Emergence of In Vitro 3D Systems to Model Human Malaria [1st ed. 2023] 9819906903, 9789819906901

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Kasem Kulkeaw

Emergence of In Vitro 3D Systems to Model Human Malaria

Emergence of In Vitro 3D Systems to Model Human Malaria

Kasem Kulkeaw

Emergence of In Vitro 3D Systems to Model Human Malaria

Kasem Kulkeaw Siriraj Integrative Center for Neglected Parasitic Diseases Department of Parasitology Faculty of Medicine Siriraj Hospital, Mahidol University Bangkok, Thailand

ISBN 978-981-99-0690-1 ISBN 978-981-99-0691-8 https://doi.org/10.1007/978-981-99-0691-8

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Malaria continues to affect global public health. The need for a safe and effective drug targeting liver-stage malaria limits the currently launched eradication program. Given the non-physiological relevance of traditional two-dimensional cell culture, the use of liver organoid, a three-dimensionally organizing cell cluster, may pave the way for studying Plasmodium spp. under human physiology. This book provides a comprehensive review of knowledge of malaria biology in a liver, and all in vitro platforms for study of liver-stage malaria, including principles, protocols, applications for disease modelling and drug screening, and their limitations. One chapter describes the basis of stem cells in liver generation during development and liver regeneration in adults. Then, it highlights recent and emerging advances in liver organoid and liver-on-a-chip for modelling malaria. The book collects all current protocols and methods to generate liver organoid and liver-on-a-chip together with their advantages and limitations. The final chapter of this book discusses the potential of these 3D models to understand the biological complexity of cellular and molecular mechanisms involved in Plasmodium development in the liver, toolboxes to investigate parasite development in the 3D models, and to implement in drug discovery. It is written for researchers and scientists in parasitology, cell biology, tissue engineering, and pharmacology, and is contributed by an author who does research in the field. I would like to thank the Department of Parasitology, Faculty of Medicine Siriraj Hospital for providing assistance in writing. I also thank Worakamol Pengsart, who assisted with all the informative illustrations. Above all, my special thanks go to my family. Without their help, I could never have undertaken the first book project, which was an extreme challenge. Bangkok, Thailand

Kasem Kulkeaw

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Contents

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Introduction to Malaria Biology in a Liver . . . . . . . . . . . . . . . . . . . . . 1.1 Global Impact of Malaria in the Past 10 Years . . . . . . . . . . . . . . . . 1.2 Liver-Stage Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Life Cycle of Plasmodium Species . . . . . . . . . . . . . . . . . . . 1.3 Mechanism of Parasite Invasion into a Liver . . . . . . . . . . . . . . . . . . 1.3.1 Liver Structure and Composition . . . . . . . . . . . . . . . . . . . . 1.3.2 Hypothesis of the Sporozoite Invasion Mechanism . . . . . . . . 1.4 Hypnozoite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Drug-Targeting Liver-Stage Malaria . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Intra-erythrocytic Parasite . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2 Intra-hepatic Parasite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 3 3 5 5 7 10 11 11 11 11

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Modeling a Liver-Stage Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Necessity of the In Vitro Models for Drug Discovery and Pre-clinical Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 In Vitro 2D Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Human Hepatoma Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Primary Human Hepatocytes (PHH) . . . . . . . . . . . . . . . . . . 2.2.3 Human Immortalized Hepatic Cells . . . . . . . . . . . . . . . . . . . 2.2.4 Human Hepatocyte-like Cells Derived from Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 Rodent Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Humanized Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 The Use of the 2D Cell Culture for Antimalarial Drug Assay . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Molecular and Cellular Basis of Liver Organoid . . . . . . . . . . . . . . . . . 3.1 Basis of Liver Generation and Regeneration . . . . . . . . . . . . . . . . . . 3.1.1 Development of a Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2 Cell Signaling in Hepatogenesis . . . . . . . . . . . . . . . . . . . . . 3.1.3 Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Biology of Liver Organoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 16 16 18 20 21 24 24 25 25 28 33 33 33 34 35 36 vii

