisation was used to identify Uroplakin3A (Upk3A) expression in mouse CLCs during development and in postnatal lungs (Guha et al. 2012, 2014, 2017), but also in human lungs (Guha et al. 2017). The presence of Upk3A in NEB-associated cells led Guha and colleagues to designate them as ‘Upk3A-expressing cells’ or ‘U-CCs’ with a Clara-like cell distribution (Guha et al. 2012, 2014, 2017). Although genetically the presence of Upk3A in CLCs is beyond doubt, no Upk3A immunostaining could be demonstrated so far in lungs, hence still hampering labelling of CLCs in routine histological experiments. Morimoto and colleagues, in a study dealing with the developing NEB ME, found stage-specific embryonic antigen-1 (SSEA-1)/CD15-labelled cells that were selectively clustered around PNECs at ED18.5 (Morimoto et al. 2012). Based on their other features, this population was designated as ‘SSEA-1-positive, peri-NEB, Notch-active, CC10-negative (SPNC)’. However, in healthy postnatal lungs, SSEA-1 disappears from the SPNC cells. Due to their specific location, these SPNC cells were suggestive of being CLCs (Morimoto et al. 2012). A subgroup of Clara cells, referred to as ‘variant’ Clara cells (v-CC) (Reynolds et al. 2000a) or variant CCSP-expressing cells (v-CE) (Hong et al. 2001), could be identified in the NEB ME by lack of the cytochrome p450 enzyme CYP2F2, and consequently survival of Clara cell eradication by naphthalene (see Sect. 4.3.1.2; Peake et al. 2000; Hong et al. 2001; Bishop 2004; Rawlins et al. 2009; Reynolds and Malkinson 2010). Cell survival, while being the most commonly used discriminating factor, is based on negative selection of SCB1A1/CCSP-immunoreactive (ir) Clara cells, and hence not very useful in functional experimental settings. A consistent literature review on CLCs is hampered by the fact that a large number of experimental procedures have been used for identifying CLCs and, in concordance, a plethora of different terms have been applied to this cell type. Moreover, over the past decade, terminological confusion has even been aggravated as a result of the policy to avoid the name ‘Clara’ for ethical reasons (Winkelmann and Noack 2010). As such, the term ‘club-like cells’ is sometimes used instead of ‘Clara-like cells’, as are the terms ‘variant Club cells (vClub)’ (Kiyokawa and Morimoto 2020) and ‘variant secretory cells (vSC)’ (Leach and Morrisey 2018). For reasons of clarity, the term ‘Clara-like cells’ (CLCs), i.e. the original term describing this cell population (Cutz et al. 1978a), will be used throughout this review article. At the moment, in the absence of a conclusive and selective marker, CLC identification in postnatal mouse lungs remains based on the unique location of CLCs, forming a rim around the PNECs in NEBs (De Proost et al. 2008; Brouns et al. 2012). Having been considered as mainly supportive elements for many years, CLCs are now regarded as additional key players in the pulmonary NEB

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ME. Although CLCs are most commonly described as an airway epithelial stem cell niche (recent reviews see Leach and Morrisey 2018; Kiyokawa and Morimoto 2020), we feel that also other functional features of CLCs will turn out to be important in the NEB ME.

2.1.3

Innervation of the NEB ME

An important feature of pulmonary NEBs, albeit definitely species- and age-dependent (Sorokin et al. 1997), is the presence of an (extensive and complex) innervation, as demonstrated in detail in rodents (for review, see (Brouns et al. 2012); rats: (Brouns et al. 2000, 2004); mice: (Brouns et al. 2009a; Lembrechts et al. 2011; Chang et al. 2015)). During development, nerve fibres are observed in contact with NEBs from ED15.5 onwards in mice (Kuo and Krasnow 2015) and from ED16 in rats (Adriaensen et al. 2001). Whereas the (often combined) afferent (sensory) and efferent features of many nerve terminals that are associated with NEBs have been described extensively at the electron microscopic level in the past (Wasano 1977; Rogers and Haller 1978; Sorokin and Hoyt 1989; Van Lommel and Lauweryns 1993), combined approaches consisting of neuronal tracing, selective denervation, multiple immunostaining, and confocal microscopy have been used more recently to unravel the main characteristics of the innervation with regard to their potential function in the NEB ME (for review, see Brouns et al. 2012). To put the available functional data (Chap. 4) into perspective, a brief summary of the most important features of the NEB innervation in mice (see Brouns et al. 2012 for detailed review) is given below (see also Fig. 2.1C for a comprehensive summary). Pulmonary NEBs in mice, just like in most species studied so far, are selectively contacted by myelinated vagal afferents, which should be regarded as a fast connection to the CNS (Brouns et al. 2009a). Unilateral infranodosal vagotomy and immunostaining revealed two different populations of vagal sensory nerve terminals, both of which are myelinated and show nerve terminals that branch between the PNECs in NEBs upon termination of the myelin sheath (Brouns et al. 2009a). One population can be labelled with antibodies against vesicular glutamate transporter 1 (VGLUT1) and calbindin D28k (Brouns et al. 2009a). This VGLUT1/CB-ir nerve fibre population seems to form cap-like nerve endings over the apical pole of at least part of the PNECs in NEBs. The second population comprises laminar nerve terminals that express P2X2/P2X3 ATP receptors. Both populations also express the mechano-gated channel TRAAK on their intraepithelial nerve terminals (Lembrechts et al. 2011), as well as the neurotrophin-4 receptor TrkB (Oztay et al. 2010). Using genetic tagging, vagal sensory nerve terminals in NEBs were found to express purinergic P2Y1 receptors, although it was not clear which populations were involved (Chang et al. 2015). Multiple immunostaining, combining markers of these intraepithelial vagal sensory nerve fibre populations and CGRP, demonstrated CGRP immunoreactivity

2.2 Receptor–Effector Properties of the Pulmonary NEB ME

13

(IR) in different groups of thin unmyelinated C-fibre-like varicose nerve fibres running close to the base of pulmonary NEBs (Brouns et al. 2009a). Vesicular acetylcholine transporter (VAChT) immunostaining showed a very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals, which may be attributed to cholinergic vesicles that have been reported in efferent-like areas of the synapses (Brouns et al. 2009a). Nitrergic nerve terminals, presumably originating from intrinsic neurons were observed close to the base of the NEBs (Brouns et al. 2009a).

2.2

Receptor–Effector Properties of the Pulmonary NEB ME

Although quantitative analysis in rodents revealed that not all NEBs are in contact with identical sets of nerve terminals (for review, see Brouns et al. 2012), NEBs were added to the group of ‘sensory airway receptors’ due to their extensive (mainly afferent) innervation about 20 years ago (Widdicombe 2001). Evidently, the nerve terminals in se may have the intrinsic capacity to perform receptor functions (for review, see Brouns et al. 2012). However, the intimate association between vagal sensory terminals and PNECs in the NEB ME certainly provides additional possibilities for sensing environmental changes and for integrating and transducing this information to the CNS (for review, see Linnoila 2006; Brouns et al. 2012; Cutz et al. 2013; Garg et al. 2019). Different receptors, channels, and other surface molecules have been identified that could potentially allow PNECs to sense subtle changes in local environmental conditions (see Sect. 2.1.1; for review, see Brouns et al. 2012; Cutz et al. 2013). Since by far the most consistently proposed function of PNECs is their ability to sense hypoxia, a large number of investigations have been dedicated to unravelling potential O2-sensing mechanisms (Sect. 4.2.1). Obvious alterations of NEBs related to smoking have led to a considerable number of studies dealing with the effects of cigarette smoke (Tabassian et al. 1988; Sorokin and Hoyt 1990; Aguayo 1993; Tabassian et al. 1993) and the localisation of nicotinic receptors (Sect. 4.2.2). Also mechanical ‘stretch’ has since long been proposed as a possible stimulus for NEBs (Lauweryns and Peuskens 1972; Wasano and Yamamoto 1978; Cutz 2015) (Sect. 4.1). To be able to react upon stimuli, it is important to confirm that PNECs within the NEB ME exhibit characteristics of excitable cells (Youngson et al. 1993). Voltageactivated potassium (Kv), calcium and sodium currents have been reported based on patch clamp (Fu et al. 1999, 2000, 2003, 2007) and live cell imaging experiments (De Proost et al. 2008; De Proost et al. 2009). The presence of the respective channels on PNECs was further substantiated by RT-PCR profiling (Cutz et al. 2009a), and in functional (Fu et al. 2001) and morphological (De Proost et al. 2007a) studies (for review, see Brouns et al. 2012).

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Based on morphological data at the ultrastructural level (Lauweryns et al. 1972), and on genetically engineered models lacking PNECs or their secretory products (Barrios et al. 2017; Sui et al. 2018), it has been presumed already for many decades that, when activated, PNECs would be able to release their secretory products. Unequivocal direct evidence of release was provided using carbon fibre amperometry in neonatal rabbit and hamster lung slices (5-HT; Fu et al. 2002; Livermore et al. 2015), reporter patching (ATP; De Proost et al. 2009), and live cell imaging (ATP: De Proost et al. 2009; Schnorbusch et al. 2012a, b; Verckist et al. 2018) in foetal and postnatal mouse lung slices. Also ELISA has been used to determine the presence of secretory products in blood/serum samples (Barrios et al. 2019) or in medium in which lung slices were incubated (CGRP; own unpublished results). The above-mentioned studies provide evidence that PNECs represent a population of airway epithelial cells that, upon appropriate stimulation, are able to secrete various bioactive substances. Possible signalling mechanisms include interaction with NEB-associated nerve terminals (for review, see Sorokin and Hoyt 1989; Adriaensen et al. 2003), uptake by nearby blood vessels and endocrine interactions, or paracrine effects on neighbouring non-endocrine epithelial cells, fibroblasts, immune cells, airway, or vascular smooth muscle (for review, see Brouns et al. 2012). The most often explored signalling mechanism is the release of 5-HT from NEBs (most recent publication Livermore et al. 2015), mainly as a neurotransmitter of hypoxic stimuli (Lauweryns and Cokelaere 1973; Cutz et al. 1993; Fu et al. 2002). Quantal 5-HT release was measured using carbon fibre amperometry in neonatal rabbit (Fu et al. 2002) and hamster (Livermore et al. 2015) lung slices, and exocytosis appeared to be dependent on extracellular Ca2+ entry (Livermore et al. 2015). These data confirm early efforts to detect 5-HT using histochemical reactions and immunohistochemistry (for review, see Adriaensen and Scheuermann 1993; Polak et al. 1993). Exogenous stimuli and modulators are able to alter 5-HT release (Fu et al. 2002; Livermore et al. 2015). However, apart from an autoreceptor function mediated by 5-HT3 receptors on the surface of NEB cells (Fu et al. 2001), no other functional implications of 5-HT release from NEBs have been confirmed. To the author’s best knowledge, no clear evidence exists for the expression of 5-HT receptors on myelinated nerve terminals that directly contact NEBs. 5-HT, however, strongly activates both nodose and jugular C-fibres in rodent airways (rats: Hsu et al. 2019). It has been proposed, but not proven, that 5-HT3 receptors may be expressed in the intrapulmonary peripheral nerve terminals, and that NEBs could be the activating cellular source (Potenzieri et al. 2012). The presence of CGRP in NEB cells has been extensively documented, especially in rodents (Uddman et al. 1985; Cadieux et al. 1986; Lauweryns and Van Ranst 1987; Shimosegawa and Said 1991; Luts et al. 1994; Verástegui et al. 1997; Brouns et al. 2009a) (Fig. 2.2 Ab). Localisation of this 37-amino acid peptide to NEB DCVs by immunoelectron microscopy (Stahlman and Gray 1997) has led to the presumption that CGRP can be released from NEB cells by exocytosis (Dakhama et al. 2002, 2004). Potential release of CGRP has mainly been studied indirectly, using

2.2 Receptor–Effector Properties of the Pulmonary NEB ME VGAT

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CGRP

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P2X3 - GAD67-GFP

Cb P2Y2 - CCSP - PGP9.5

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Fig. 2.2 (A) Double immunolabelling for the vesicular GABA transporter (VGAT; red fluorescence) and calcitonin gene-related peptide (CGRP; green fluorescence), as a marker for NEB cells. (a) VGAT-ir intraepithelial cell group. (b) CGRP-ir NEB cells. (c) Combination of the two channels showing that VGAT is selectively expressed in pulmonary neuroendocrine cells (PNECs) of the NEB ME, and that there is a considerable co-localisation with CGRP (arrowheads). L: airway lumen; E: epithelium. (B) Single confocal optical section of a NEB in a live GAD67-GFP (green GFP fluorescence) mouse lung slice, after incubation with BODIPY TMR-X muscimol (red TMR fluorescence), a selective GABAA receptor ligand. (a) Green channel showing a GFP-expressing NEB, due to the presence of GAD67 in PNECs. (b) Combination of the green and red channels shows localisation of the muscimol-labelled GABAA receptors on the surface of the PNECs. L: airway lumen. (C) Immunostaining for the GABABR1 receptor (red fluorescence) in a GAD67GFP (green GFP fluorescence) mouse lung cryosection. (a) Red channel showing GABABR1 IR on intrapulmonary airway epithelial cells. (b) Combination of the red and green channels allows localisation of GABABR1 on GFP-fluorescent NEB cells. L: airway lumen. (D) Histochemical detection of ATP accumulation by systemic injection of quinacrine allows to identify pulmonary NEBs (arrowhead) in mouse lungs as structures that store high levels of ATP in their secretory vesicles. L: lumen of the airway. (E) Immunohistochemical staining for the ATP receptor P2X3 (blue fluorescence) shows that intraepithelial terminals of P2X3-ir nerve fibres extensively branch between NEB cells (green GFP fluorescence) in a GAD67-GFP mouse. L: lumen of the airway. (F) Single confocal optical section of a mouse intrapulmonary airway, triple stained for the P2Y2 ATP receptor (red fluorescence), CCSP (blue fluorescence) as a marker for Clara cells and CLCs, and PGP9.5 (green fluorescence) as a pan-neuronal and neuroendocrine marker. The P2Y2 ATP receptor is localised on Clara cells and on CLCs, the latter surrounding NEB cells. L: lumen of the airway

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supraoptimal dilution immunocytochemistry (Springall et al. 1988; McBride et al. 1990; Roncalli et al. 1993; Sorhaug et al. 2008), qRT-PCR of whole lungs (Sui et al. 2018), or by radioimmunoassay of perfusate collected from the pulmonary circulation (Helset et al. 1995; Sorhaug et al. 2008). Data obtained by CGRP ELISA of live murine vibratome lung slices kept in physiological solution, and subsequent correlation with the number of NEB cells in the fixed slices, allowed us to confirm that NEBs are able to release CGRP (own unpublished data). The most often reported action of CGRP is a vasodilator effect (Brain et al. 1985; Smillie and Brain 2011), by which endogenous CGRP may play an important role in pulmonary pressure homeostasis by directly dilating precontracted pulmonary blood vessels (Martling et al. 1988; Tjen-A-Looi et al. 1998; Springer et al. 2004). CGRP has also been reported to induce proliferation of airway epithelial cells (White et al. 1993) and to be an anti-inflammatory agent (Gomes et al. 2005; Rochlitzer et al. 2011; Lo et al. 2018). Recently, the close proximity of PNECs to group 2 innate lymphoid cells (ILC2) was suggested to be indicative of PNEC-derived CGRP involvement in eliciting immune responses (Sui et al. 2018). The high-affinity receptor for CGRP is a heterodimer of calcitonin receptor-like receptor (CRLR) and receptor activitymodifying protein 1 (Ramp1). These receptors have been localised to pulmonary vascular smooth muscle (Qing et al. 2001), endothelial and epithelial cells (Zhao et al. 2016), fibroblasts and immune cells (for review, see Dakhama et al. 2004), but, to our knowledge, so far not on airway-related nerve terminals. Thorough investigation of the role of CGRP in airway homeostasis and disease should always take into account CGRP/SP-ir sensory C-fibres, which are known to play afferent as well as efferent roles (for recent review, see Lo et al. 2018). In human and non-human primate lungs, the major neuropeptide reported to be produced by PNECs and NEBs is gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian peptide bombesin (Li et al. 1994). GRP belongs to the family of bombesin-like peptides (BLPs) and is associated with lung development in mice, rats, and humans. It appears to have a prominent role in a number of lung diseases, particularly those associated with decreased alveolarisation, such as bronchopulmonary dysplasia and emphysema (for review, see Sunday 2014; Ramos-Alvarez et al. 2015). As a proinflammatory peptide, it functions as an inflammatory cell activator, mitogen, and cell differentiation factor (for review, see Sunday 2014; Atanasova and Reznikov 2018). With regard to signalling mechanisms, GRP exerts its function by binding to the GRP receptor (also called BB2), a G protein-coupled receptor and member of the bombesin (BB) receptor family (for review, see Jensen et al. 2008; Ramos-Alvarez et al. 2015). Although receptor expression has been detected and is functional in pulmonary epithelial cells, fibroblasts, endothelial cells, and macrophages (for review, see Sunday 2014), direct GRP release by PNECs has not been documented so far. GRP secretion, suggested to originate from PNECs, has been reported to be induced by reactive oxygen species (ROS) from exposure to hyperoxia, ozone, or ionising radiation (Sunday 2014), but the observations are indirect and based on determination of urine or bronchoalveolar lavage (BAL) BLP levels and PNEC counts. In small cell lung carcinoma (SCLC), GRP seems to be important as a growth factor, exerting its effects via