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3.2.1 Definition and Characteristics of Organoid . . . . . . . . . . . . . 3.2.2 Sources of Stem Cells for Liver Organoid Generation . . . . . 3.3 Types of Liver Organoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Single Cell Type-Derived Hepatic Organoids . . . . . . . . . . . . 3.3.2 Unknown Cell Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3 Multiple Cell Types for Heterogenous Organoids . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Generation of Liver Organoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Culture of Human iPSCs (Si-Tayeb et al. 2010) . . . . . . . . . . . . . . . 4.1.1 Preparation of the Matrigel-Coated Well . . . . . . . . . . . . . . . 4.1.2 Preparation of the Culture Medium for Human iPSCs . . . . . . 4.1.3 Thawing the Frozen iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4 Cell Passaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5 Evaluation of Cell Pluripotency . . . . . . . . . . . . . . . . . . . . . 4.2 Generation of Hepatic Endoderms from Human iPSCs (Si-Tayeb et al. 2010) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Step 1: Monolayers of Human iPSCs (Day 5–Day 0) . . . . . . 4.2.2 Step 2: Endoderm Induction (Day 0–5) . . . . . . . . . . . . . . . . 4.2.3 Step 3: Specification of Hepatic Endoderm (Day 6–10) . . . . 4.2.4 Step 4: Differentiation of the Hepatic Endoderm into Hepatoblasts (Day 11–15) . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5 Step 4: Hepatocyte Maturation (Day 16–20) . . . . . . . . . . . . 4.3 Generation of Hepatic Organoid from the iPSC-Derived Hepatic Endoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Matrigel Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Cell Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3 Preparation of a Cell–Matrigel Mixture . . . . . . . . . . . . . . . . 4.3.4 Formation of the Hepatic Organoid . . . . . . . . . . . . . . . . . . . 4.3.5 Split of the Hepatic Organoid . . . . . . . . . . . . . . . . . . . . . . . 4.3.6 Expected Result of the Hepatic Organoid . . . . . . . . . . . . . . 4.4 Generation of Liver Bud from the Human iPSCs . . . . . . . . . . . . . . 4.4.1 Generation of Endoderm and Hepatic Endoderm (Takebe et al. 2013) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2 Generation of Septum Transversum Mesenchyme from Human iPSCs (Sundaram et al. 2014; Takebe et al. 2013) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3 Generation of Endothelial Progenitor Cells from Human iPS Cells (Adams et al. 2013; Takebe et al. 2013) . . . . . . . . 4.4.4 Formation of Liver Bud (Takebe et al. 2013) . . . . . . . . . . . . 4.4.5 Expected Result of the Liver Bud Formation . . . . . . . . . . . . 4.5 Characterization of Human iPSC-Derived Hepatic Endoderm, Septum Transversum Mesenchyme and Endothelial Cells . . . . . . . . 4.5.1 Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . .

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4.5.2 4.5.3

Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Characterization of the Hepatic Organoid and the Liver Bud . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 5

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Design of a Liver-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Liver-on-a-Chip and Its Applications for the Hepatotropic Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Personalized Liver-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Liver Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 Transdifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 Mesenchymal Stem Cells (MSCs) . . . . . . . . . . . . . . . . . . . . 5.2.4 Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.5 Hematopoietic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.6 Human-Induced Pluripotent Stem Cells (iPSCs) . . . . . . . . . . 5.3 Design of the Liver-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 Artificial Fenestrae of the Sinusoid Endothelial Cells . . . . . . 5.3.2 Radial Pattern of Hepatocytes and SECs . . . . . . . . . . . . . . . 5.3.3 Microfluidic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Use of 3D In Vitro Systems to Model Human Malaria . . . . . . . . . 6.1 Application of Organoid Technology for the Pre-Clinical Study of Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Proposed Ways to Use the Organoid Technology as a Liver-Stage Malaria Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Potential Applications of Liver Organoid . . . . . . . . . . . . . . . . . . . . 6.3.1 Hepatitis C Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 The Use of the Hepatic Organoid and Liver Bud as a Model of the Liver-Stage Malaria . . . . . . . . . . . . . . . . . . . . 6.4 The Use of Human Spheroids as a Model of the Liver-Stage Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Human Hepatoma and Immortalized Hepatic Cell-Derived Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.2 Human Primary Hepatocyte-Derived Spheroids . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 67 69 69 69 71 72 72 74 75 75 76 77 78 78 83 83 85 87 87 88 90 91 92 92