2.2 Receptor–Effector Properties of the Pulmonary NEB ME

17

autoreceptor signalling (for review, see Moody et al. 2018). In one report, the effect of bombesin on peripheral cardiorespiratory vagal reflexes has been tested, leading the authors to suggest that in the NEB ME the vagal pathway is essential for the respiratory response to bombesin (Kaczynska and Szereda-Przestaszewska 2009). Expression of bombesin receptors on vagal pulmonary afferents, however, has not been demonstrated. A few years ago, GABA-ergic signalling was added as a potential functional mechanism in the NEB ME for mouse (Yabumoto et al. 2008; Schnorbusch et al. 2013; Sui et al. 2018), monkey (Fu and Spindel 2009; Barrios et al. 2019), and human (Barrios et al. 2019) PNECs. The system comprises a selective expression site for glutamic acid decarboxylase (i.e. GAD67) (Fig. 2.2Ba), the rate-limiting enzyme in the biosynthesis of GABA, in NEB cells (Yabumoto et al. 2008; Schnorbusch et al. 2013). The presence of vesicular GABA transporter (VGAT) in the basal compartment of the PNECs (Schnorbusch et al. 2013) (Fig. 2.2A) is suggestive of storage of GABA in DCVs and its potential release by stimulated exocytosis. GABA signalling is impaired in mice in which the GAD1 or VGAT genes are disrupted (Sui et al. 2018). GABA release has been measured by ELISA in serum samples (Barrios et al. 2019). GABAA receptors (mouse: (Schnorbusch et al. 2013); Fig. 2.2Bb), as well as both types of GABAB receptors were shown to be localised to PNEC surface membranes (mouse: Schnorbusch et al. 2013) (GABABR1 Fig. 2.2C); monkey: Fu and Spindel 2009). The GABABR1 receptor was also reported in ILC2 (Sui et al. 2018), mediating an immune-response effect. GABA signalling in the NEB ME has been suggested to be involved in mucus overproduction and goblet cell hyperplasia (Barrios et al. 2017; Sui et al. 2018; Barrios et al. 2019), in which the nerve–PNEC–GABA axis plays an important role (Barrios et al. 2017, 2019). A well-characterised mechanism by which PNEC transmitters can influence their target vagal sensory nerve terminals relies on the expression of P2X2/3 receptors on the myelinated vagal sensory components of the innervation of PNECs in rodents (Fig. 2.2E) (Brouns et al. 2000; Van Genechten et al. 2004; Brouns et al. 2009a). The extensive intraepithelial arborisations of these vagal afferent terminals are in close contact with ATP-storing NEB cells (Fig. 2.2D), strongly suggesting that ATP secreted by NEB cells via Ca2+-dependent exocytosis may act as a neurotransmitter/neuromodulator in the vagal transduction pathway. In this way, the NEBs would be ‘functional units’ that allow fast transduction of local pulmonary information to the CNS. In lung slice preparations, PNECs were indeed shown to release ATP when activated (De Proost et al. 2009; Schnorbusch et al. 2012b, 2013; Verckist et al. 2018). Similar complexes of functional ATP-containing receptor cells and associated ATP receptor-expressing nerve terminals have been reported for taste buds (for review, see Housley et al. 2009; Kinnamon and Finger 2013) and carotid bodies (Piskuric and Nurse 2013). Apart from exerting a role in interactions between PNECs and nerve terminals, ATP also appears to be involved in direct interactions between PNECs and CLCs

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2 The Pulmonary NEB ME Is a Complex Intraepithelial Unit

(De Proost et al. 2009; Burnstock et al. 2012; Lembrechts et al. 2012) in the NEB ME. Upon PNEC stimulation, release of ATP was demonstrated to activate the surrounding CLCs via functional G protein-coupled P2Y2 ATP receptors (Fig. 2.2F), resulting in a rise of the intracellular calcium concentration ([Ca2+]i) in CLCs (De Proost et al. 2009; Schnorbusch et al. 2012b, 2013; Verckist et al. 2018). Given that the NEB ME is considered a stem cell niche for CLCs (see Sect. 4.3), this interaction may also be of importance for further unravelling the NEB ME in its entire complexity. Following an overview of the methodology used to explore the NEB ME, in this review we will elaborate on the advances that have been made in unravelling its functions, with main focus on potential stimuli for PNECs and CLCs.

Chapter 3

Studying the Pulmonary NEB ME: A Multidisciplinary Approach

In order to unravel the function of the pulmonary NEB ME, the last decades multidisciplinary approaches involving multiple immunohistochemistry, genetic tagging, live cell imaging, laser microdissection, and gene expression analysis have been developed. An important prerequisite for all above-mentioned morphology-based techniques is a clear and reliable identification and visualisation of the pulmonary NEB ME, since pulmonary NEBs show a widespread distribution in the airway epithelium. For all below-mentioned methods, a clear outline of NEB identification is comprised in the respective paragraphs.

3.1

Functional Morphological Characteristics of the NEB ME

Historically, functional studies of the NEB ME focussed on the more-or-less selective identification and functional morphological characteristics of PNECs and NEBs in experimental circumstances (for review, see Sorokin et al. 1983; Adriaensen and Scheuermann 1993; Adriaensen et al. 2003; Brouns et al. 2012). As a result, a plethora of methods have been developed for NEB identification. Whereas in brightfield microscopy, NEBs are hardly recognisable after routine fixation and classic light microscopic staining, immunohistochemical methods or genetic labelling (see Sect. 3.2) nowadays enables the easy and unambiguous identification of NEBs. Fluorescent NEB labelling combined with visualisation by confocal microscopy, has proven to be a very successful qualitative tool to explore the detailed functional morphology of the entire pulmonary NEB ME. NEB identification can be combined with immunohistochemistry or in situ hybridisation to unravel PNEC characteristics. A combined approach consisting of NEB labelling, immunohistochemistry for nerve terminal features, neuronal tracing, or denervation experiments is instrumental in © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 I. Brouns et al., The Pulmonary Neuroepithelial Body Microenvironment, Advances in Anatomy, Embryology and Cell Biology 233, https://doi.org/10.1007/978-3-030-65817-5_3

19

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3 Studying the Pulmonary NEB ME: A Multidisciplinary Approach

further unravelling the relationship of PNEC clusters with their complex innervation (for review, see Brouns et al. 2012). Although the intercellular relationship between NEBs and the overlying CLCs can be visualised based on their unique location and SCGB1A1 immunohistochemistry, in postnatal mouse lungs immunohistochemical markers that can exclusively identify CLCs are still lacking (see Sect. 2.1.2). An easy-to-perform, selective, and reliable method to identify NEBs in morphological experiments is based on immunohistochemical staining with antibodies against (selective) markers for PNECs (see Table 2.1) and on their organisation as clustered cells in the airway epithelium (for review, see Brouns et al. 2012). Markers of choice are those for the abundant transmitters stored in DCVs (like CGRP), or for molecules that are present in the PNEC cytoplasm related to their neuron-like differentiation. Worth mentioning in this current overview is protein gene product 9.5 (PGP9.5), also known as ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), which has species-wide marker properties for both NEBs and the majority of contacting nerve fibres. Other cytoplasmic markers used for PNEC identification in mice are synaptophysin (SYN) and synaptic vesicle protein 2 (SV2).

3.2

Genetically Engineered Mouse Models to Study the NEB ME

Over the past few decades, genetically engineered mouse models have gained interest to study the functional morphology of the NEB ME. Especially during development or differentiation (see Sect. 4.3.1.1), selective ablation of genes important for PNECs (Borges et al. 1997; Ito et al. 2000; Guha et al. 2012; Song et al. 2012; Sui et al. 2018) or CLCs (Tsao et al. 2009; Morimoto et al. 2012; Guha et al. 2014; Kuo and Krasnow 2015; Guha et al. 2017), or ectopic expression of some of these genes (Linnoila et al. 2000), has been shown to be reliable new tools to study the behaviour of the NEB ME. Lineage tracing studies, in which cells are genetically tagged, have been proven important for the follow-up of lung repair after injury (Li and Linnoila 2012; Song et al. 2012; Yao et al. 2018). Whereas 20 years ago genetical engineering used to be reserved for a limited cohort of researchers and research labs, nowadays several interesting commercially available mouse lines are available for relatively easy Cre recombinase-driven knock-out or knock-in gene manipulation (for review about Cre recombinase, see Kim et al. 2018). Cre recombinase recognises a specific DNA fragment called loxP and is able to delete DNA sequences between two loxP sites. After recognition of the loxP sites, Cre excises the loxP-flanked (¼floxed) DNA, thereby removing (part of) and hence inactivating the gene of interest. This technique can be applied to generate conditional knock-out mice by breeding a Cre driver mouse strain—in which Cre recombinase is expressed by a promoter that specifically targets the cell or tissue of interest—with a floxed mouse strain (for review, see Kim et al. 2018). Mouse models based on a tamoxifen-inducible Cre system (CreERT) use a modified Cre protein

3.3 3D-Imaging and -Analysis of NEBs in Airway Whole Mount and in Cleared. . .

21

fused with an oestrogen receptor. Upon tamoxifen administration, CreERT translocates to the nucleus where it can bind with loxP sites, in this way providing a Cre-loxP system that allows Cre activation at a precise time and in a specific cell type. Cre recombinase systems combined with Rosa reporter lines allow tracing of targeted cells using fluorescence (confocal) microscopy. A number of genetically engineered models have proven useful to study conditional changes or follow specific cell types after lung injury. Such conditionally targeted knock-out mice, lineage tracing studies, conditioned reporter cell lines, etc., have now also been applied in NEB ME-related studies (Borges et al. 1997; Ito et al. 2000; Shan et al. 2007; Sutherland et al. 2011; Noguchi et al. 2015; Barrios et al. 2017; Sui et al. 2018; Ouadah et al. 2019). Often non-selective Cre mouse lines are used for breeding (e.g. the ShhCre+ line), because in these conditions Cre acts in the entire epithelium (Harris et al. 2006). Regarding overall NEB research, it is certainly worth mentioning here that for about 10 years ‘GAD-GFP’ mice are commercially available, in which PNECs in the NEB ME can be easily recognised because they are genetically tagged with GFP (Figs. Sect. 2.2). The mouse model is based on the selective expression of GAD65/67 in PNECs, as part of a GABAergic signalling mechanism (Yabumoto et al. 2008; Schnorbusch et al. 2013; Barrios et al. 2017). eGFP is expressed under the control of GAD67 regulatory elements, having a single-copy gene for GAD67 expression, hence avoiding overexpression, and a single-copy gene for GFP expression (Feng et al. 2004; Zhao et al. 2010). In this GAD67-GFP mouse strain, GFP diffuses freely in the cytoplasm and fills the whole GFP-expressing cell (Tamamaki et al. 2003). Since in mouse intrapulmonary airways PNECs in the NEB ME appear to be the only GAD expressing (GABA-producing) cell type (Yabumoto et al. 2008; Schnorbusch et al. 2013; Barrios et al. 2017), unequivocal, fast and straightforward identification of NEBs is possible. Hence, elaborate methods are not necessary to identify PNECs in the airway epithelium of GAD67-GFP mice, which is an asset for morphological, neurochemical, functional, and gene expression analysis experiments (Lembrechts et al. 2013; Schnorbusch et al. 2013; Barrios et al. 2017; Verckist et al. 2017, 2018).

3.3

3D-Imaging and -Analysis of NEBs in Airway Whole Mount and in Cleared Whole Lungs

The functional morphological characteristics of the pulmonary NEB ME (Sect. 3.1) have been and are still mainly investigated using lung cryostat sections (for review, see Brouns et al. 2012). Although certainly valuable for qualitative interpretation, thorough quantification is tricky and at best highly labour intensive (for review, see Brouns et al. 2012). Consequently, information concerning the total population of pulmonary NEBs in

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3 Studying the Pulmonary NEB ME: A Multidisciplinary Approach Cleared whole mouse lungs

3 mm

A

Cleared whole mouse lungs Vagal sensory innervation - GAD67-GFP

10 μm

B

Fig. 3.1 (A) Transparently reconstructed light sheet microscopic low magnification overview of cleared whole mouse lungs (grey background fluorescence). Proof-of-principle multiple immunostaining showing the potential of this model for visualising the entire population of NEBs (green GFP fluorescence) and myelinated vagal afferents (red fluorescence). (B) 3D-reconstructed high magnification detail of a single NEB (green GFP fluorescent PNECs) in a cleared whole mouse lung. Terminals of a myelinated vagal afferent nerve fibre (red fluorescence) are seen to extensively branch between the NEB cells

their typical microenvironment remains fragmentary due to their relatively low number and widespread distribution in the airway epithelium. Therefore, additional elegant methods have been developed that allow a more consistent overview of the population of NEB MEs. Airway whole mounts, in which alveolar tissue has been carefully removed under a dissection microscope using watchmaker’s forceps and microsurgical scissors, leave a number of branching levels of the airway tree more-or-less intact, and have been applied for the localisation of NEBs using immunohistochemistry (Avadhanam et al. 1997; De Proost et al. 2007). Also, endobronchial biopsies (West et al. 2015) and precisioncut vibratome slices (De Proost et al. 2009) allow (immuno)histochemical staining and functional studies of individual NEBs and thorough analysis of the immediately surrounding tissues. The past few years, advances in optical clearing techniques have enabled the use of intact whole lungs or lung lobes of different species for robust whole-organ (immuno)labelling and 3D imaging for a variety of research questions (Liu et al. 2020). Adopting a combined approach consisting of immunostaining, tissue clearing with a (modified) iDisco+ protocol and light sheet microscopy, allowed us to visualise and image the entire population of NEB MEs in whole mouse lungs (own observations to be published soon; Fig. 3.1A). A challenging multilabel protocol was applied for immunostaining of the NEBs, their vagal sensory innervation and myelinated nerve fibres. It could be shown that the staining, clearing, and

3.4 Models and Techniques for Functional Live Cell Imaging (LCI) of the NEB ME

23

imaging protocols allowed for 3D rendering of the complete lungs. All NEBs and associated myelinated vagal afferent nerve terminals were included, introducing possibilities for 3D quantification (own observations to be published soon). Moreover, selected individual NEB/nerve terminal complexes in these lungs can be further analysed in detail using high-resolution confocal imaging and 3D reconstruction (own observations to be published soon; Fig. 3.1B).