About the Author

Kasem Kulkeaw is a director of the Siriraj Integrative Center for Neglected Parasitic Diseases at the Faculty of Medicine Siriraj Hospital, Mahidol University in Thailand. He has earlier served as a visiting scientist under “Exchange Program for East Asia Young Researchers” of the Japan Society for Promotion of Science at Kyushu University, Japan; an assistant professor in the Department of Research and Development of Next Generation Medicine, Faculty of Medical Sciences, Kyushu University, Japan (2014–2018; and assistant professor at the Department of Parasitology at Mahidol University, Thailand (2021–present). His research interest is in cell biology and molecular biology specializing in stem cell-based disease models, cell differentiation of pluripotent stem cells, and malaria biology. He has been conferred with various prestigious fellowships, notably, the Royal Golden Jubilee PhD program of Thailand Research Fund (2002–2008), the postdoctoral fellowship of Ajinomoto Scholarship Foundation (2009–2010), the postdoctoral fellowship of the Tokyo Biochemical Research Foundation (2010–2012), and the postdoctoral fellowship of the Japan Society for the Promotion of Science (2012–2014). He has more than 10 years of research experience in cell biology, stem cell science, and animal ethics, and more than 3 years of teaching experience in stem cell biology and medical parasitology. Since 2008, he has also published more than 30 research articles and 5 review articles in peer-reviewed international journals.

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The emergence of zoonosis and drug resistance in infectious pathogens are evidence supporting the necessity of vaccines and treatments. Not to overclaim, humans still face a significant global threat caused by infectious diseases to their health. Particularly, vaccine development in aging people has setbacks relevant to deteriorated immunity. The lack of effective treatment and prevention exacerbates this problem and emphasizes the necessity of drug and vaccine development. This chapter overviews the global impact of malaria and the biology of the Plasmodium parasite. Then, comprehensive knowledge of liver-stage malaria is reviewed, including the mechanism underlying the parasite entry into a hepatocyte, parasitic vacuole formation, proliferation, and hypnozoite formation. Following the basis of malaria, the actions of current antimalarial drugs targeting liver-stage malaria are explained.

1.1

Global Impact of Malaria in the Past 10 Years

Malaria remains a major threat to public health, affecting hundreds of million people worldwide yearly. Progress in the prevention and treatment of malaria leads to a significant decrease in morbidity and mortality in many endemic areas, including Southeast Asia, South Asia, and Africa. According to the World malaria report during 2010–2019, more than 200 million malaria cases are reported yearly, resulting in more than 400,000 deaths worldwide (World malaria report 2019). Over the past 10 years, malaria cases and fatalities have remained unchanged. Most malaria patients (90%) are prevalent in the African, Southeast Asia, and Eastern Mediterranean zones. Children less than 5 years of age and pregnant women have the highest morbidity and death risks due to malaria (Lalloo et al. 2006). Moreover, drug resistance and insufficient drug against liver-stage malaria challenge our efforts to eradicate malaria. Nevertheless, history has shown the survival mechanism of the Plasmodium parasites to escape from almost all

# The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 K. Kulkeaw, Emergence of In Vitro 3D Systems to Model Human Malaria, https://doi.org/10.1007/978-981-99-0691-8_1

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antimalarial drugs we have ever developed. Therefore, the malaria eradication program launched by World Health Organization worldwide has been challenging. Plasmodium species, the obligate intracellular protozoa, parasitizes animals, including birds, mice, monkeys, and humans. An insect vector transmits malaria via the blood meal bites of a female mosquito of Anopheles species. Four species in the genus of Plasmodium cause human malaria: P. falciparum, P. vivax, P. ovale, and P. marariae, and an emerging zoonotic P. knowlesi. Among the five human malaria-causing species, P. falciparum and P. vivax are the widespread species across the continents of Asia, Africa, and South America. The most virulent and predominant species is P. falciparum, which causes severe malaria. By contrast, P. vivax is relatively less virulent but distributed in many geographical areas. Thus, vivax malaria exhibits significant economic impacts. Nevertheless, there was an increased number of severe cases and deaths due to P. vivax in the WHO Southeast Asia Region, according to the World Malaria Report 2015, an emerging challenge that needs to be monitored. All Plasmodium species causing human malaria must first develop in a hepatocyte. After multiple rounds of cell replication (a process known as schizogony) in the hepatocyte, the Plasmodium parasite will invade erythrocytes, causing lifethreatening clinical manifestations. Thus, the intra-hepatic development of the Plasmodium species is an intervention target for blocking a progression to clinical malaria. Also, such intervention could lead to a prophylactic regimen for people at risk of high morbidity or traveling to an endemic area. Unlike others, P. vivax and P. ovale malaria lead to relapse due to dormancy in the hepatocytes. Significantly, liver-stage malaria is a drug target for malaria prevention (prophylactic) and antirelapse. Currently, only four drugs are available to target liver-stage malaria, e.g., atovaquone, proguanil, primaquine, and tafenoquine. Atovaquone and proguanil target only liver-stage parasites but cannot kill the dormant parasites (hypnozoites). By contrast, primaquine and tafenoquine could prevent relapse, indicating a capability in hypnozoite killing (Wells et al. 2010). However, prevention of vivax malaria relapse needs a complete 14-day drug administration, risking a loss of coherence and failure in the relapse prevention. Moreover, primaquine and tafenoquine cause hemolysis in individuals who are deficient in G6PD (Lacerda et al. 2019; LlanosCuentas et al. 2019). Thus, the adverse hemolytic effect restricts mass drug administration in at-risk patients. Along the pipeline of drug discovery and development, the preclinical studies require in vitro cell culture systems to test the efficacies and toxicities of drug candidates. Most in vitro cell cultures are based on cell growth in a two-dimensional (2D) setting. Cells adhere to the stationary phase of the plastic plate and interact with other adjacent cells side by side. Advances in stem cell biology, tissue engineering, and biomaterials allow the in vitro cell culture in three-dimensional (3D) systems, e.g., spheroid and organoid. Despite the mainstay of the cell culture in many labs, the in vitro 2D platforms cannot recapitulate the complex microarchitecture and functions of the tissue counterpart, a long-standing hurdle in drug discovery (Arrowsmith and Miller 2013). Hence, the host–parasite interaction in a given tissue is important to identify therapeutic targets.