3.4

Models and Techniques for Functional Live Cell Imaging (LCI) of the NEB ME

Although the presence of live pulmonary NEBs has been demonstrated in intrapulmonary micro-dissected airways (De Proost et al. 2007), direct measurements and manipulation remain impossible because NEBs are embedded in the inaccessible airway epithelium. Precision-cut vibratome slices are useful tools for studying lung physiology (Liberati et al. 2010; Sanderson 2011; Akram et al. 2019; Liu et al. 2019). Vibratome sectioning of agarose-filled mouse lungs results in 100-300-μm-thick slices that contain branching airways, blood vessels, and alveolar areas, with a normal airway physiology (Perez and Sanderson 2005; Delmotte and Sanderson 2006). Cell types are present in the same ratio and with the same cell–cell and cell–matrix interactions as seen in vivo, offering many advantages over both in vivo experiments and cell culture systems. The highly reproducible and controllable setting has resulted in increased use of human and mouse ex vivo lung slices to study different aspects of lung biology (Sanderson 2011; Neuhaus et al. 2018; Akram et al. 2019). Compared to systemic in vivo work, tissue slices are cost- and time-effective, and still harbour many of the features of the intact organ. Initially, lung slices of rabbits (Fu et al. 1999, 2000), hamsters (Fu et al. 2001, 2003, 2004), and mice (Fu et al. 2000) were mainly applied for electrophysiology (patch clamp) of individual PNECs and for amperometry. To keep and investigate clusters of PNECs in their ‘natural environment’, i.e. located between a variety of other epithelial cells such as their covering Clara-like cells, Clara cells, and ciliated cells (Pintelon et al. 2005; De Proost et al. 2008, 2009), the ex vivo lung slice model was fully optimised to perform microscopic functional fluorescent dye-based physiological studies of the NEB ME in postnatal mice, rats, and hamsters about fifteen years ago (Pintelon et al. 2005; De Proost et al. 2008, 2009). In the latter lung slice model, based on our previous experience with intestinal whole mounts (Cornelissen et al. 1996), the styryl pyridinium dye 4-(4-diethylaminostyryl)-N-methylpyridum iodide (4-Di-2-ASP) was found to accumulate in the mitochondria of PNECs (Pintelon et al. 2005) and intact live NEBs could be unambiguously identified in their natural environment (for review, see Brouns et al. 2012). Because of the often claimed importance of NEBs during the transition from foetal to neonatal life (Bollé et al. 2000; Pan et al. 2004; Linnoila 2006; Cutz 2015), the lung vibratome slice

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3 Studying the Pulmonary NEB ME: A Multidisciplinary Approach GAD67-GFP

Fluoforte T0

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Fig. 3.2 (a) Image of a vibratome slice of a GAD67-GFP mouse lung showing a GFP-fluorescent NEB (ROI 1), surrounded by a rim of non-fluorescent CLCs (examples: ROI 2 and 3). The airway epithelium contains intermingled rounded non-fluorescent Clara cells and polygonal fluorescent ciliated cells. (b–d) The lung slice is loaded with the red-fluorescent calcium indicator FluoForte, and shows the NEB ME at different time points before (T0), during (T1), and after (T2) a 5s challenge with 50 mM [K+]o (e) Graph plotting the time course of the changes in FluoForte fluorescence intensity. The ROIs in the graph correspond to the NEB (ROI 1) and 2 CLCs (ROI 2–3) as outlined in image a (Reproduced with permission from Schnorbusch et al. 2013)

technology was adapted a few years later to selectively visualise prenatal NEBs in a foetal mouse ex vivo lung slice model (Schnorbusch et al. 2012b). More recently, the use of GAD67-GFP mice even allowed easy visualisation of GFP-fluorescent NEBs in live lung slices without time-consuming pre-treatments that might influence NEB physiology (Schnorbusch et al. 2013) (Fig. 3.2a). To perform physiological experiments, the ex vivo lung slice model was optimised in a confocal ‘live cell imaging (LCI)’ set-up (Pintelon et al. 2005; De Proost et al. 2008, 2009; Lembrechts et al. 2012; Schnorbusch et al. 2012a, b, 2013; Lembrechts et al. 2013), in which a dual spinning disk confocal microscope enables fast capturing of high-resolution images with minimal photobleaching and low phototoxicity. Physiological and pharmacological LCI experiments are performed by transferring the lung slice to a chamber mounted on the stage of the microscope so that experimental stimuli can be applied via an attached perfusion system during imaging. In the LCI set-up, both 4-Di-2-ASP labelling in wild-type mice and GFP fluorescence in GAD67-GFP mice (Fig. 3.2a) reveal NEBs as clusters of small

3.4 Models and Techniques for Functional Live Cell Imaging (LCI) of the NEB ME

25

fluorescent PNECs, surrounded by a continuous layer of much larger rounded and almost non-fluorescent CLCs (De Proost et al. 2008; De Proost et al. 2009; Schnorbusch et al. 2012b, 2013; Verckist et al. 2018). Additionally, ciliated cells are morphologically distinguishable as polygonal fluorescent cells that are intermingled with large, rounded, and virtually non-fluorescent Clara cells (Fig. 3.2) (De Proost et al. 2008, 2009). Straightforward discrimination between the different epithelial cell types makes it possible to visualise physiological reactions upon stimulation in any specific cell type (Fig. 3.2b-e) (for review, see Brouns et al. 2012). The ex vivo lung slices can be loaded with functional fluorescent probes (e.g. Ca2+ indicators, membrane potential indicators, mitochondrial membrane potential probes) that translate responses of cells to applied stimuli in visual/measurable changes in fluorescence intensity (De Proost et al. 2008, 2009; Brouns et al. 2012; Schnorbusch et al. 2012b, 2013; Verckist et al. 2018). Since activation of (excitable) cells is often accompanied by a rise in the [Ca2+]i, fluorescent calcium indicators (e.g. green Fluo-4, red FluoForte (Fig. 3.2b-d)) are often used in LCI experiments. Upon activation of calcium indicator-loaded cells, a rise in [Ca2+]i is reflected by increasing fluorescence intensity, which can be plotted against time for selected regions of interest (ROIs; Fig. 3.2e). Forced depolarisation of NEB cells in lung slices by short-term application of a physiological solution that contains a high extracellular potassium concentration ([K+]o) invariably results in a fast, reversible, and reproducible rise in calciumindicator fluorescence in NEB cells approximately 1.5 s after stimulation (Fig. 3.2), reflecting the influx of extracellular Ca2+. Since no adverse effects are apparent on the physiological properties, high [K+]o has typically been used as a positive control stimulus to test the viability and proper loading of NEBs in ex vivo lung slices (De Proost et al. 2008, 2009; Schnorbusch et al. 2012b, 2013; Verckist et al. 2018). Remarkably, following stimulation of the NEBs with high [K+]o, the surrounding CLCs showed a slightly delayed Ca2+ response, with a variable onset of the [Ca2+]i rise in the individual CLCs (Fig. 3.2b-e). This response of CLCs results from quantal exocytosis of ATP by the activated NEB cells, which in turn activates nearby CLCs via P2Y2 receptors (De Proost et al. 2009). The possibility of visualisation of this secondary paracrine activation of CLCs interestingly indicates that LCI of NEBs in lung slices not only enables real-time visualisation of PNEC activation, but also the consecutive exocytosis of bioactive substances and therefore potential signalling within the NEB ME niche. For Ca2+ imaging experiments of the different epithelial cell types of intrapulmonary airways, appropriate and reliable control stimuli have been optimised (De Proost et al. 2008; Brouns et al. 2012). In this way, the confocal multipoint analysis method is perfectly suitable to address the complexity of the NEB ME in lung slices, since it is capable of discriminating between recordings from NEBs and those of other nearby cell types in the airway epithelium, based on morphological characteristics and the specific fluorescent patterns obtained with the in vivo NEB markers and functional fluorescent probe loading. A few years ago, LCI of lung slices was optimised for hamsters, in which NEBs were visualised with

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3 Studying the Pulmonary NEB ME: A Multidisciplinary Approach

FM4-64FX and calcium imaging was performed using Oregon Green Bapta-2 AM (Livermore et al. 2015). When, after LCI, agarose-filled lung slices are fixed and subjected to multiple immunostaining procedures, NEBs and their extensive innervation can still be visualised, offering additional possibilities for elucidating the function(s) of NEBs in integrated approaches.

3.5

Laser Microdissection and Selective Gene Expression Analysis of the NEB ME

Like in other cells and systems, the unique origin, structure, and function of the NEB ME is related to the specific expression of distinct genes, as compared to the surrounding airway epithelium. Methods for selective mRNA expression analysis, such as reverse transcription polymerase chain reaction (RT-PCR), quantitative (real-time) RT-PCR (qRT-PCR; qPCR), and microarrays yield valuable information to unravel NEB ME functions. Gene expression analysis of the pulmonary NEB ME, however, has always been challenging, as a result of the low number and dispersed distribution of NEBs in the airway epithelium. Most of the molecular information has, therefore, been gathered from pure populations of PNECs using primary cell lines, induced PNECs from pluripotent stem cells (Hor et al. 2020), immortalised SCLC cell lines (O’Kelly et al. 1998), or from embryonic lungs (Morimoto et al. 2012; Guha et al. 2014). Cell lines, however, may differ significantly from their counterparts in the natural environment. Therefore, a method that has been designed to dissect cell groups selectively, precisely, and directly from freshly isolated organs, like laser microdissection (LMD) (Emmert-Buck et al. 1996), seems to be a better option to collect genetic information from the pulmonary NEB ME. This technology can identify pure cell populations or tissues of interest from fixed tissues by direct visualisation under a microscope. This microscope is coupled to a laser that excises selected areas, which are subsequently collected in a tube and used for further molecular analysis. Sporadic efforts have been made to isolate mRNA after LMD from NEBs in human lungs (Cutz et al. 2004; Livermore et al. 2015). The majority of available data, however, concern laboratory animals. Complex mRNA expression analysis depends on good RNA integrity (PerezNovo et al. 2005), but degradation of RNA by exogenous and endogenous RNase enzymes may occur rapidly (Fleige and Pfaffl 2006; De Spiegelaere et al. 2011). Using a standard protocol for LMD and an RNA quality check with determination of the RNA integrity number (RIN), our lab demonstrated that RNA in postnatal lungs seems to be highly degraded, in contrast to that in pooled LMD samples of cryosections of brain or embryonic lung tissue (Fig. 3.3A), which both show a low endogenous RNase activity (Verckist et al. 2017). These observations are in line

3.5 Laser Microdissection and Selective Gene Expression Analysis of the NEB ME Brain

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LMD-NEB ME 28S rRNA

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Fig. 3.3 (A) RNA quality of pooled laser microdissection (LMD) samples of cryostat sections of mouse brain (PD21; a), embryonic lungs (ED14; b), and postnatal lungs (PD21; c), visualised in electropherograms by the 18S and 28S rRNA peaks. Although the same (unadapted) protocol is used, RNA in postnatal lungs appears to be degraded (RNA integrity number (RIN) ¼ 3.2), while high-quality intact RNA can be detected in brain (RIN ¼ 7.9) and embryonic lungs (RIN ¼ 8.9) (Reproduced with permission from Verckist et al. 2017). (B) Optimisation of tissue processing and RNA isolation considerably improved the RIN values for whole lung cryostat sections (RIN ¼ 9.3; c). Use of these optimised protocols for isolation of the NEB ME (a) and control airway epithelium (CAE) (b) also resulted in high RIN values for both samples (RIN NEB ¼ 8; RIN CAE ¼ 9.3). (C) Example of the isolation of a pulmonary NEB ME from a GAD67-GFP mouse lung cryostat section by LMD. (a) A GFP-fluorescent NEB (arrowhead) was software-encircled. (b) After automated LMD, the NEB is collected (gravity-based) in an Eppendorf tube, leaving a hole in the cryostat section. (D) Non-amplified RNA samples of the NEB ME (after LMD collection; NEB) and of whole lung (WL) sections. The gel electrophoresis image shows expression of the PCR products.

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3 Studying the Pulmonary NEB ME: A Multidisciplinary Approach

with literature data, mentioning that endogenous RNase activity in lungs is about 5000 times higher compared to that in brain (Cox et al. 2008). An adapted LMD protocol has, therefore, been developed that allows easy identification of NEBs, a maximal reduction of the time that the tissue is exposed to an aqueous environment and inhibition of endogenous RNase enzyme activity (Verckist et al. 2017). Standard immunohistochemical staining protocols usually require several hours of incubation in aqueous media, resulting in significant degradation and loss of RNA through the activation of tissue RNAses or other factors (Wang et al. 2006). Eliminating immunohistochemical staining for pulmonary NEB identification is only possible by using the adopted GAD67-GFP mouse model (Schnorbusch et al. 2013) that selectively expresses GFP in PNECs. Since in GAD67-GFP mice, eGFP is expressed as a free protein in the cytoplasm (Tamamaki et al. 2003) and RNA integrity is subject to excessive fixation (Medeiros et al. 2007; Cox et al. 2008), a gentle (0.1% paraformaldehyde for 5 minutes) intrapulmonary fixation protocol was designed that still allows the fast and straightforward visualisation of the GFP-fluorescent NEBs in non-coverslipped cryostat sections. When positioned on glass membrane slides (Verckist et al. 2017), a laser microdissection system with upright research microscope and UV-laser excision beam can cut out the desired area. Samples are gravity-based collected in fluid-filled Eppendorf tubes (Fig. 3.3C) and can be further processed for total RNA isolation. Evaluation of RNA integrity after the LMD procedure showed that pooled NEB ME samples yielded sufficient high quality (RIN value higher than 6.5) for further molecular analysis (Fig. 3.3Ba). To allow comparative analysis, control airway epithelium (CAE) (Fig. 3.3Bb) is also collected by LMD from the same cryostat sections. Whole lung LMD samples (Fig. 3.3Bc) processed by the same protocol are always collected as an internal control. qPCR analysis of the NEB ME, CAE, or whole lung sections showed clear expression of housekeeping genes (Fig. 3.3D and E), confirming that the LMD procedure did not harm the possibility to get access to molecular information from the different sample types (Fig. 3.3D and E). The NEB ME represents only a minor part of the airway epithelium and evidently of the whole lung tissue. Consequently, qPCR analysis performed on airway epithelium or whole lung tissue may mask the presence of the very little amounts of

Fig. 3.3 (continued) Although the reference genes (Rpl4, eEF2, Rpl8) are equally present in both the NEB ME and whole lung sample, both CGRP (amplicon length 105 bp) and GAD (102 bp), as selective markers for NEBs, were only visible as a distinct band in the LMD-collected NEB sample. These experiments clearly revealed that low doses of mRNA, such as those present in less frequent NEB cells, can only be detected in pooled concentrated samples and not in whole lung tissue. (E) Amplified RNA samples of LMD-collected NEB ME (NEB) and of control airway epithelium (CAE). Expression of CGRP (as a selective marker for the NEB ME only), CCSP (as a marker for both Clara-cells and CLCs), and Flt-1 (as a marker for ciliated cells). While CGRP is present in the NEB ME only, CAE can easily be identified based on the Flt-1 expression. CCSP appears to be present in both the NEB ME and CAE samples, although expression seems much lower in the NEB ME

3.5 Laser Microdissection and Selective Gene Expression Analysis of the NEB ME

29

mRNA expressed in the NEB ME. The transcripts of the NEB ME may be diluted by the presence of excessive mRNA from the surrounding tissue. CGRP (Uddman et al. 1985; Hong et al. 2001; Brouns et al. 2009) and GAD (low expression enzyme) (Yabumoto et al. 2008; Schnorbusch et al. 2013) are both selectively expressed in mouse PNECs, and, are, therefore, used as marker molecules to identify pulmonary NEBs (see Sect. 3.1). qPCR analysis on non-amplified samples of LMD-selected and pooled NEB MEs showed strong CGRP and somewhat weaker GAD expression (Fig. 3.3D). In contrast, neither CGRP nor GAD expression could be detected in whole lung RNA samples (Fig. 3.3D). These observations underline the need that molecular information on pulmonary NEBs can only be accessed when ‘bulk’ information is eliminated. Since reliable detection and quantification of weakly expressed genes using very small amounts of degraded DNA are challenging (Kerman et al. 2006), all NEB-pooled samples and CAE samples were treated for RNA amplification before further use (Verckist et al. 2017). To finally confirm whether the optimised LMD protocols allow selective isolation of either NEB ME samples or CAE samples from postnatal airway epithelium, qPCR was performed for genes that are known to be selectively expressed in the NEB ME (i.e. PNECs and CLCs), and CAE (i.e. ciliated cells and Clara cells). Clear mRNA expression for CGRP (and GAD) can be detected in the NEB ME but not in CAE (Fig. 3.3E), while Flt-1 (marker for mouse ciliated cells; Thebaud et al. 2005) mRNA expression is present in samples of CAE but not in NEB ME samples (Fig. 3.3E). CCSP mRNA is expressed both in CAE, due to the presence of Clara cells, and in NEB ME samples, due to the presence of CLCs (Hong et al. 2001). These data show that differential gene expression of CGRP, Flt-1, and CCSP is sufficient to evaluate the straightforward discrimination and isolation of pooled NEB ME and CAE samples from postnatal lung sections by our optimised LMD protocols. These three marker genes are now routinely used to test the selectivity/purity of all newly isolated and pooled NEB ME and CAE samples. The LMD technique for isolation of GFP-fluorescent NEBs is a promising tool for further unravelling the molecular basis of NEB ME physiology. Collected mRNA samples from the NEB ME can be used to obtain reliable gene expression data using single qPCR and large-scale PCR array experiments.

Chapter 4

Functional Exploration of the Pulmonary NEB ME

Since the discovery of pulmonary NEBs (Fröhlich 1949), a quest to determine their exact function has been going on. Traditionally, NEBs are regarded as sensory organoids able to perform chemoreception (especially sensing low oxygen levels) and mechanoreception, and are likely involved in regulation of lung maturation and growth (Sorokin and Hoyt 1990, 1993; Sorokin et al. 1997; Linnoila 2006). Whereas many studies focussed on their role during ontogeny and epithelial regeneration, others have tried to unravel the postnatal receptor function ascribed to NEBs, mainly on the basis of their connection with vagal sensory nerve terminals. Nowadays, it is generally accepted that the clustered PNECs, together with surrounding Clara-like cells and extensive nerve fibre populations (the majority of which are sensory) make up the NEB ME, a unique complex with intrinsic capacities to selectively sense and respond to intrapulmonary stimuli. The current review focusses on the multimodal (sensing) features of the NEB ME, which have been widely assumed but never truly validated. The power of the studies discussed below all start from in situ visualisation of NEBs, further explored by confocal LCI, molecular investigations, and functional morphological studies in integrated approaches.

4.1

Mechanosensing in the NEB ME

During passive breathing, airway ‘stretch’ sensation is mediated by sensory neurons residing in the nodose ganglion (Mazzone and Undem 2016; Williams et al. 2016; Umans and Liberles 2018) with terminals travelling in the vagal nerve, i.e. the major sensory innervation to the pulmonary system (for recent reviews, see Mazzone and Undem 2016; Nonomura et al. 2017; Umans and Liberles 2018). Whereas the respiratory input from the vagal nodose neurons to the brainstem region has been clearly determined (for review, see Umans and Liberles 2018), the morphology of © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 I. Brouns et al., The Pulmonary Neuroepithelial Body Microenvironment, Advances in Anatomy, Embryology and Cell Biology 233, https://doi.org/10.1007/978-3-030-65817-5_4

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terminals of force-sensing neurons in the airways is still a matter of debate (Lee and Yu 2014; Mazzone and Undem 2016; Umans and Liberles 2018) and generally based on determining the counterpart of ‘slowly adapting stretch receptor’ activity that can be measured in the vagal nerve. Knowledge of the exact location, and of the morphological and neurochemical features of intrapulmonary sensory receptors, however, is crucial for the functional identification of the distinct populations of electrophysiologically identified sensory airway receptors (Yu 2009). The availability of antibodies against vesicular glutamate transporters (VGLUTs), calcium-binding proteins, P2X2/3 receptors, and Na+/ K+-ATPase α3 already has allowed to identify vagal sensory nerve terminals in rodent airways and lungs with presumed mechanosensory activity (for review, see Adriaensen et al. 2006; Brouns et al. 2009b, 2012) forming, as investigated in rodents, smooth muscle-associated airway receptors (SMARs) (Brouns et al. 2006b; Lembrechts et al. 2011) and terminals in connection with pulmonary NEBs (Adriaensen et al. 2006; Brouns et al. 2012). Sensitivity of cells—including neuronal terminals—to mechanical stimuli is mediated by the expression of specific mechanosensitive receptors. These receptors may directly or indirectly interact with cytosolic force-sensing elements, such as microtubules and actin filaments (Sinha et al. 2018; Cox et al. 2019), and can rapidly convert mechanical stimuli into electromechanical intracellular signals (Martinac 2004; Cox et al. 2017). Well-known superfamilies of mechano-gated ion channels are the K2P-type (TREK1/2, TRAAK), transient receptor potential (TRP), degenerin and epithelial sodium channel (DEG/ENaC), and Piezo1/2 ion channels (for review, see Cox et al. 2019). These channels have been studied for more than 30 years and are widely expressed in both specialised cells and non-specialised cell types in the body (for review, see Cox et al. 2019). Vagal sensory neuronal cell bodies can express mechano-gated K2P ion channels TREK-1 and TRAAK in rats (Zhao et al. 2010a) and mice (Fernandez-Fernandez et al. 2018). TRAAK belongs to the TREK clade (¼ the lipid and mechanosensitive channels; for review, see Renigunta et al. 2015) of the eukaryotic mechano-gated ‘K2P ion channels’ (Fink et al. 1998; Maingret et al. 1999). They possess the unique functional property to be directly activated by mechanical stimuli, such as membrane tension, cell swelling and stretch, or by ‘chemical stimuli’ that change the curvature of the cell membrane, such as polyunsaturated fatty acids and membrane crenators (Maingret et al. 1999; Lesage and Lazdunski 2000; Patel and Honore 2001; Kim 2003). Immunohistochemical labelling of TRAAK in mouse lungs, localised TRAAK on SMAR-endings (Lembrechts et al. 2011; Brouns et al. 2012), and in two neurochemically distinct populations of myelinated vagal sensory nerve fibres populations terminating between PNECs in the pulmonary NEB ME: (1) immunopositive for VGLUTs/CB and Na+/K+-ATPase α3; (2) expressing P2X2/3 ATP receptors (Fig. 4.1A) (Lembrechts et al. 2011). Both populations, which could already be identified using ‘mechanosensory’ markers, hence possess intrinsic mechanosensitive capacities. Although mechanosensory roles have been proposed for NEBs for many years (Lauweryns and Peuskens 1972; Wasano and Yamamoto 1978; Cutz 2015), the K2P channel TRAAK found in this study was the

4.1 Mechanosensing in the NEB ME

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TRAAK

TRAAK - P2X3

TRAAK - P2X3 - PGP9.5

Aa

Ab

Ac

E L 10 μm

Ba

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4-Di-2-ASP

4 3

2 1

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Bb

Bc

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Fluo-4 T3

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TRPC5 - SV2 L

L

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Cb

Fig. 4.1 (A) Mouse pulmonary NEB, triple immunostained for TRAAK (red fluorescence), P2X3 (green fluorescence), and PGP9.5 (blue fluorescence). (a) TRAAK-ir nerve terminals (arrowheads) in the airway epithelium. (b) Combination of red and green channels showing a vagal sensory intraepithelial P2X3-ir arborisation. The intraepithelial nerve endings appear to co-express P2X3 receptors and TRAAK (arrowheads). (c) Combination of red, green, and blue channels showing that the P2X3/TRAAK-ir nerve endings coincide with the presence of a PGP9.5-ir NEB. L: lumen

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first actual mechanosensitive channel reported on their vagal afferents. Because TRAAK is responsible for ‘leak’ K+ currents, TRAAK K+ channel opening drives the cell membrane towards hyperpolarising potentials, thereby leading to decreased cell excitability (Bayliss and Barret 2008). The observation that TRAAK is expressed on vagal afferent nerve terminals in NEBs (and SMARs) has allowed us to suggest that mechanical activation of TRAAK channels modulates the excitability of the respective sensory airway receptors (Lembrechts et al. 2011). A few years ago, Piezo2 ion channels were reported to be present on nodose neuronal cell bodies in mice (Nonomura et al. 2017). Piezo proteins have been characterised as the pore-forming subunits of non-selective cationic mechanosensitive ion channels, whose expression is required for many mechanotransduction processes (Wu et al. 2017). When exposed to a step-like mechanical stimulus, Piezo channels activate (open) rapidly in a dose-dependent manner, and subsequently inactivate (entering a non-conducting state) within tens of milliseconds (Coste et al. 2010). In genetically tagged mice, Piezo2 mechanoreceptor channels were shown to be expressed in PNECs in the NEB ME (Nonomura et al. 2017), which could also be visualised using immunohistochemistry (own unpublished observations). Developmental lineage tracing also revealed localisation of Piezo2 in nerve fibres innervating the pulmonary NEB ME (Nonomura et al. 2017). In Piezo2 knock-out mice, the vagal nerve response to inflation was abolished, indicating that Piezo2 functions as an essential stretch sensor in pulmonary vagal afferents (Nonomura et al. 2017).

Fig. 4.1 (continued) of the airway; E: epithelium. (B) Representative time courses of changes in Fluo-4 fluorescence intensity measured in NEB cells, surrounding CLCs, and control epithelial cells in 4-Di-2-ASP-stained lung slices during stimulation with a hypo-osmotic solution (230 mOsm; 30s). (a) Traces obtained from the ROIs marked in panel b. NEB cells (ROI 1) respond to application of 230 mOsm with an increase in Fluo-4 fluorescence intensity, and a typical delayed activation of the CLCs (ROI 2 and 3), while other epithelial cells (ROI 4) remain unaffected. (b–e) Fluorescence images showing (b) the 4-Di-2-ASP image (NEB cell staining; red fluorescence in ROI 1), and (c–e) time-lapse images of Fluo-4 in the NEB, surrounding CLCs and nearby control epithelial cells at different time points, before (T1) and during (T2, T3) administration of the hypoosmotic solution (Adapted with permission of the American Thoracic Society.Copyright © 2020 American Thoracic Society. All rights reserved. Cite: Robrecht Lembrechts et al. 2012/ Neuroepithelial Bodies as Mechanotransducers in the Intrapulmonary Airway EpitheliumInvolvement of TRPC5/ Am J Respir Cell Mol Biol Vol 47, pp 315–323. The American Journal of Respiratory Cell and Molecular Biology is an official journal of the American Thoracic Society. Readers are encouraged to read the entire article for the correct context at https://www.atsjournals. org/doi/full/10.1165/rcmb.2012-0068OC. The authors, editors, and The American Thoracic Society are not responsible for errors or omissions in adaptations). (C) Single confocal optical section of a pulmonary NEB in a mouse lung, immunostained for TRPC5 (red, fluorescence) and for synaptic vesicle protein 2 (SV2; green fluorescence), a pan-neuronal and neuroendocrine marker. (a) The red channel reveals a group of epithelial cells in an intrapulmonary airway that express TRPC5 exclusively on their apical plasma membrane (arrowheads). (b) Combination of the red and green channels shows that TRPC5 expression is invariably located in the apical membrane of NEB cells. E: airway epithelium; L: lumen of the airway

4.1 Mechanosensing in the NEB ME

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Both Piezo2 (Nonomura et al. 2017) and TRAAK channels (Lembrechts et al. 2011) have been suggested to be involved in the Hering–Breuer reflex (Hering 1868), which was recorded in mice (Zhang et al. 2006; Nonomura et al. 2017). When during inspiration a certain lung volume threshold is reached, an inspiratory off-switch is induced, resulting in termination of inspiration and the initiation of expiration. Both Piezo2, a major stretch sensor (Nonomura et al. 2017), and TRAAK, an inhibitory stretch-activated channel, appear to be expressed on nerve terminals in the NEB ME. Together with elegantly combined ablation and optogenetic activation, it has been suggested that these vagal afferents, and (by extension) the targeted NEB ME may be important in this reflex mechanism (Zhang et al. 2006; Nonomura et al. 2017). Although it is clear that the sensory TRAAK- and Piezo2-expressing vagal afferents connected to NEBs are capable of carrying out functions of their own (the ‘physical’ hypothesis; Taylor-Clark and Undem 2006), their close association with specialised groups of excitable neuroendocrine cells is highly suggestive of more complex signal transduction pathways. According to the ‘chemical’ hypothesis of mechanotransduction (Taylor-Clark and Undem 2006), NEB cells should be able to respond to mechanical stimuli, thereby secreting (chemical) substances that would be able to activate their respective molecular receptors on myelinated vagal sensory terminals (Lembrechts et al. 2012). Using freshly isolated PNECs from rabbit foetal lungs and a PNEC-related tumour cell line, Pan and colleagues showed that PNECs release 5-HT via mechanosensitive channels (Pan et al. 2006). Although the precise mechanism is still unclear, it was presumed that the stretch-induced 5-HT release from PNECs/ NEBs is independent of exocytosis of DCVs but rather originates from their cytoplasmic pool (Pan et al. 2006). The presence of Piezo-2 on PNECs (Nonomura et al. 2017), on the other hand, is reminiscent of the mechanism uncovered in enterochromaffin cells in the intestine, where Piezo2-dependent mechano-sensation results in intracellular Ca2+ increase, and subsequent 5-HT release (Wang et al. 2017; Alcaino et al. 2018). Ca2+-dependent activation (De Proost et al. 2008, 2009) and subsequent potential neurotransmitter release (De Proost et al. 2009) of PNECs in the pulmonary NEB ME can be directly visualised using the optimised ex vivo lung slice model for confocal LCI of the NEB ME (see Sect. 3.4). To explore physiologically relevant mechanosensory reflexes carried out by the NEB ME, the well-known and technically easy method to mimic ‘mechanical’ stimulation, namely the use of hypoosmotic solutions, was applied at the level of the lung slice (Lembrechts et al. 2012). Extracellular application of hypo-osmotic solutions causes osmotic water influx across the plasma membrane in most cell types, and consequently induces cell swelling, which in turn increases membrane tension and activates force-sensitive ion channels (Gomis et al. 2008). Since hypo-osmotic solutions permit simultaneous activation of many cells, this method has often been applied in the past to examine mechanosensitivity of potential ‘mechanoreceptor’ cells (Cunningham et al. 1995; Viana et al. 2001; Gomis et al. 2008; Haeberle et al. 2008). Swelling of airway epithelial cells in a lung slice model implies that for an individual cell ‘mechanical’

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stimulation due to extracellular hypo-osmosis is derived both from membrane deformation by its own cell swelling, and from cell compression caused by swelling of neighbouring epithelial cells. Therefore, hypo-osmotic solutions are used to reproduce the ‘compressive stress’ that is induced in the airway wall during in vivo inspiration. Progressive hypo-osmotic stimulation (below 200 mOsmol) led to an [Ca2+]i rise is induced in most airway epithelial cells. Milder hypo-osmotic stimulation (230 mOsmol; 30s), however, resulted in a clear, reversible, and reproducible Ca2 + -mediated activation of NEB cells only (Fig. 4.1B) (Lembrechts et al. 2012). Because the [Ca2+]i rise was completely abolished in the absence of extracellular free Ca2+, it was assumed that the ion channel responsible for osmomechanical activation of NEB cells should show mechano-gated Ca2+ permeability (Lembrechts et al. 2012). The best characterised channels that are permeable for Ca2+ after mechanical activation, and thus allow for a fast rise in [Ca2+]i, are members of the transient receptor potential (TRP) channel family (Beech 2007; Harteneck and Reiter 2007; Gomis et al. 2008; Lorenzo et al. 2008). Immunocytochemical staining showed that NEB cells selectively express the Ca2+ permeable osmo-/mechanosensitive ion channel TRPC5 on their apical membrane (Fig. 4.1C). In contrast to vagal mechanosensory nodose nerve terminals in the aortic arch area (Glazebrook et al. 2005), NEB-related vagal mechanosensory nerve terminals did not express TRPC5. Experimental evidence for the functional expression of TRPC5 in NEB cells was further provided using the spider peptide toxin GsMTx-4, a well-illustrated selective blocker of mechanosensitive ion channels, including TRPC5 (Suchyna et al. 2000; Oswald et al. 2002; Bowman et al. 2007). Hypo-osmotic stimulation of NEB cells in lung slices in the presence of GsMTx-4 led to strong inhibition of the osmomechanical activation of NEB cells, supporting the notion that TRPC5 is responsible for the cytoplasmic Ca2+ rise in NEB cells after hypo-osmotic stimulation (Lembrechts et al. 2012). The latter hypothesis was strengthened using SKF96365, a known blocker of several Ca2+ entry channels, including the group of TRPC channels and TRPC5 (Okada et al. 1998; Beech 2007; Ding et al. 2011). Hypo-osmotic activation of TRPC5 expressed by NEB cells appeared to be irreversibly blocked by SKF96365. Since TRPC5 can be transiently activated by shortterm external challenge with lanthanides (Zeng et al. 2004; Beech 2007), the observation that application of gadolinium (Gd3+) resulted in a quick and reversible Ca2+ mediated activation of NEB cells, provided further evidence for the hypothesis that TRPC5 is the channel involved in osmo-/mechanosensing in NEB cells (Lembrechts et al. 2012). In addition to the hypo-osmotically induced [Ca2+]i rise in NEB cells, a delayed increase in [Ca2+]i was also observed in CLCs adjacent to stimulated NEB cells (Fig. 4.1B), and was shown to be the result of Ca2+-mediated ATP release from NEB cells and activation of P2Y2 ATP receptors on CLCs (Lembrechts et al. 2012). As such, it was illustrated for the first time that NEB cells can be directly activated by a physiological hypo-osmotic stimulus, including opening of the mechanosensitive Ca2+ channel TRPC5, subsequent [Ca2+]i increase, and Ca2+-dependent release of

4.2 Chemosensing in the NEB ME

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ATP (Lembrechts et al. 2012). Apart from the Ca2+-dependent P2Y2 ATP receptor mediated activation of CLCs, it is clear that released ATP may also interact with the P2X2/3 ATP receptors expressed on the massive intraepithelial terminals of myelinated vagal afferents that surround NEB cells in the mouse pulmonary NEB ME (Brouns et al. 2009a). In conclusion, PNECs in the NEB ME are fully equipped to initiate mechanosensory signal transduction from the airway epithelium to the CNS via the vagal nerve. Purinergic signalling mechanisms have been implicated in mechanosensory transduction in several other visceral organs, relying on ATP receptors and ATP release upon mechanical epithelial distortion (Burnstock 1999, 2000, 2001a, b, c, 2006; Burnstock et al. 2012). These observations strongly support the view that also in the pulmonary NEB ME mechanotransduction acts via purinergic signalling pathways (Adriaensen and Timmermans 2004; Burnstock et al. 2012). Together, these data provide vital evidence that the NEB ME accomplishes indeed a role as a complex mechanosensory receptor end-organ in intrapulmonary airways, as has been suggested for many years (earliest hypotheses: Lauweryns and Peuskens 1972; Wasano and Yamamoto 1978; Borges et al. 1997). When NEBs are mechanically stimulated by increasing ‘pressure’ in the airway epithelium via acute mild hypo-osmotic stimulation, the mechano-gated Ca2+ permeable ion channel TRPC5, expressed on the apical membrane of NEB cells, is opened, resulting in an increase in [Ca2+]i in NEB cells. The Ca2+-dependent activation of NEB cells subsequently evokes exocytosis of neurotransmitters, as shown for ATP. The released ATP is then able to activate P2X2/3 ATP receptors present on the closely associated terminals of a subpopulation of myelinated vagal sensory nerve fibres. In this way, mechanical information primary sensed by NEB cells can be transduced to the CNS via myelinated vagal nerve fibres and a purinergic signalling pathway. The mechanosensitive K2P channel TRAAK and the Piezo2 stretch channels expressed on myelinated vagal sensory nerve terminals in the airways are strongly believed to play a regulatory role in the transmission of mechanical information through these fibres. It may be concluded that the NEB ME should be seen as an airway mechano-transducer unit, and, therefore, as the morphological counterpart of at least subpopulations of the electrophysiologically characterised SAR and RAR airway mechanoreceptors (Mazzone and Undem 2016).