1.2 Liver-Stage Malaria

1.2

Liver-Stage Malaria

1.2.1

Life Cycle of Plasmodium Species

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Plasmodium species could survive in highly distinct biological niches, covering invertebrate and vertebrate organisms. In Anopheles mosquitoes, all Plasmodium species undergo sexual development in the gut, giving rise to progenies infectious to the vertebrates, e.g., birds, rodents, primates, and humans. In the vertebrates, Plasmodium parasites switch the sexual development to complicated asexual development in hepatocytes and erythrocytes. Intra-hepatic development of Plasmodium parasites causes no illness. In contrast, intra-erythrocyte development leads to a cycle of febrile illness, and some Plasmodium species cause severe clinical symptoms: cerebral malaria, multi-organ failure, and severe anemia.

1.2.1.1 Liver-Stage Malaria While suctioning blood, female Anopheles mosquitoes inoculate hepatocyteinfective sporozoites into human skin. Immediately, the inoculated sporozoites enter human blood vessels before the macrophages engulf them. After entering blood circulation, the sporozoites translocate across the liver sinusoid. To invade the hepatocytes, it has been hypothesized that sporozoites invade Kupffer cells, the resident macrophages, before infecting hepatocytes. Nevertheless, the direct hepatocyte invasion was documented, raising a debatable mechanism underlying the hepatocyte entry. In the liver, the sporozoites migrate across the firstly exposed hepatocyte to the adjacent hepatocyte (a process known as cell transversal). Upon arrival, the sporozoites undergo schizogony, in which the Plasmodium parasites multiply several times, generating tens of thousands of merozoites (a stage known as schizont). Merozoite is derived from two Greek words; meros means a part, and zoon means animal or life. The schizont-containing hepatocytes rupture, releasing hepatic merozoites into blood vessels. In blood circulation, the hepatic merozoites can infect erythrocytes to initiate intra-erythrocytic schizogony, which causes clinical manifestation. In hepatocytes, the sporozoite-to-merozoite development is referred to as the pre-erythrocytic stages. Notably, exoerythrocytic schizogony causes no symptoms. P. vivax and P. ovale can undergo a secondary exoerythrocytic phase. Without known factors, some parasites undergo dormancy, the stage in which the Plasmodium parasites become inactive in cell metabolism and proliferation, called hypnozoite. Hypnos is a Greek word, which means “sleep.” The dormant parasites can be activated to undergo multiplication months or years later in the infected individuals. Activation of the hypnozoites results in clinical manifestations known as relapse malaria. In Africa, a significant proportion of P. vivax malaria resulted from hypnozoites activation, indicating the potential reservoir of malaria transmission. The hypnozoites cause substantial clinical and financial burdens due to chronic and relapsing potential (Price et al. 2007). Here, there are several unsolved questions regarding the liver-stage development of the Plasmodium species:

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1. Why do sporozoites need to invade the adjacent hepatocytes after an initial invasion (a process known as cell transversal)? 2. Which host factors regulate a transition of sporozoites to schizonts in a hepatocyte? 3. How do thousands of merozoites break the cell membrane of hepatocytes to enter the blood circulation? 4. What are the mechanisms underlying the formation of hypnozoites?

1.2.1.2 Erythrocyte Stage Malaria Given the encounter of the merozoite to host immune cells, this stage causes symptoms, and some patients become more severe, known as complicated malaria, leading to death. After entering into blood circulation, the hepatocyte-derived merozoites invade erythrocytes and undergo an intra-erythrocytic growth. Upon uptake of human hemoglobin, the parasites collect hemoglobin in a food vacuole. Based on the standard Giemsa-stain, the parasites appear as a ring that the nucleus is a head ring with red-blue color. At the same time, the Plasmodium cytoplasm is fully occupied with hemoglobin (color similar to hemoglobin in erythrocytes), while the hemoglobin-free cytoplasm of the parasite remains blue. Thus, the appearance of the thin parasite cytoplasm looks identical to the whole of a ring. Later, the parasites digest hemoglobin, use globin as an amino acid source, and discard heme by forming the yellow-brown granule, known as hemozoin. Food vacuoles become smaller than the ring-like form, and the parasite cytoplasm expands. The by-product of the parasite metabolism appears as fine or rough dots in the cytoplasm of erythrocytes. Following the growing trophozoite, the parasites subsequently undergo nuclear division, generating multiple nuclei, so-called early schizogony (The Greek words of schizis mean division, while gone refers to generation). Due to loss of cell membrane integrity, the schizonts are prone to rupture, releasing more merozoites infectious to other erythrocytes. Through several rounds of the enter-proliferation-release process, Plasmodium-infected individuals face a cycle of clinical symptoms of malaria. Under the stress of the host body (immune response or drug), the parasites develop into gametocytes: microgametes (male gametocytes) and macrogametes (female gametocytes). These two stages will undergo sexual reproduction in a midgut of Anopheles spp. 1.2.1.3 Mosquito Stage Following an infestation by a female Anopheles mosquito, microgametocytes, and macrogamete undergo fertilization, known as sporogony. The fertilized zygotes invade through a gut wall, where they further develop into oocysts. Many thousands of sporozoites are generated through multiple rounds of cell division. The sporozoites migrate along the Anopheline gut to a salivary gland, where they are infectious to a human. During a blood meal, the female Anopheles mosquitoes inoculate the sporozoites into a human host to initiate a cycle of asexual reproduction.

1.3 Mechanism of Parasite Invasion into a Liver

1.3

Mechanism of Parasite Invasion into a Liver

1.3.1

Liver Structure and Composition

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As part of the digestive system, the human liver is the largest organ, where two in the third part of blood flows. The liver is capable of functioning in various roles. The liver removes toxic substances in blood via catabolism. To support food digestion, the liver provides enzymes and non-enzymatic emulsifiers. Moreover, the liver secrets homeostasis-regulating factors, e.g., coagulation factors and albumin. Thus, the liver is regarded as a gland. Given its largest size, it also stores carbohydrates as glycogen and lipids. A human liver can be divided into two lobes, grossly separated into eight segments (Fig. 1.1a, left panel). The individual segment has blocks of hexagon shape, called lobules (Fig. 1.1a, middle panel). In each lobule, several cell types are classified into parenchymal and non-parenchymal cells. Hepatocytes are parenchymal epithelial cells that constitute a solid liver mass (60% of the total number of liver cells) (Stanger 2015). Non-parenchymal cells maintain the microstructure and do other functions. In the liver, the non-parenchymal cells are cholangiocytes (forming a bile duct for bile secretion), sinusoidal endothelial cells (forming sinusoid capillary for molecule transportation between blood and hepatocytes), Kupffer cells (resident macrophages), and hepatic stellate cells (vitamin A storage and collagen production).

Fig. 1.1 Microstructure and cellular compartments of a liver lobule. (a) A human liver is composed of many hexagon-shaped structures, called liver lobules. The center of each liver lobule is a central vein, while the apical area consists of the hepatic artery, hepatic portal vein, and bile duct, collectively called a portal triad. The three-dimensional structure of a liver lobule shows a blood flow direction. (b) Hepatic cells arranged in a radial pattern from the central vein (rectangle from panel a). Venous blood enters the liver via the hepatic portal vein, and flows to the central vein via sinusoid capillary. Molecules in the blood pass across the sinusoid capillary through the fenestrae of the sinusoidal endothelial cells (SECs) to the hepatocytes. Bile canaliculi is a space between the apical sides of adjacent hepatocytes. Hepatocytes secret bile acid into the bile canaliculi, which then flow to a bile duct comprising cholangiocytes. (c) Fenestration of SECs (rectangle from panel b) allows molecules entering the perisinusoidal space (space of Disse). Here, the basolateral side of hepatocytes is exposed to nutrients, gases, and chemical substances. This illustration was modified from a review of Kasem and Pengsart, 2021 (Kulkeaw and Pengsart 2021)