4.2

Chemosensing in the NEB ME

Since the 1940s, the clustered PNECs identified by Fröhlich have been suggested to function as chemoreceptors (Fröhlich 1949), a feature attributed to them that was merely based on their location in the airway epithelium and their connection to nerve terminals (for review, see Linnoila 2006).

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Shielded by CLCs, and with processes of NEB cells in contact with the airway lumen, NEBs can indeed be regarded as excellent candidates to register alterations in local environmental conditions and transmit this information to the CNS (Adriaensen et al. 2003, 2006; Brouns et al. 2009b, 2012). Attempts to identify the potential chemical stimuli are found throughout literature from the 70s onwards and range from cigarette smoke (Lauweryns et al. 1977) to olfactory activation (Gu et al. 2014), with oxygen sensing certainly being the most extensively explored stimulus (see Sect. 4.2.1).

4.2.1

Oxygen Sensing in the NEB ME

The by far most frequently proposed and extensively illustrated function of NEB cells is their ability to sense hypoxia in the airway lumen (for review, see Youngson et al. 1993; Linnoila 2006; Cutz et al. 2009a; Domnik and Cutz 2011; Cutz et al. 2013; Cutz 2015). Neatly embedded in the airway epithelium, with surface specialisations that contact the lumen, pulmonary NEB cells are ideally located to register potential changes in the airway gas composition (Cutz et al. 2013). The oxygen-sensing capacity of NEBs is originally based on morphological studies from the 1970s on neonatal rabbits, in which Lauweryns and co-workers showed that after hypoxia, but not hypoxaemia (Lauweryns et al. 1978), NEB cells show an increased exocytosis of DCVs at their basal pole (as judged by TEM-images), and have a decreased cytoplasmic amine fluorescence (due to serotonin reduction) (Lauweryns and Cokelaere 1973; Lauweryns et al. 1973; Lauweryns and Van Lommel 1982). The hypoxia-induced secretion of serotonin from NEBs was suggested to evoke local vasoconstriction in hypoxic lung areas, shunting blood from poorly to better ventilated parts of the lungs (Lauweryns and Cokelaere 1973; Lauweryns et al. 1977, 1978; Van Lommel 2001). In NEB cells (Youngson et al. 1993; Fu et al. 1999) and in the immortalised SCLC cell line H146 (O’Kelly et al. 1998; Hartness et al. 2001) acute inhibition of O2-sensitive K+ channels by hypoxia seems to be central to O2 chemosensing (López-Barneo 1994; Cutz et al. 2013). The proposed signalling cascade involves closure of background K+ channels (Fu et al. 1999; Peers and Kemp 2001; Kemp and Peers 2009), consequent membrane depolarisation and Ca2+ influx via CaV channels (De Proost et al. 2007a), eventually triggering neurotransmitter exocytosis from NEB cells (Cutz and Jackson 1999; Fu et al. 2002; Kemp et al. 2003). An important aspect of this O2-sensing model is the presence of the full array of O2 signal transduction proteins, including an O2 sensor. A considerable amount of evidence suggests that the main O2 sensor would be NADPH oxidase (Youngson et al. 1993; Wang et al. 1996; Youngson et al. 1997; Fu et al. 2000; O’Kelly et al. 2000) which consists of a catalytic membrane-bound subunit, cytochrome b558, an integral membrane heterodimer containing gp91phox and p22phox, and the regulatory subunits p47phox, p67phox, and rac2, which together constitute the functional NADPH oxidase. Evidence in support of NADPH oxidase as a principal O2 sensor

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in NEB cells stems from gene expression profiling experiments (Cutz et al. 2009b), in situ hybridisation (Wang et al. 1996), and immunohistochemical localisation of gp91phox and/or p22phox in mouse (Fu et al. 2000), neonatal rat (Cutz et al. 2009b), foetal rabbit (Youngson et al. 1993, 1997), and SCLC (Wang et al. 1996). Immunolocalisation of gp91phox (NOX2) in neonatal rat lung has been reported to be restricted to the apical surface facing the airway lumen, as would be expected for a sensor monitoring the intraluminal airway O2 concentration (Cutz et al. 2009b). In NADPH oxidase-deficient mice, NEBs failed to respond to hypoxia, whereas in control wild-type mice this response was intact (Fu et al. 2000). Although NADPH oxidase has generally been the main focus of studies on O2-sensing compounds in pulmonary NEBs (for review, see Cutz et al. 2009a; Kemp and Peers 2009), also other players may be involved in this pulmonary sensory mechanism. Electrophysiological studies on cultured NEB cells (Youngson et al. 1993), PNEC cell lines (O’Kelly et al. 2000), and NEB cells in situ (Fu et al. 1999), in addition to fluorescence assays with dihydro-rhodamine 123 as a probe to detect H2O2 generation (Youngson et al. 1993; Wang et al. 1996), support the hypothesis that the O2 concentration of inhaled air is continuously sensed by NADPH oxidase, which converts molecular O2 into O2 and H2O2, the dismuted by-product of O2. The involvement and nature of O2 radical-sensitive K+ channels have been (intensively) investigated in PNEC/SCLC cell lines (O’Kelly et al. 1998; Hartness et al. 2001), NEB cultures (Wang et al. 1996), and NEBs in situ (Fu et al. 2007). Functional as well as morphological studies reported that pulmonary NEB cells express Kv3.3a (rabbit, rat) (Wang et al. 1996; Cutz et al. 2003), Kv3.4, and KV4.3 (neonatal rabbit) (Fu et al. 2007), all potentially O2-sensitive K+ channels. These data confirm the findings of Youngson et al. (1993), who implicated the involvement of functional O2-sensitive channels in NEB hypoxia sensing three decades ago. While KV4.3 IR is found in the apical membrane of NEBs in rabbit neonatal lungs (Fu et al. 2007), it is apparently lacking in human NEBs, indicative of possible species differences (Cutz et al. 2009b). Experiments using PNEC/SCLC cell lines suggested either hTASK3 or the heterodimer hTASK1/hTASK3, acid sensitive members of K2P channel family, as putative NADPH oxidase-associated K+ channels (Hartness et al. 2001; Kemp et al. 2003). The latter is supported by the fact that K2P channels represent background K+ channels that influence the resting membrane potential (Kim 2005) and by the observation that both hTASK1 (Lewis et al. 2001) and hTASK3 (Lewis et al. 2002) are O2-sensitive when expressed in HEK293 cells. RT-PCR gene profiling of laser-captured human NEBs also revealed TASK1-3 expression (Cutz et al. 2009b). The hypoxia-mediated closure of O2 radical-sensitive K+ channels has been suggested, without direct evidence, to induce changes in membrane potential that are sufficient to cause depolarisation, which in turn leads to opening of Cav channels, followed by an influx of extracellular Ca2+. The increase in [Ca2+]i would then trigger exocytosis of neurotransmitters (Youngson et al. 1993; Cutz and Jackson 1999). Evidence obtained from NEBs in situ suggests that hypoxia evokes the release of serotonin and ATP (Fu et al. 2002, 2004), whereas preliminary

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experiments using co-cultures of PNECs and nodose neurons suggest that 5-HT, ACh, and ATP are neurotransmitters involved in signalling hypoxia in the NEB ME (Cutz et al. 2013). To measure the influence of acute changes in partial O2 pressure (pO2) on the NEB ME, Fu and co-workers (2000) performed patch-clamp experiments on intact NEBs identified by neutral red staining in fresh lung slices from wild-type and NADPH oxidase-deficient mice. NEB cells in wild-type mice showed a reversible inhibition of both Ca2+-independent and Ca2+-dependent K+ currents upon exposure to hypoxia. In contrast, hypoxia had no effect on K+ current in NEB cells of NADPH-oxidase-deficient mice, even though both K+ current components were expressed. Evidence for the stimulation of NEBs by acute hypoxia, and for the resulting transmitter release, was provided by a study that used a fresh slice preparation of neonatal rabbit lungs and carbon fibre amperometry (Fu et al. 2002). The latter method is able to detect in real-time the quantal release of oxidisable molecules, such as 5-HT, and provided evidence for a dose-dependent hypoxic release of 5-HT from NEB cells within the physiological range expected in the airways (i.e. pO2 < 95 mmHg;  12% O2). The effects of chronic hypoxia on NEBs have been examined in animals at high altitude, in hypobaric chambers to simulate hypobaric hypoxia, or after exposure to normobaric hypoxia in a chamber with diminished O2 concentration by mixing air with nitrogen. The findings of these studies were rather diverse, likely due to high variations in experimental design and species differences, but the overall consensus was that chronic hypoxia leads to increases in the number of NEBs, the number of cells per NEB (Pack et al. 1986), and/or the amount of releasable bioactive substances (Cutz 1997). In Wistar rats exposed to hypoxic conditions for 1–3 weeks, NEBs showed elevated levels of CGRP, without a change in CGRP mRNA levels or NEB cell numbers (McBride et al. 1990; Roncalli et al. 1993). Furthermore, the number of NEB cells with a detectable immunoreactivity for CGRP is augmented after chronic or intermittent hypoxia (McBride et al. 1990; Springall and Polak 1993; Sorhaug et al. 2008). Several other studies described that (chronic) hypoxia may actually lead to inhibition of the release of NEB cell products (Springall and Polak 1993; Helset et al. 1995; Springall and Polak 1997; Sorhaug et al. 2008), resulting in a decrease in neuroendocrine secretory products (e.g. CGRP, bombesin-like peptides) in perfusate from isolated blood-perfused lungs (Helset et al. 1995; Sorhaug et al. 2008).

4.2.2

Activation of the NEB ME by Cigarette Smoke and Nicotine

Based on the strong aetiologic association of PNECs and SCLC (see Sect. 4.3.2), and on SCLC and smoking (Cook et al. 1993) (for review, see Schuller 2019), the potential effects of cigarette smoke components/nicotine on PNECs have been

4.2 Chemosensing in the NEB ME

41

extensively explored (Schuller et al. 1988; Tabassian et al. 1988; Schuller et al. 1990; Aguayo 1993; Schuller et al. 2003). Normal and neoplastic PNECs express α7 nicotinic acetylcholine receptors (α7 nAChR), as demonstrated in hamsters (Plummer et al. 2000), lungs of foetal rhesus monkeys (Sekhon et al. 1999), and in several SCLC cell lines (for reviews, see Schuller et al. 2003; Plummer et al. 2005; Schuller 2019). Upon binding of an agonist to α7 nAChR, the ion channels open, resulting in influx of extracellular Ca2+, membrane depolarisation, and opening of voltage-gated Ca2+-channels (Sher et al. 1998; Sheppard et al. 2000). The resulting increase in intracellular Ca2+ triggers the release of autocrine growth factors, such as 5-HT (Cattaneo et al. 1993) and neuropeptides (bombesin), in this way mediating proliferation of PNECs and SCLC cells. In a neonatal rabbit model, ultrastructural analysis of NEBs demonstrated increased exocytosis of DCVs after intratracheal nicotine challenge (Lauweryns et al. 1977). Agonists for the α7 nAChR include nicotine and the tobacco-specific carcinogenic nitrosamine NNK. It has been shown that in vitro, nicotine and NNK activate the Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc (Jull et al. 2001). Because of the frequently observed overexpression of myc family genes in SCLC (Wistuba et al. 2001), it has been suggested that this intracellular α7 nAChR signalling pathway is chronically activated in smokers and may underlie the obvious association between smoking and SCLC (for review, see Schuller 2019). However, a direct causal relationship has not yet been demonstrated.

4.2.3

Sensing Extracellular Ca2+ in the NEB ME

The ‘extracellular Ca2+’-sensing receptor (CaSR) is a cell-surface protein, belonging to the G protein coupled receptor superfamily (GPCRs) that can be activated by many di-, tri-, and polyvalent cations (for review, see Riccardi and Kemp 2012). Furthermore, CaSR function can also be modulated by several other physiologically relevant stimuli, including ionic strength, extracellular pH, L-aromatic amino acids, and naturally occurring polyamines such as spermine (Riccardi et al. 2009; Riccardi and Kemp 2012; Conigrave and Ward 2013; Gerbino and Colella 2018; ChavezAbiega et al. 2020). It is, therefore, not surprising that, apart from its best-documented role in systemic homeostasis of the extracellular calcium concentration [Ca2+]o (for review, see Conigrave 2016; Hannan et al. 2016), the CaSR has also been reported to be expressed in several other specialised cells and tissues (for review, see Riccardi et al. 2009; Riccardi and Kemp 2012) in which the receptor acts as a multimodal sensor that can integrate inputs deriving from changes in divalent cation concentrations with a variety of different physiological stimuli, including ionic strength, pH, and redox status. One of these tissues is the lung epithelium. In foetal mice (Finney et al. 2008) and in human lungs (Brennan et al. 2016), CaSR expression in airway epithelium

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4 Functional Exploration of the Pulmonary NEB ME

appeared to be confined to embryonic days (ED) 11.5 to 16.5 (mouse) and weeks 9 to 16 (human), respectively. It was found that CaSR activation in the developing lung ensures that the fluid secretion rate matches the extent of branching morphogenesis (Riccardi and Kemp 2012). The CaSR was also detected in airway smooth muscle and implicated in inflammation and hyperresponsiveness in allergic asthma (Yarova et al. 2015). Because CaSR has been reported in several members of the diffuse neuroendocrine system, such as parathyroid cells (Conigrave 2016), endocrine pancreatic cells (Gray et al. 2006), intestinal cholecystokinin secreting cells (Wang et al. 2011), and in a subset of innervated sensory epithelial cells in taste buds (San Gabriel et al. 2009; Bystrova et al. 2010; Brennan et al. 2014), the functional expression and physiological impact of the CaSR in PNECs of the pulmonary NEB ME were also explored. Laser microdissection of the NEB ME from lung cryostat sections of postnatal mice, and subsequent RT-PCR showed CaSR expression, while control airway epithelium was negative (Lembrechts et al. 2013). Immunostaining in postnatal mouse lungs showed that, in the airway epithelium, CaSR expression is restricted to the plasma membrane of NEB cells (Fig. 4.2A) (Lembrechts et al. 2013). In foetal mouse lungs, a general CaSR expression has been reported in airway epithelium (see higher) and was shown to be important in developmental airway branching (Finney et al. 2008). The observation that the CaSR is absent from NEBs during the pre- and perinatal period (Lembrechts et al. 2013), but is detected on the plasma membrane of NEB cells of postnatal mice (Lembrechts et al. 2013), implicates an exclusive role for the CaSR in NEB cell physiology after birth. Also in adult human lungs, NEBs could be shown to express the CaSR (Fig. 4.2B). Acute exposure of calcium indicator-loaded lung slices to several compounds known to activate or modulate the CaSR—e.g. cations such as Ca2+ (Fig. 4.2C), La3 + , the polyamine spermine, and the calcimimetic NPS-R568 (Fig. 4.2D)—was shown to induce a Ca2+-mediated activation of NEB cells in postnatal mouse lungs (Lembrechts et al. 2013). Since NEB cell activation was consistently inhibited by the selective CaSR blocker Calhex-231 (Lembrechts et al. 2013), it could be concluded that the observed reactions are CaSR-mediated. Activation of CaSR expressed on NEB cells was shown to also result in a delayed [Ca2+]i rise in CLCs, indicating that CaSR activation in NEB cells leads to the Ca2+-mediated release of ATP (Lembrechts et al. 2013). In various cell types (e.g. smooth muscle cells, breast cancer cells, ventricular cardiomyocytes), CaSR activation has been reported to mediate Ca2+ entry via TRPC-encoded receptor- and store-operated channels (El Hiani et al. 2009; Sun et al. 2010; Chow et al. 2011). In the airway epithelium, NEB cells selectively express the osmo-/mechanosensitive TRPC5 channel in their apical membrane (Lembrechts et al. 2012). The general TRPC channel blocker SKF96365 was shown to inhibit the [Ca2+]i rise in NEB cells by CaSR dependent stimuli, thereby suggesting cross-talk between TRPC5 and the CaSR (Lembrechts et al. 2013).

4.2 Chemosensing in the NEB ME

43

CaSR

CaSR GAD67-GFP

L

L

E

E 10 μm

50 μm

Aa

Ab

CaSR

GRP

Ba

Bb

5 mM Ca2+

7

100 μM NPS-R568

6

NEB (average of 5 NEB cells)

5

Clara-like cell

4 3 10s 2

10s

1 0

C

D

Fig. 4.2 (A) Single confocal optical section immunostained for the CaSR (red fluorescence) of a pulmonary NEB (green GFP fluorescence) in a postnatal (PD14) GAD67-GFP mouse lung cryosection. (a) Group of intraepithelial cells showing plasma membrane expression of the CaSR (arrowheads). (b) Combination of the red and green channels revealing selective expression of the CaSR on NEB cells (arrowheads) in the airway epithelium (E) (arrowheads). L: lumen of an airway. (B) Consecutive paraffin sections of adult human lung, immunostained (brown stain) for the CaSR (in a) and gastrin-releasing peptide (GRP in b; as a marker for pulmonary NEBs). Note CaSR expression in the NEB (arrows in a and b) in the epithelium of a terminal bronchiole. Nuclei are counterstained with haematoxylin (purple-blue colour). (C) Graph plotting the changes in Fluo-4 fluorescence intensity (ΔFluo-4) measured in a pulmonary NEB in a 4-Di-2-ASP-stained live WT mouse lung slice that was loaded with Fluo-4. During acute challenge with a solution containing a high concentration (5 mM) of extracellular Ca2+, the NEB cells react with a rise in the intracellular Ca2+ concentration ([Ca2+]i). With a short delay, a [Ca2+]i rise can also be observed in CLCs, indicative of neurotransmitter release from NEB cells. (D) Graph plotting the changes in fluo-4

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4 Functional Exploration of the Pulmonary NEB ME

Ca2+ mobilisation in one cell may produce local extracellular Ca2+ changes that can be detected by closely associated cells, sharing the same microenvironment and expressing the CaSR (for recent review, see Gerbino and Colella 2018). ATP-evoked intracellular Ca2+ signalling in CLCs, and the subsequent active extrusion of Ca2+ across the plasma membrane, causes a local [Ca2+]o increase in the NEB ME that appears to be detectable by closely associated NEB cells expressing the CaSR (Lembrechts et al. 2013). When the sensitivity of the CaSR was increased experimentally, a Ca2+-mediated activation of CLCs occasionally seemed to cause the consecutive activation of neighbouring NEB cells, as such lending further support to the hypothesis (Lembrechts et al. 2013). In addition to revealing a purinergic paracrine signalling mechanism, by which activated NEB cells release ATP that activates CLCs (De Proost et al. 2009), these data also point to the possibility of bilateral communication between these two closely associated endocrine and stem-like cells within the NEB ME (Lembrechts et al. 2013). In many species, including humans, NEBs express CGRP (Uddman et al. 1985; Johnson and Wobken 1987; Brouns et al. 2009a) and calcitonin (Cutz et al. 1981; Luts et al. 1991). As a secretory product of thyroid C-cells, calcitonin is an important mediator in thyroid/parathyroid control of [Ca2+]o homeostasis, in which the CaSR is the key [Ca2+]o sensor (Garrett et al. 1995; Freichel et al. 1996). Literature data indicate that the effects of thyroid and parathyroid gland removal on Ca2+ homeostasis and plasma calcitonin levels are limited, that lungs seem to represent an important secondary source of calcitonin, and that the number of pulmonary endocrine cells is upregulated (Becker et al. 1980; Becker and Silva 1981; Kasacka et al. 2001). It is, therefore, not unlikely that NEBs, which express both [Ca2+]o sensing and regulating proteins, besides their involvement in multiple mechanisms regulating both [Ca2+]o and [Ca2+]i homeostasis and sensing in NEB ME physiology, may also serve as a potential back-up system for [Ca2+]o homeostasis in case of thyroid dysfunction. Keeping in mind that the CaSR is generally accepted as a polymodal sensor for a broad range of physiologically relevant extracellular molecules, the functional plasma membrane expression of the CaSR in a complex sensory receptor unit such as the NEB ME, with both direct access to the environment and extensive central connections, may also allow sensing/integrating changes in the concentration of a variety of chemical substances in the NEB ME and transmission of information to the CNS.

Fig. 4.2 (continued) fluorescence intensity (ΔFluo-4) measured in a pulmonary NEB challenged with a solution containing the calcimimetic NPS-R568 (100 μM), a CaSR agonist. The experiment shows activation of the NEB cells, visualised by a rise in [Ca2+]i , and the subsequent activation of CLCs

4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

45

Hypercapnia and H+ Sensing

4.2.4

In search for evidence of a polymodal airway chemosensory function of the NEB ME, attempts have been made to explore the reaction of NEB cells to CO2/H+ stimuli for already more than 40 years. In the 1970s, it was described that NEBs in neonatal rabbit lungs produced an increased amine fluorescence and exocytosis of DCVs after changes in pCO2 (Lauweryns et al. 1977), an observation that was challenged in a later study (Lauweryns et al. 1990). In NEB cell-derived tumour cell lines, an elevated CO2 level was found to evoke a significant effect on 5-HT release from PNECs (Schuller 1994). In organ cultures of whole foetal rat lungs, H+/HCO3 ions appeared to modulate CGRP secretion, indicative of NEB involvement in this reaction (Ebina et al. 1997). A few years ago, Livermore and colleagues (Livermore et al. 2015), in a study using carbon fibre amperometry and calcium imaging in a hamster lung slice model, provided evidence that hypercapnia (10%CO2) and acidosis (pH 6.8-7) elicit 5-HT release from NEBs. Since the effect was abolished by acetazolamide, an inhibitor of carbonic anhydrase (CA), and by blocking voltage-gated Ca2+-channels, it was concluded that H+/HCO3- sensing is dependent on carbonic anhydrase-dependent voltage-gated entry of extracellular Ca2+. Carbonic anhydrases are zinc-containing metalloenzymes that catalyse the reversible hydration of CO2 and participate in various biological processes. mRNAs for several CA isozymes were detected in extracts of NEB cells isolated from human lung by laser capture microdissection (Livermore et al. 2015), and the CAII protein was visualised in NEB cells of both human and hamster lungs by immunohistochemistry (Livermore et al. 2015). NEB cells, therefore, indeed may be seen as polymodal sensors of airway gas concentrations (pO2/pCO2).

4.3

The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

4.3.1

The NEB ME During Lung Development and After Airway Injury

4.3.1.1

Ontogenetic Development and Importance of the NEB ME

Lung development, including growth and differentiation, is a complex process involving epithelial–epithelial or epithelial–mesenchymal interactions, functional cross-talk between different cell types and the sequential action of regulatory factors such as hormones, growth and diffusible factors, extracellular matrix components, etc. (for reviews, see Montuenga et al. 2003; Whitsett et al. 2019).

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4 Functional Exploration of the Pulmonary NEB ME

In the primitive airway epithelium, PNECs are the first cells to become recognisably differentiated (i.e. 6th week of gestation in humans (Cutz et al. 1985; Gosney 1993); ED12.5 in mice for solitary PNECs and ED13.5 for PNEC clusters (Song et al. 2012; Kuo and Krasnow 2015)) (for review, see Cutz 2015). PNEC clusters are formed in a proximal to distal centrifugal pattern (Hoyt et al. 1990; Wuenschell et al. 1996; Avadhanam et al. 1997; Sorokin et al. 1997; Noguchi et al. 2015), in which cell migration may play an important role in cluster formation (Kuo and Krasnow 2015; Noguchi et al. 2015). The establishment of any cell phenotype requires the activation or repression of differentiation-specific genes by transcription factors. Several ‘neurogenic’ genes belonging to the family of basic helix-loop-helix (bHLH) transcription factors have been identified to be involved in the ontogeny and differentiation of PNEC/NEBs (for review, see Ito et al. 2001; Cutz 2015). The activation of mammalian achaete– scute complex homologue-1 (Mash1; also termed Ascl1) in mice, and its human counterpart (Hash1), appeared to be essential for PNEC development (Borges et al. 1997; Ito et al. 2000) (for review, see Ito et al. 2001; Linnoila 2006), as supported by the observation that no PNECs can be detected in Mash1-deficient mice (Borges et al. 1997; Sui et al. 2018). Immunostaining of mouse lungs revealed that Mash1 is expressed in nuclei of PNECs from ED13.5 onwards (Borges et al. 1997; Guha et al. 2012). It has also been shown that the hairy and enhancer of Split-1 (Hes1) transcriptional repressors play an opposite role in the differentiation of PNECs (Ito et al. 2000; Morimoto et al. 2012), due to targeted silencing of Mash1 (Ito et al. 2000; Ball 2004). Enhanced differentiation of PNECs was demonstrated in Hes1deficient mice, by the precocious appearance of PNECs at ED13 and relative hyperplasia at ED18. Histologically, the lung architecture appears normal, except for nodular lesions that consist of PNECs (for review, see Ito et al. 2001). The Hes1-mediated restriction of neuroendocrine cell differentiation is facilitated by canonical Notch signalling (Ito et al. 2000; Collins et al. 2004; Shan et al. 2007; Tsao et al. 2009; Kiyokawa and Morimoto 2020). Notch signalling is a highly conserved cell–cell signalling pathway that is ideally suited for very short-range cellular communication, because it is transmitted to adjacent cells through direct contact (for review, see Kiyokawa and Morimoto 2020). The mammalian genome encodes four transmembrane Notch receptors (named Notch 1-4) and at least 5 ligands (Jagged1,2 and Delta-like ligand (Dll) 1,3,4). Direct interaction between a ligand-expressing cell and a receptor-expressing cell triggers two successive proteolytic cleavages in the receptor-expressing cell, mediated by the γ-secretase complex and triggers the release of the Notch intracellular domain (NICD). Upon its release, NICD enters the nucleus where it interacts with a transcription factor of the CSL family (RBPjk/CBF-1 in mammals) to activate transcription of target genes. Among the best known targets of Notch/RBPjk signalling are the Hes/Hey family of bHLH transcription repressors, which, in turn, repress expression of downstream neural/neuroendocrine cell genes (Lewis 1998; Kiyokawa and Morimoto 2020). Efforts have been made to document Notch signalling in developing mouse lungs, including in the NEB ME (for review, see Kiyokawa and Morimoto 2020). Most findings are based on observations of the NEB ME after selective/conditional

4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

47

gene deletion. Deleting Notch1; Notch2; Notch 3 (Morimoto et al. 2012) or modification by altering their modulatory enzymes (Pofut1: Tsao et al. 2009; Lnfg Xu et al. 2010) resulted in the expansion of PNEC clusters. Mutation in the DNA-binding domain Rbpjk (Tsao et al. 2009), or in the Notch-dependent repressor Hes1 (Ito et al. 2000; Morimoto et al. 2012; Noguchi et al. 2015), equally led to expanded PNEC clusters at later stages in development. Deletion of Notch-ligands revealed that only Delta-like ligands (Dll)—and not Jags—are important in PNEC expansion (Stupnikov et al. 2019). Conversely, reduced neuroendocrine cell numbers have been reported in transgenic mice expressing activated Notch1 driven by the calcitonin (Cgrp; Calca) promoter (Shan et al. 2007). The current vision is that Mash1-expressing PNEC precursors differentiate from the primitive airway epithelium around ~ED12.5-13 in mice (Li and Linnoila 2012; Kuo and Krasnow 2015; Noguchi et al. 2015). Since Mash1 is known to regulate Dll expression, the Notch ligands Dll1 and Dll4 appear in the PNEC precursors on ED13.5 (Stupnikov et al. 2019). Dll expression then activates Dll-Notch signalling in adjacent cells (Stupnikov et al. 2019), leading to the formation of NEB-associated CLC progenitors around ED14.5 (Guha et al. 2012; Morimoto et al. 2012). Immunostaining for NICD1 underpins that the Notch pathway is activated in the CLC progenitors (Guha et al. 2012; Morimoto et al. 2012). This local activation of Notch signalling shields the NEB ME from the neighbouring epithelium, preventing aberrant NEB expansion (Stupnikov et al. 2019). At least in early development, the close interaction between PNECs and adjacent CLCs supports the notion that the pulmonary NEB ME serves as a niche for CLCs (Guha et al. 2012). Since NEBs are claimed to be prominently present in prenatal lungs, they are believed to be involved in airway growth and development, during which they may regulate branching morphogenesis, cellular growth and maturation (for reviews, see Sorokin et al. 1997; Bishop 2004; Linnoila 2006). In impaired lung development, NEBs and solitary PNECs often show strong hypertrophy and an enhanced secretion of bioactive substances (Van Lommel 2001). The increased number of PNECs has been hypothesised to be a compensatory change for the abnormal lung development (Ijsselstijn et al. 1997). These assumptions are supported by the observation that PNECs store and release different bioactive substances that can influence epithelial cell proliferation (Wharton et al. 1978; Kauffman 1980; Breuer et al. 1990; Montuenga et al. 2003; Shan et al. 2007; Sunday 2014). However, the normal branching pattern observed in MASH1-deficient mice, which lack PNECs (Ito et al. 2000; Sui et al. 2018), raises the question whether PNECs are required for primary aspects of lung development (Garg et al. 2019). A recent review suggests that there is a time overlap of lineage specification, branching morphogenesis, sacculation, and alveologenesis (Basil et al. 2020).

48

4.3.1.2

4 Functional Exploration of the Pulmonary NEB ME

Importance of the NEB ME for Epithelial Regeneration After Injury

Although studies of foetal lung development have provided fundamental insights for advancing our understanding of the NEB ME in terms of cellular and molecular aspects of (human) lung disease and cancer, additional information has been gleaned from studying regeneration of airway epithelial cells after injury. The healthy postnatal lung shows a remarkably low turnover of epithelial cells. However, upon acute damage, lung epithelium can react quickly, as a result of which different spatially controlled stem and/or progenitor cells that reside at specific anatomical loci will re-enter the cell cycle and promote repair (Kiefer 2011; Hogan et al. 2014; Stabler and Morrisey 2017; Ouadah et al. 2019). Lungs have, therefore, been classified into a group of organs that contain cells that are considered physiologically functional and fully differentiated during homeostasis, but may exhibit stem/progenitor-like activity after injury or in disease states (Leach and Morrisey 2018). To unravel the mechanisms that underlie lung regeneration, several models that induce severe experimental damage to the airway epithelium have been developed (Bertoncello and McQualter 2013; Vaughan and Chapman 2013; Wansleeben et al. 2013; Lynch and Engelhardt 2014; Stabler and Morrisey 2017). These injury models have been combined with DNA labelling techniques such as H3-thymidine labelling ((Reynolds et al. 2000a), bromo deoxyuridine (BrdU) labelling (for review, see Liu et al. 2006), or with animals models that express distinct genetically labelled cell populations to follow their migration, proliferation, and differentiation (Rock et al. 2009; Li et al. 2015; Vaughan et al. 2015; Ouadah et al. 2019). The majority of the applied injury methods tend to be drastic and included complete ablation of abundant cell populations (for reviews, see Rawlins et al. 2008; Blaisdell et al. 2009; Asselin-Labat and Filby 2012), while differentiation of certain cell types appeared to be dependent on the type and severity of the insult (Teisanu et al. 2011; Nabhan et al. 2018). To study the regenerative potency of the NEB ME, naphthalene ablation of Clara cells has been and still is a very popular method (see, for e.g., recent review Ouadah et al. 2019). Exposure of mice to this xenobiotic leads to a massive loss of Clara cells because of conversion of naphthalene to a cytotoxic product by the cytochrome P450 enzyme Cyp2f2 (Reynolds et al. 2000b). After naphthalene challenge, epithelial regeneration (in the form of nascent Clara cells) is preferentially associated with NEBs, which typically also showed hyperplasia (Stevens et al. 1997; Reynolds et al. 2000a). Since in many lung disorders (see Sect. 4.3.2) PNECs show aberrant numbers and/or morphology, several studies have focused on the way PNECs behave after naphthalene exposure. The classical view is that in postnatal lungs, PNECs are terminally differentiated and mitotically inert (Hoyt et al. 1990; Gosney 1997; Montuenga et al. 2003), but under specific conditions may have the ability to selfrenew or differentiate (Van Lommel 2001), albeit with a limited capacity (Peake

4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

49

et al. 2000; Reynolds et al. 2000a, b; Song et al. 2012). As a consequence of severe epithelial injury, PNECs may revert to their initial role as lung growth regulators, because of their potency to locally produce mitogenic factors. Recent lineage tracing studies confirmed the ability of PNECs to transdifferentiate into Clara cells and ciliated cells, after naphthalene eradication of Clara cells (Yao et al. 2018; Ouadah et al. 2019). Repeated naphthalene ablation even revealed that only a minor population of the neuroendocrine cells (named NEstem) in NEBs harbours a reserve stem cell activity, and is able to reprogram to Clara cells, ciliated cells and perhaps also other cell types that were lost during injury (Ouadah et al. 2019). Although the NEstem appear to be fully differentiated and likely function normally during homeostasis, they display features of classical stem cells upon severe airway injury. Molecular pathways that control the stem cell program include Rb/p53 which constitutively suppresses self-renewal (Ouadah et al. 2019). Deprogramming and the switch to ‘transient amplifying’ are controlled by the Notch pathway (Yao et al. 2018; Ouadah et al. 2019). In adults, the Notch pathway is quiescent in neuroendocrine cells in normal healthy homeostatic conditions (Ito et al. 2000; Morimoto et al. 2012; Guha et al. 2017; Yao et al. 2018). Building on observations that PNECs were not able to repopulate the entire airway epithelium following ablation of Clara cells (Hong et al. 2001), and that CLCs are resistant to naphthalene injury because they lack the expression of cytochrome Cyp2f2 (Stripp et al. 1995; Reynolds et al. 2000a), the possibility of CLCs as potential stem/progenitor cells in specific regions of the airways has been explored in many subsequent studies (e.g. see Guha et al. 2017). Following naphthalene ablation of Clara cells, also CLCs were found to proliferate (Reynolds et al. 2000a, b; Guha et al. 2017), and at least some proliferating cells expressing both CCSP and neuroendocrine markers could be detected in the NEB ME (Reynolds et al. 2000b; Linnoila 2006). Other studies have shown that CLCs are able to self-renew, and differentiate into Clara cells and ciliated cells (Plopper et al. 1992a, b; Giangreco et al. 2002; Chen and Fine 2016; Guha et al. 2017). At present, CLCs are, therefore, regarded as a quiescent cell population that, following severe injury, may re-enter the cell cycle and produce nascent differentiated cells. After repair, these ‘progenitor cells’ return to their original state and may be regarded as ‘facultative progenitor cells’ (for recent review, see Leach and Morrisey 2018). CLCs may, therefore, be put forward as the true ‘stem cells’ in the NEB ME (Linnoila 2006; Snyder et al. 2009; Kratz et al. 2010; Reynolds and Malkinson 2010; Roomans 2010; Sullivan et al. 2010; Glaser et al. 2012; Guha et al. 2012; Chen and Fine 2016; Leach and Morrisey 2018; Kiyokawa and Morimoto 2020). Being a source of potential stem/progenitor cells (Rawlins et al. 2008; AsselinLabat and Filby 2012; Guha et al. 2012; Li et al. 2015; Chen and Fine 2016) in which Notch signalling also plays an important role in local interactions between PNECs and CLCs (Kiyokawa and Morimoto 2020), the NEB ME is now proposed to be an important stem cell niche in the intrapulmonary airways, in which intercellular communication between stem cells and surrounding cells is crucial with regard to potential dysregulation and consequent tumour genesis (Kiefer 2011). A possible

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4 Functional Exploration of the Pulmonary NEB ME

hypothesis could be that the NEB ME behaves as a reservoir for the maintenance of a stem and/or progenitor cell type, e.g. CLCs, which can be influenced by bioactive substances that are secreted by invariably closely associated PNECs, which are additionally connected to the CNS via vagal afferents.