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Fig. 1.2 Cellular heterogeneity in a liver lobule. Hepatocytes polarize into apical and basolateral domains. The basolateral membrane contains microvilli proximal to the space of Disse, while the apical membrane connects the adjacent hepatocytes. Bile canaliculi form between the hepatocytes and function in draining bile acid into bile duct. There are stellate cells, Kupffer cells, and pit cells in the sinusoid capillary. Fenestrae of the sinusoid endothelial cells act as selective filters for transporting molecules from blood to the hepatocytes

At the apex of a hexagon-shaped lobule, there are three vessels: the hepatic artery, portal vein, and bile duct, collectively called the portal triad (Fig. 1.1a, middle panel). Blood from the gastrointestinal tract enters the liver via the hepatic portal vein and drains into the central vein at the center of the lobules. The hepatocytes arrange as plates radiated in the central vein-to-portal vein direction. The basolateral membrane of the two juxtaposed hepatocytes forms the small channel (bile canaliculi) for secreting bile acid. The apical membrane of hepatocytes has microvilli and exposes it to a perisinusoidal space termed the space of Disse (Wojciech Pawlina 2006) (Fig. 1.1a, right panel). The microvilli increase the area for molecules transportation. Along the hepatic portal to the central vein, sinusoidal endothelial cells (SECs) line as a single layer to form liver sinusoid capillary. The SECs allow molecules to pass from blood through their cytoplasmic holes (fenestrae) to the space of Disse (Fig. 1.1c). Nutrients and gases pass across the fenestrated SECs to the hepatocytes in a gradient manner (Lautt 2009). Polarization of the hepatocyte is necessary for the absorption and transportation of biomolecules. The microvilli-containing apical membrane of the hepatocyte takes up low-density lipoprotein via receptor-mediated endocytosis. By contrast, bile salt is secreted via ATP-binding cassette proteins at the basolateral membrane of two juxtaposed hepatocytes. Moreover, tight junction ZO1 proteins control the molecule flux through the cell membrane of adjacent hepatocytes (Fig. 1.2). Extracellular matrix proteins surround the hepatocytes (e.g., laminin, collagen type IV, and fibronectin), a scaffold supporting hepatocyte adhesion (Gissen and Arias 2015).

1.3 Mechanism of Parasite Invasion into a Liver

1.3.2

7

Hypothesis of the Sporozoite Invasion Mechanism

1.3.2.1 Motility of Sporozoites Unlike other flagellate or ciliate parasitic protozoa, the Plasmodium sporozoites lack a locomotion-supporting cellular compartment. The Plasmodium sporozoites utilize the microneme, an organelle, for movement (Menard 2001). At the apical end, Plasmodium microneme secrets proteins, which are moved backward along the actomyosin on the cell surface, resulting in traction motility (Mota and Rodriguez 2002). In vitro studies categorized the patterns of sporozoite movement into gliding, infection-ending motility, and transversal motility. The sporozoites usually glide in a circular pattern on a glass slide. By using the reflection interference contrast microscopy and the traction force microscopy, the imaging at high temporal resolution reveals a similar gliding of the P. bergehei sporozoites on the glass surface and the cultured human hepatocellular carcinoma Huh7 cells (Munter et al. 2009). Thus, the gliding motility of the sporozoites is likely caused by internal factors. Other patterns of sporozoite motility are invasive, leading to a transversal migration and a hepatic infection (Mota et al. 2001). In transversal migration, the sporozoites break the cell membrane before invading the adjacent cells. To develop in the hepatocyte, it has been hypothesized that such transversal motility is essential for this process (Mota et al. 2002). Probably, the transversal migration precedes the infection of the invaded hepatocyte (Fig. 1.3). 1.3.2.2 Passage of Sporozoites from the Blood Circulation Through the Liver Sinusoid Endothelium The sporozoites access the hepatocyte via migration through the SECs. There are several hypotheses regarding the mechanism of the hepatocyte infection of a given sporozoite, mainly from the rodent malaria, P. bergihei. After 2 min of an intravenous inoculation, Shin demonstrated the presence of the P. berghei sporozoites in some hepatocytes, while the majority of the sporozoites were in the phagolysosome of Kupffer cells. This data suggest a direct infection of the hepatocyte by the sporozoite (Shin et al. 1982). Similarly, Meis et al. reported that the P. berghei sporozoites were in the phagolysosome of Kupffer cells (Meis et al. 1985). In addition to potential lysis in the phagolysosome, the Kupffer cells may engulf the sporozoites without destruction, followed by translocation of the sporozoite to the hepatocytes (Meis et al. 1983). In agreement with this notion, the in vitro invasion assay shows that the rodent Plasmodium sporozoites enter the Kupffer cells without localization within the lysosome, followed by a transverse to hepatocyte. Thus, it is hypothesized that the sporozoite’s active motility likely allows them to infect the hepatocytes via Kupffer cells by avoiding intracellular lysis (Pradel and Frevert 2001). Thus, it remains uncertain whether the Kupffer cells are the gate of the sporozoite entry (Fig. 1.3). Despite the tiny diameter of the fenestrae of the SECs, Stewart proposed a penetration of P. berghei sporozoites in the fenestrated cytoplasm of the SEC layer using the in vitro rodent malaria model (Steffan et al. 1987). Since the contractile microfilament enveloped the fenestrae, it is possible that the larger cells could

8

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Introduction to Malaria Biology in a Liver

Fig. 1.3 Mechanisms of the Plasmodium sporozoite invasion and infection in the hepatocytes. Gliding of the sporozoites allows interaction with hepatic stellate cells. The sporozoites may bind to heparan sulfate proteoglycans (HSPGCs) on the hepatic stellate cells or CD68 on Kupffer cells. These two ways may trap the sporozoites that are circulating in the sinusoid capillary. Next, the sporozoites migrate across the endothelial cells to invade hepatocytes (upper part of the illustration). Despite cell lysis in phagolysosome, studies suggest that Kupffer cells act as a gate for hepatocyte entry. SPECT1 and SPECT2 are secreted from the sporozoite’s microneme and used for migrating across the hepatocyte known as cell traversal. Following the cell traversal, the sporozoites invade the adjacent hepatocyte. SR-BI, CD81, and SPHA2 are proteins expressed on the basolateral membrane of the hepatocytes and are involved in cell invasion. The parasites rely on P36 and P52 for cell invasion

penetrate, a phenomenon of cancer metastasis into the liver. After passing through the SECs, the sporozoites undergo hepatocyte transversal migration. Mota et al. propose migration-activating exocytosis of the sporozoites, in which the sporozoites penetrate several hepatocytes before vacuole formation. It has been hypothesized that the thrombospondin-related anonymous protein is essential for sporozoite invasion (Mota and Rodriguez 2002). Following the intravenous inoculation into a rat, the hepatocyte infectivity of the P. berghei sporozoites deficient for the sporozoite microneme protein essential for cell traversal (SPECT) decreased. By contrast, direct inoculation of the spect-deficient sporozoites allowed the successful infection in the in vitro cultured hepatocytes, implying the importance of the SPECT in the penetration through the SECs but not direct invasion into a hepatocyte. What factors play a role in the hepatocyte infectivity of the sporozoite in vivo? Depletion of Kupffer cells in the rat model restored the spect-mutant sporozoite infectivity. Thus, the Kupffer cells are targets of the sporozoites to transverse the SECs into the liver parenchyma (Fig. 1.3).

1.3 Mechanism of Parasite Invasion into a Liver

9

1.3.2.3 Host Response to Hepatocyte Invading Parasite Most cells use the pattern recognition receptors to recognize the invading pathogens, including Toll-like receptors on the cell membrane or RNA and DNA sensors in the cytoplasm. These molecules bind to the pathogen-associated molecular pattern (PAMP), which is a pattern or motif of molecules conserved among groups of pathogenic microbes. Following the PAMP-mediated recognition, the host cells use several intracellular adaptor molecules to activate the cellular responses, such as the transcription of effector molecules. Type I interferons (IFN) are effector molecules responsible for inhibiting viral replication in a given cell in an autocrine or paracrine manner. The type I IFNs bind to the transmembranous IFN receptor, which is widely displayed on the cell surface, and upregulate the transcription of interferon-stimulated genes (ISGs). In gene trasncription, the IFN-α and IFN-β gene is regulated by transcription factors, e.g., interferon regulatory factor (IRF) 3 and IRF7 (Fig. 1.4).

Fig. 1.4 A proposed mode of action of the IFN in regulation of a liver-stage malaria. A Plasmodium-infected hepatocyte releases Plasmodium RNA, which is recognized by RNA sensor Mad5 and the unknown molecules. The Mad5 needs an adaptor Mavs to transduce a signal to activating transcription factors Irf3 and Irf7, which regulate transcription of IFN-α and IFN-β genes. The IFN-α and IFN-β genes are secreted from the Plasmodium-infected hepatocytes. As an autocrine, IFN-α and IFN-β bind to the IFN receptor on the adjacent hepatocyte, resulting in the upregulation of ISG transcription, i.e., leukocyte-mobilizing chemokines. Following the chemoattractant release, macrophages and neutrophils accumulate in the infected hepatocytes, probably eliminating the Plasmodium parasites. The illustration is modified from Haque et al. (Haque and Engwerda 2014)

10

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Introduction to Malaria Biology in a Liver

Unlike intra-erythrocytic growth, liver-stage malaria is asymptomatic. Although the hepatocytes are damaged during the parasite growth, the immune privilege and immune tolerance may reduce symptoms of liver-stage malaria. However, there is evidence of hepatocyte response to the invading sporozoite. Transcriptome analysis of the mouse liver infected with P. berghei sporozoite identified an increase of interferon-stimulated genes (ISGs), particularly those functioning in the type I interferon (IFN) signaling pathway (Portugal et al. 2011; Portugal 2011). The IFN receptor-deficient mice failed to produce ISGs in response to P. berghei and P. yoelli sporozoite infection, confirming the involvement of the IFN signal. Transcription of the IFN-α and -β is regulated by two transcription factors, the interferon regulatory factor (IRF3) and IRF7. The Irf3-/- or Irf7-/- mice could not produce IFN-α and -β in response to the P. berghei sporozoite infection. Haque et al. identified the PAMP transcript in the P. beghei-infected hepatocytes in a murine malaria model. To activate type I IFN response, the mitochondria-derived cytosolic RNA sensors, and a cytoplasmic pattern recognition receptor are reportedly essential for recognizing the Plasmodium RNA. The IFN receptor-deficient mice had higher parasite load as assessed by amplification of Plasmodium 18S rRNA. Consequently, the percentage of the P. beghei-infected erythrocytes increased in IFN receptordeficient mice. This inhibitory effect of IFN-α and -β is not directly against the sporozoites. Instead, recruitment of macrophages and neutrophils to the IFN-producing hepatocytes is likely the mechanism underlying low parasite load in a liver and low parasitemia compared to the mice lacking the IFN receptor (Fig. 1.4, Haque and Engwerda 2014).

1.4

Hypnozoite

After the cell invasion, the sporozoites of P. vivax and P. ovale develop into trophozoites. Some sporozoites do not precede schizogony to produce the hepatic merozoites. Instead of progressing to clinical malaria, the parasites delay their growth and development. They are becoming quiescence, resulting in a latency of the clinical manifestation. The quiescent parasite is called “hypnozoite,” meaning a sleeping animal, and appears as a small size in a hepatocyte (Markus 2011). The hypnozoites can survive in a liver for a long period of time. Several months or years later, some factors activate the hypnozoite to resume the schizogony, causing malaria relapse (White 2011; White and Imwong 2012). The mechanism of hypnozoite formation is unknown. A mechanism underlying vivax malaria relapse in endemic malaria areas has been proposed (White 2011). A subject carries hypnozoites that are derived from inoculation with two distinct genotypes of the sporozoites. Later, the same individual is infected with the new strain of Plasmodium parasites. Half of the parasites undergo schizogony, generating the merozoites, while the remaining parasites become hypnozoites. The new infection-caused clinical phase of erythrocyte-stage malaria may activate one pre-existing hypnozoite of each genotype.

References

1.5

Drug-Targeting Liver-Stage Malaria

1.5.1

Intra-erythrocytic Parasite

11

The current frontline antimalarial drug is based on a combination of ring-stage trophozoite targeting artemisinins with other companion drugs, known as artemisinin-based combination therapy. For vivax malaria, chloroquine remain effective. Dihydroartemisinin, artesunate, and artemether are artemisinin derivatives, which are combined with companion drugs, including lumefantrine, mefloquine, amodiaquine, sulfadoxine/pyrimethamine, piperaquine, and chlorproguanil/dapsone (White et al. 2014). P. falciparum could resist almost all available antimalarial drugs. Human cases infected with artemisinin-resistant