4.3.2

Modification of the NEB ME Related to Perinatal and Postnatal Disorders

The NEB ME seems to be implicated in a wide array of paediatric and adult lung diseases, including congenital lung disorders, disorders of respiratory control, bronchial asthma, and pulmonary hypertension (for review, see Linnoila 2006; Cutz et al. 2007a; Cutz 2015; Garg et al. 2019). Although CLCs of the NEB ME clearly have potential to be important in airway diseases, pathologies have so far more often been linked to changes in PNECs (see Table 4.1). In the diseased lungs, the pulmonary diffuse neuroendocrine system, which is normally sparse but evenly distributed throughout the respiratory tract, may be dramatically affected (for review, see Gosney 1992; Cutz et al. 1995; Cutz 1997; Gosney 1997; Linnoila 2006; Cutz et al. 2007b; Cutz 2015; Garg et al. 2019). Compared to normal healthy lungs, most of the reported disorders/diseases show increased numbers and altered arrangements of PNECs or NEBs. In a variety of congenital and acquired (paediatric) pulmonary disorders, hyperplasia of PNECs/ NEB cells appears to be a relatively common phenomenon (for review, see Cutz 2015; Garg et al. 2019). In the healthy adult human lung, the vast majority of PNECs are solitary and evenly distributed along the bronchial epithelium, while in infants and neonates a proportionally higher number appear in the form of NEBs. In literature, a distinction between responses of solitary PNECs and NEBs has not been made consistently, which may obscure structure–function correlations in diseased lungs (Linnoila 2006). During the disease progress, also the nature of the secretory products of NEBs/PNECs may be altered (e.g. GRP: Sunday 2014). Most studies of PNECs in human pulmonary disease, however, have to deal with conditions characterised or complicated by acute or chronic hypoxia, local inflammation or infectious processes. Whether the changes in PNECs are primary (¼causative) or secondary (¼consequence) remains a matter of debate, but the local production of a variety of bioactive substances by these cells is likely to play an important role in the response to injury and repair, in order to restore vital local homeostasis. Genetic mouse models indeed show that PNECs are able to elicit immune responses via neuropeptide secretion (Branchfield et al. 2016; Sui et al. 2018). The most commonly reported clinical relationship of PNECs with lung-related diseases stems from the observation that characteristic markers of PNECs are typically also expressed in human SCLC (Sung et al. 2020), leading to the speculation that the target cells of malignant transformation could be PNECs (Linnoila 2006; Sutherland et al. 2011; Song et al. 2012). SCLC accounts for 15% of all lung

Bronchopulmonary dysplasia Congenital diaphragmatic hernia (CDH) Congenital malformations of the lung Congenital central hypoventilation syndrome Congenital pneumonia Cystic fibrosis Hyaline membrane disease Interstitial pneumonia Neuroendocrine cell hyperplasia of infancy (NEHI) Pulmonary dysmaturity (Wilson-Mikity Syndrome) Pulmonary hypertension Sudden infant death syndrome

Bronchial asthma pathophysiology

Cutz et al. 2015 Johnson et al. 1982, 1985, 1993; Johnson and Wobken 1987; Hyperplasia and increased IR for neurotransmitters in PNECs Cutz 2015 (review); Sunday et al 2014 (review) PNEC hyperplasia, increased bombesin-like peptide expression Asabe et al. 1999; Ijsselstijn et al. 1997 low bombesin, high 5-HT in PNECs Cutz et al. 1995 atrophy of carotid bodies and compensatory increase in NEBs Cutz et al. 1997 ‘hyperfunction’ of PNECs Saad et al. 2003 Johnson et al 1988; Wolf et al. 1986; Cutz et al. 2015 PNEC hyperplasia after lung transplantation Decrease in PNECs Johnson et al. 1982; Ghatei et al. 1983; Johnson et al. 1985 PNECs hyperplasia Jiramethee et al. 2017, Chatterjee et al. 2016, Ito et al 2002 Deterding et al. 2005; Young et al. 2011; Guha et al. 2017 (PNECs; CLCs); Popler et al 2010 Increased number of PNECs and NEBs; NEB hyperplasia Hyperplasia of PNECs Gillan and Cutz 1993; Saad et al. 2003 ? involvement of the vasoconstrictor 5-HT or the vasodilator CGRP; Gosney et al. 1989; Shenberger et al. 1997; Heath et al 1990; hyperplasia of PNECs/NEBs Gillan et al. 1989; Perrin et al. 1991; Cutz et al. 1996, 2007, 2015; Porzionato et al. 2008

? No abnormalities, but involvement of 5-HT

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) Eosinophilic granuloma Plexogenic pulmonary arteriopathy Pneumonia Pulmonary hypertension

Neonatal/pediatric disorders

Aguayo et al. 1992; Koliakos et al. 2017; Guha et al. 2017 (PNECs; CLCs); (nodular) PNEC hyperplasia but no other per-existing lung disease review: Marchevsky and Walts 2015; review: Rossi et al. 2016 Increase number of BOM-ir PNECs Aguayo 1990 increase in PNECs Gosney et al. 1989a Allibone and Gosney 1990 Increase in PNECs Increase in GRP-positive PNECs Gosney et al. 1989a

Asthma Bronchiectasis Chronic bronchitis, emphysema Cigarette smoking Gosney et al. 1989b Aguayo 1993, 1994

References Sui et al. 2018; Stanislawski et al 1981; Gould et al. 1983;

Effects

Increase in bombesin and CGRP-positive PNECs increase in PNECs increase in PNECs increase in PNECs

Adult disorders

Table 4.1 Overview of human diseases associated with alternations in PNECs/NEBs

4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium? 51

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cancer cases and belongs to the broader class of pulmonary neuroendocrine tumours (for review, see Borczuk 2020). It is a highly aggressive form of lung cancer, characterised by a poor prognosis and rapid development of resistance to treatment, and is strongly related to cigarette smoking (for review, see Hamilton and Rath 2015). Transformation of lung cells into SCLC is promoted by the inactivation of both Trp53 and Rb1 (Meuwissen and Berns 2005; George et al. 2015). Using cell typerestricted Adeno-Cre viruses, several research groups have shown that loss of these tumour suppressors may result in the efficient transformation of PNECs, leading to SCLC after a few months (Park et al. 2011; Sutherland et al. 2011). These findings were elegantly confirmed in a CGRPCRE ER mouse model (Song et al. 2012). The recent finding of an NEstem cell population in the NEB ME has led to the hypothesis that these NEstem cells may be the prominent cell of origin of SCLC (Ouadah et al. 2019), and that tumour initiation results from the almost immediate and persistent activation of NEstem cell renewal following loss of Rb and p53 (Ouadah et al. 2019). Several neurogenic transcription factors that are important for the developmental maturation of neuroendocrine cells of the lungs are expressed in SCLC. Notably, Ascl1 contributes to pulmonary neuroendocrine tumour genesis through regulation of the atypical inhibitory Notch ligand Delta-like 3 (Dll3), which is overexpressed in more than 80% of SCLC, and plays a critical role in inhibiting Notch tumour suppressor signalling (for reviews, see Leonetti et al. 2019; Reguart et al. 2020). Notch signalling can also promote intratumoural cellular heterogeneity, in which Notch-activated SCLC cells are relatively chemoresistant and provide trophic support to neuroendocrine tumour cells (Lim et al. 2017). It has been proposed that inactivated Notch signalling is involved in an ‘epithelial-to-mesenchymal transition’-like phenotype of SCLC (Ito et al. 2017). Molecular pathogenesis involving Notch signalling, therefore, seems to be highly complex and context-dependent (for review, see Leonetti et al. 2019; Kiyokawa and Morimoto 2020). Overexpression of sonic Hedgehog (Shh) is another common developmental pathway alteration in SCLC that markedly accelerates tumour progression (for review, see Giroux-Leprieur et al. 2018). Also Wnt signalling may be involved (for review, see Lundin and Driscoll 2013; Rapp et al. 2017). Taken together, it is very likely that non-physiological triggers of the same signal transduction pathways that drive lung development (Sect. 4.3.1.1) and lung repair after injury (Sect. 4.3.1.2) are involved in the origin of lung cancer.

4.3.3

Selective Gene Expression in the Postnatal NEB ME: Special Focus on Stem Cell Characteristics

The NEB ME is nowadays seen as one of the ‘stem cell niches’ dispersed along the respiratory tract that is activated during development and in regeneration after severe epithelial injury (Asselin-Labat and Filby 2012; Wansleeben et al. 2013; Donne et al.

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2015; Li et al. 2015; Leach and Morrisey 2018), and whose potential residing ‘stem cells’ are CLCs and PNECs. Recent insights consider CLCs as cells that exhibit ‘facultative progenitor’ activity (Leach and Morrisey 2018). ‘Facultative progenitors’ are fully differentiated cells that carry out a defined physiological function during homeostasis, but may contribute to restoration of functional tissues due to their ability to re-enter the cell cycle and differentiate into a limited number of daughter cells. In many respects, the facultative stem/progenitor cell is a functionally mature cell waiting for tissue injury or disease initiation to activate its regenerative response (Leach and Morrisey 2018). The recent discovery of NEstem cells in the NEB ME (Ouadah et al. 2019) suggests that the NEB ME is even a more important residency or ‘niche’ in the airway epithelium. For easy reading, the NEB ME is referred to as a ‘stem cell niche’ throughout this section. Although every single epithelial ‘stem cell niche’ possesses unique features facilitating its specialised functionality, they share many common aspects of regulation. Several signalling pathways such as Wnt, Hedgehog (Hh), and Notch have emerged as key regulators of stem cells (Campelo et al. 2011). Next to the Notchsignalling pathway (see Sect. 4.3.1), which seems to play an important role in local interactions between PNECs and CLCs (Kiyokawa and Morimoto 2020), keeping the adult lung quiescent seems to be regulated by Hh signalling (Peng et al. 2015). Also the TGFβ/BMP and Wnt pathways harbour dynamic and reciprocal interactions during lung development, that are re-established to drive regeneration (Attisano and Wrana 2013; Volckaert and De Langhe 2015). The pathway is reactivated for repair after adult lung tissue injury (Sountoulidis et al. 2012). Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus, such as injury. Although previous studies have demonstrated that distinct embryonic developmental programs are reactivated aberrantly in adult organs to drive repair and regeneration, it is not well understood how quiescence is maintained in organs such as the lung, which displays a remarkably low level of cellular turnover. Insight into the molecular mechanisms that keep the ‘facultative progenitor’ cells quiet would require thorough investigation of the niche in healthy postnatal airways. However, the latter have not been available until recently. Our lab performed a large-scale selective gene expression analysis of NEBs aiming at unravelling unique features of molecular pathways for the NEB ME acting as a potential ‘stem cell niche’ in healthy postnatal mouse lungs. To this end, a set of commercially available PCR arrays, related to development, stem cell behaviour, and cell signalling was selected (Verckist et al. 2017), allowing access to a panel of more than 600 genes. In view of thorough comparison between the NEB ME and intrapulmonary control airway epithelium (CAE), each PCR array was performed for both a pooled NEB ME sample and a CAE sample collected from the same mouse lungs by laser microdissection (LMD). Relative gene expression differences were calculated using fold regulation differences of more than two as a threshold to define ‘higher’ or ‘lower’ gene expression levels (Verckist et al. 2017). The complete gene expression dataset (unpublished data) is added as an addendum (supplementary files S1-S5).

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Fig. 4.3 (A) Single qPCR experiments for the expression of Dll3. The mean of ‘normalised relative quantification (NRQ)’ levels of the NEB ME of 6 different mice was compared to the levels in CAE and normalized to reference gene expression. Dll3 expression levels are significantly higher ( p < 0.05 (*), using Mann–Whitney U test) in the NEB ME than in CAE (Reproduced with permission from Verckist et al. 2017). (B) (a) Dll3 immunostaining (red fluorescence) of a postnatal mouse (PD 21) lung cryosection, showing a spot-like staining (arrowheads) at distinct locations in the airway epithelium (E). (b) Combination of the red and green channels reveals that Dll3 is covering the surface membrane of a group of intraepithelial cells that can be identified as NEB cells by their CGRP IR (green fluorescence). L: airway lumen

In healthy control mice, analysis of PCR array data unravelled differential expression levels (high: Verckist et al. 2017; low: own unpublished results) for a large percentage of stem cell-related genes in the NEB ME compared to CAE, supporting the idea that the pulmonary NEB ME harbours intrinsic molecular characteristics of a postnatal stem cell niche (Verckist et al. 2017). When looking for pathways that appeared to be most heavily involved in this stem cell behaviour, the Notch-signalling pathway popped up. Delta-like ligand 3 (Dll3), as a key player of pulmonary Notch signalling (also see Sect. 4.3.1), showed the highest overexpression level in the NEB ME compared to CAE, when data were ranked in a top 20 (Verckist et al. 2017). Further quantitative qPCR analysis proved that Dll3 expression was virtually absent in the intrapulmonary CAE, but relatively high in the NEB ME (Fig. 4.3A). mRNA levels were normalised based on expression levels of an optimised panel of housekeeping genes. Co-immunostaining for CGRP, a marker for PNECs which are an integral part of the NEB ME, revealed that Dll3 protein was localised in a dotted pattern on the surface membrane of PNECs (Fig. 4.3B) (Verckist et al. 2017). This location could be expected because Dll3 is a transmembrane ligand of the Notch-signalling pathway. In general, it is known that binding of Dll3 on a Notch receptor results in downstream activation of the signalling pathway (Collins et al. 2004). It is clear that interactions between the niche and stem cells are crucial in controlling stem cell behaviour, such as the balance between self-renewal, proliferation and differentiation, and consequent tissue repair (Borok et al. 2006; Cabarcas et al. 2011; Bertoncello and McQualter 2013). Deregulation of these interactions has been demonstrated to drive tumour genesis in known stem cell niches in other organs (Kiefer 2011).

4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

4.3.4

55

Selective Activation and Proliferation of a Quiescent Stem Cell Population in the NEB ME by Transient Acute Lung Inflammation

To detect and study activation of pulmonary ‘stem cell niches’, a variety of models that use experimental damage to monitor airway epithelial repair, have been implemented (for reviews, see Bertoncello and McQualter 2013; Vaughan and Chapman 2013; Wansleeben et al. 2013; Lynch and Engelhardt 2014; Stabler and Morrisey 2017). Since most of these models require eradication of complete epithelial populations or genetic manipulation (also see Sect. 4.3.1.2), a minimally invasive model that mimics common diseases would seem more relevant to explore the functional significance of the NEB ME in healthy and diseased lungs. Models based on instillation of the bacterial endotoxin lipopolysaccharide (LPS) allow to mimic all levels of severity of inflammation-related acute lung injury (ALI) (Vernooy et al. 2001), but until recently (Verckist et al. 2018) have not been applied in NEB research. ALI is caused by lower respiratory tract infections and remains the third cause of mortality worldwide (Basil et al. 2020). Intratracheal instillation of a low dose (1 mg/kg body weight) of LPS in mouse lungs did result in an influx of neutrophils, reminiscent of airway inflammation, but the resulting injury appeared to be very mild and transient, as was proven by wholebody plethysmography and the lack of visible epithelial damage (Verckist et al. 2018). This was in accordance with literature data showing that low-dose intratracheal LPS instillation in mice results in a rapid (few hours) intrapulmonary inflammatory reaction, potentially without causing important endothelial or epithelial injury (Vernooy et al. 2001; Matute-Bello et al. 2008; Alm et al. 2010). The potential effects of soluble mediators in broncho-alveolar lavage fluid (BALF) on healthy mouse airway epithelium were evaluated using BALF collected from LPS-challenged mice, 16 hours after instillation. BALF was acutely administered to ex vivo lung slices of healthy control mice, and application was followed in live cell calcium imaging experiments. A reversible and reproducible selective calcium-mediated activation was observed in CLCs, but not in NEB cells, Clara cells, or ciliated cells (Fig. 4.4A) (Verckist et al. 2018). Although the identity of the molecule responsible for activation is so far undetermined, it is reasonable to assume that activation of CLCs is triggered by one of the soluble inflammatory mediators released by activated neutrophils/macrophages. Since in LPS-challenged mice the NEB ME is continuously exposed to BALF, the long-term exposure effect of LPS on CLCs, as a potential quiescent stem cell population, was evaluated by using LPS instillation in combination with BrdU as a marker for cells that divide during the experimental window. In healthy control mice, BrdU incorporation during the S-phase of the cell division cycle and subsequent BrdU immunostaining revealed that in a 48h time frame only very few cells were divided in the airway epithelium (Verckist et al. 2018), which is in accordance with literature data (Stabler and Morrisey 2017).

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Fig. 4.4 (A) Live cell imaging experiment in which broncho-alveolar lavage fluid (BALF) collected from an LPS-challenged mouse, was applied to a live vibratome cut lung slice of a control mouse. Recording of calcium changes in the NEB ME. (a) Graphs plotting the time course of Fluo-4 fluorescence intensity (ΔFluo-4; A.U. ¼ arbitrary units), as an indicator for [Ca2+]i, in different cell types in the airway epithelium. After application of BALF (¼T0), CLCs that surround the NEB cells show a calcium-mediated activation within about 10 s (¼T1). The oscillating [Ca2+]i rise in CLCs continues (¼T2) for more than a minute post-exposure. No changes in [Ca2+]i can be observed in NEB cells or CCs. (b, c) Corresponding pseudo-colour time-lapse images of Fluo-4 fluorescence at two time points (B ¼ T0; C ¼ T2) after the application of BALF. Note that the ROIs corresponding to the graphs in (a) are represented in the same colour code in image b (Reproduced with permission from Verckist et al. 2018). (B) Immunostaining for BrdU (as a marker for cell

4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

57

Combination of a single LPS challenge and BrdU treatment for 48 h resulted in strongly enhanced proliferation of airway epithelial cells, the vast majority of which appeared to be located around NEB cells (Fig. 4.4B-D). The number of divided cells in the NEB ME was found to be almost 25 fold higher than those in untreated controls. More than 70% of all counted NEBs harboured divided (BrdU-positive) cells in their ME (Fig. 4.4E), with 40% of them showing clusters of 3-8 BrdUpositive cells. In contrast, BrdU-positive cells were rare in healthy control (10% of the counted NEBs) and sham-treated (23% of the counted NEBs) animals, and, if present, the divided cells mainly appeared solitary instead of clustered (Fig. 4.4E) (Verckist et al. 2018).  ⁄ Fig. 4.4 (continued) division) in cryosections of the lungs of a GAD67-GFP mouse, 48 h after intratracheal instillation with LPS and intraperitoneal injection with BrdU. (a) A cluster of BrdUpositive nuclei (arrowheads; red fluorescence) is present in the airway epithelium (E). (b) Combination with GAD67-GFP fluorescence (green) shows that the BrdU-positive nuclei surround GFP-fluorescent NEB cells. L: airway lumen. (C) Cryosection of a mouse lung 48 h after LPS challenge, double stained for BrdU (red fluorescence) and CGRP (green fluorescence). BrdU-ir (divided) cells (arrowheads) are observed in the NEB ME, in close proximity to the CGRP-ir PNEC cluster. L: airway lumen, E: airway epithelium. (D) Single confocal optical section of airway epithelium (E) in a cryosection of the lungs of a GAD67-GFP mouse, 48 h after challenge with LPS. (a) BrdU-positive nuclei (arrowheads; red fluorescence) in the airway epithelium. (b) Combination with GAD67-GFP fluorescence (green), marking NEB cells in the NEB ME. Note that the NEB cells do not show BrdU-positive nuclei. (c) CCSP immunostaining (blue fluorescence) marks Clara cells (open arrowheads) and, more faint, also CLCs (arrowheads). (d) Image combining the three channels. Note that the divided epithelial cells (BrdU-labelled nuclei; arrowheads) are located adjacent to the PNECs, are co-stained with CCSP, and can, therefore, be identified as CLCs. L: airway lumen. (E) Percentage of the total number of counted NEBs that harbour cells in their ME which have divided during the 48 h experimental window after LPS challenge. The red part of the pie charts represents the percentage of NEBs with BrdU-positive cells, and is further subdivided based on the number of BrdU-labelled cells per NEB (see framed areas). The data in the framed areas represent the percentages of NEBs with one or two, three to eight, or nine and more BrdU-positive cells. In the untreated controls (n ¼ 5), less than 10% of the NEB MEs harbour divided cells. Interestingly, both the percentage of NEBs with BrdU-positive cells (about 23%) and the number of divided cells per NEB ME appear somewhat higher in sham-treated mice (n ¼ 10) than in untreated control mice. After LPS instillation (n ¼ 10), however, about 72% of the NEBs show divided cells in their ME. The mean number of BrdU-positive cells is significantly higher (Dunn’s multiple comparisons test) in the LPS-treated group compared to both the untreated controls (****, p < 0.0001) and the sham-treated group (**, p < 0.01) (adapted from Verckist et al. 2018). (F) Ranking of performed PCR arrays, based on the number of genes (of the total number of 84 genes in each array) that showed at least a two-fold (higher/lower) differential mRNA expression. Arrays are ranked from the lowest (top) to the highest number of genes (bottom) that are selectively changed in the NEB ME. (a) Based on the differential expression between NEB MELPS and CAELPS. (b) Based on the differential expression between NEB MEctrl and the CAEctrl (c) Based on the differential expression between NEB MELPS and the NEB MEctrl. (d) Table that includes the actual numbers of genes that minimally showed a two-fold higher or lower mRNA expression in the NEB ME compared to CAE; columns represent the different experimental comparisons. The colour code is in accordance with the percentage of the 84 genes; more than 50% (dark green), more than 40% (green or red), and more than 25% (light green or light red)

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4.3 The Pulmonary NEB ME: A Unique Stem Cell Niche in the Airway Epithelium?

59

Double staining for CCSP (¼SGB1A1), as a marker for Clara cells and CLCs, and the typical location of the divided cell type in the NEB ME, allowed identification of the majority of the proliferated cells as CLCs (Fig. 4.4D). The number of divided PNECs was very limited compared to that of CLCs and did not differ between experimental and control animals (Verckist et al. 2018). In contrast to studies based on full depletion of Clara cells (Reynolds et al. 2000a, b; Hong et al. 2001), in which PNECs proliferation was observed, the LPS-based mild injury model apparently induces cell division in CLCs only. Since CLCs in the NEB ME were shown to selectively re-enter the cell cycle, this minimally invasive model of LPS-induced ALI was further used to analyse gene expression of the NEB ME in LPS-challenged mice (results specified in the following paragraphs mainly concern new so far unpublished data). Samples of NEB ME and CAE of LPS-instilled GAD67-GFP (Schnorbusch et al. 2013) mice were processed in the same way as described for LMD and gene expression experiments in healthy postnatal mice (Verckist et al. 2017; Sect. 4.3.3). PCR arrays, previously employed to detect the stem cell characteristics of the NEB ME and CAE in healthy control animals (further indicated as NEB MEctrl and CAEctrl), were also used for NEB ME and CAE samples from LPS-treated mice (referred to as NEB MELPS and CAELPS). mRNA expression levels were compared between NEB MELPS and CAELPS, and between the NEB MELPS and NEB MEctrl (complete gene expression dataset (unpublished data) is added as an addendum (supplementary files S1–S5)). Changes in gene expression between NEB MELPS/CAELPS and NEB MEctrl/ CAEctrl were obvious (Fig. 4.4Fa-d), suggesting that specific panels of genes are involved in the function of the NEB ME in healthy controls and LPS-treated animals and that the induced mild inflammation has a major impact on gene expression in the NEB ME/CAE. LPS challenge appeared to result in both upregulation and downregulation of high numbers of genes. The performed PCR arrays also allowed comparison of gene expression levels in the NEB MELPS with the initial expression levels in NEB MEctrl samples. Multiple genes displayed a differential expression in the activated NEB MELPS compared to the NEB MEctrl (Fig. 4.4Fc). Of the analysed genes, almost 23% showed an at least two-fold higher expression in the NEB MELPS compared to NEB MEctrl and might be important in the stem cell behaviour of the NEB ME. Comparison between the NEB MELPS and NEB MEctrl also showed that 17% of the genes within the niche get downregulated after induction of proliferation (own unpublished observations). The observed differential expression is likely involved in regulating and maintaining a balance between facultative progenitor cell silencing/proliferation and commitment of daughter cells to differentiate and restore airway epithelial integrity. Since the 600 genes-counting panel was obtained originally from ten different pathway-focused gene arrays, quick analysis could be performed by ranking the array results according to the number of differentially expressed genes (Fig. 4.4Fac). Of the 84 genes in each panel, those that showed a differential gene expression (fold regulation higher than 2/lower than 2) in NEB ME compared to CAE or between NEB MELPS and NEB MEctrl were counted (number of genes between

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brackets). Figure 4.4Fd shows a table with the number of genes that showed a higher and lower expression level per pathway and for all compared conditions (complete gene expression dataset (unpublished data) is added as an addendum (supplementary files S1-S5)). When comparing the NEB MELPS and CAELPS, overview of the pathways (Fig. 4.4Fa, d) revealed that the ‘Inflammatory Cytokines and Receptors’ (52), ‘Hedgehog’ (49), and ‘Stem cell’ (48) PCR arrays contained the highest numbers of differentially expressed genes. Also for comparison between NEB MEctrl/CAEctrl, most changes in gene expression were found in the ‘Inflammatory Cytokines and Receptors’ array (Fig. 4.4Fb). The above data suggest that the NEB ME may be implicated in the regulation of inflammation and immune responses. Comparison between the NEB MELPS and the NEB MEctrl, the PCR array ranking based on the number of affected genes interestingly showed that the ‘Stem Cell’ (59) and ‘Cancer Stem Cells’ (58) arrays had the highest number of differentially expressed genes (Fig. 4.4Fc), pointing out that—at the gene level—the NEB MELPS appeared to be in an activated ‘stem cell’ state. This observation supports the interpretation that, in the LPS-induced ALI model, the selective proliferation of CLCs in the NEB ME is the result of (re)activation of the stem cell capacity in this niche. The number of upregulated genes in NEB MELPS/NEB MEctrl seemed to be mainly due to the higher upregulation for NEB MELPS compared to CAELPS, both for the Cancer Stem Cells (23 genes) and for the Stem Cell arrays (41 genes; almost 50%). The lower number of differentially expressed genes in healthy control animals (NEB MEctrl/CAEctrl; see table in Fig. 4.4Fd) for both the Cancer Stem Cell and the Stem Cell array further supports the observations that the NEB ME appears to be quiescent in healthy control animals and that LPS-induced ALI causes activation of the stem cell properties of the NEB ME, with selective proliferation of CLCs. Further analysis of the data available from the ten PCR arrays (complete gene expression dataset (unpublished data) is added as an addendum (supplementary files S1-S5)), showed that in the NEB MELPS compared to the NEB MEctrl at least some of the pathways clearly seem to be influenced by the LPS-induced activation of CLCs (Fig. 4.4Fd). While the Notch pathway contained a high number of upregulated genes, the TGFβ/BMP pathway had the most downregulated genes. These results corroborate literature data on other lung stem cells, indicating that BMP/Notch signalling may be involved in the homeostatic maintenance, and plays key roles during development and remodelling after tissue injury (Borok et al. 2006; Hogan et al. 2014; Kiyokawa and Morimoto 2020). Based on a single low-dose intratracheal LPS instillation, a highly reproducible, minimally invasive and physiologically relevant transient ALI model was generated and validated for inducing selective activation of a quiescent airway stem cell population (CLCs) in the NEB ME of postnatal mouse lungs (Verckist et al. 2018). The fact that CLCs can be activated from a silent to a dividing stem cell population, in the absence of identifiable damage to the airway epithelium and without additional proliferation of PNECs, creates new opportunities for unravelling the cellular mechanisms/pathways regulating silencing, activation, proliferation, and

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61

differentiation of this unique postnatal airway epithelial stem cell population. Highend gene expression analysis used to compare mRNA from NEB ME and CAE from healthy control and LPS-challenged mice led to the conclusion that the NEB ME in postnatal mouse lungs is a quiescent ‘stem cell niche’ activated upon appropriate stimulation. Several pathways are likely involved in signalling within the NEB ME ‘stem cell niche’.

4.3.5

Silencing Stem Cells in the Healthy Pulmonary NEB ME: Involvement of BMP Signalling

Bone Morphogenetic Proteins (BMPs) are members of an evolutionarily conserved family of signalling molecules (for review, see Sieber et al. 2009; Conidi et al. 2011; Wang et al. 2014), with over 20 different known BMP ligands identified involved in stem cell maintenance, cell proliferation, migration, growth, differentiation, and apoptosis. BMP signalling is effectuated by type I (BMPRI) and type II receptors (BMPRII), which are serine/threonine kinases. BMP ligands bind to a complex composed of a pair of BMPRI and a pair of BMPRII. The constitutively active BMPRII activates the BMPRI, which results in the phosphorylation of cytoplasmic targets and subsequent activation through Smad-dependent and Smad-independent pathways (Miyazono et al. 2005; Sieber et al. 2009). In the nucleus, Smads bind directly to specific DNA sequences, interact with other DNA-binding proteins or cause the recruitment of transcriptional co-activators or co-repressors. In this way, BMPs regulate the transcription of target genes (Miyazono et al. 2010). BMP signalling can be modulated extracellularly (e.g. the antagonist noggin), intracellularly (e.g. microRNAs and phosphatases), and by other receptors in the plasma membrane (e.g. endoglin) (Wang et al. 2014). In lungs, BMP signalling has been reported to be implicated in both development (Bellusci et al. 1996; Eblaghie et al. 2006; Domyan et al. 2011; Sountoulidis et al. 2012; Wang et al. 2014) and postnatal lung homeostasis and repair after injury (Sountoulidis et al. 2012; Tadokoro et al. 2016; Chung et al. 2018). Using a transgenic reporter mouse line harbouring a BMP-responsive eGFP reporter allele, Sountoulidis et al. (2012) reported eGFP expression, and hence BMP signalling, in the NEB ME. eGFPpos-CC10low cells form “caps” over the NEB cells during development, whereas from PD15 onwards BMP signalling seemed to be restricted to the NEB cells. These intriguing observations prompted us to explore BMP signalling in the pulmonary NEB ME in more detail. Analysis of PCR array data obtained in postnatal healthy control (ctrl) mice (Verckist et al. 2017) taught us that BMP2 and BMP7 are highly expressed in the NEB ME compared to CAE, an observation that was confirmed by single qPCR experiments (own new data) (Fig. 4.5A).

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62 4 Functional Exploration of the Pulmonary NEB ME

expression of the reference genes, both BMP2 and BMP7 revealed a significantly ( p < 0.05 (*) and p < 0.005 (**), using Mann–Whitney U test) higher expression in the NEB ME (own new data). NRQ: normalized relative quantification. (B) Immunostaining for bone morphogenetic protein 2 (BMP2) (red fluorescence) in a GAD67-GFP (green GFP fluorescence) mouse lung cryostat section. (a) A BMP2-ir nerve fibre (open arrowhead) gives rise to an intraepithelial arborisation of laminar BMP2-ir nerve terminals (arrowheads). (b) Combination of the red and green channels reveals that it is a GFP-expressing NEB that is selectively innervated by the BMP2-ir nerve terminals. (c) Single confocal optical section, demonstrating the localisation of the BMP2-ir nerve terminals between the NEB cells, reminiscent of the vagal sensory innervation of NEBs. E: airway epithelium; L: airway lumen (own new data). (C) Results of single qPCR experiments for the expression of BMP2 and BMP7 in LPS-challenged mice compared to control mice. The graph shows that the mean mRNA levels in LMD samples of the activated NEB MELPS are significantly ( p < 0.001 (**)) downregulated compared to the NEB MEctrl. mRNA levels are normalized to expression of reference genes in the same samples. NRQ: normalized relative quantification, n ¼ 3 (own new data). (D) Comparison of the distribution of BrdU-labelled (red fluorescence) airway epithelial cells after challenge of WT-Bl6 mice with the BMP receptor antagonist LDN-193189. (a) Clustered BrdU-positive nuclei (arrowheads) can be observed at certain locations in the airway epithelium (E). Combination with CGRP immunostaining (b; green fluorescence) reveals that many divided cells that have incorporated BrdU are found in the NEB ME and are reminiscent of CLCs. L: airway lumen (own new data). (E–F) Schemes representing the KEGG–TGFβ/BMP pathway, showing the majority of genes, i.e. ligands, receptors, target genes, and antagonists, that are involved with a colour-coded expression (green: >2 upregulation; red: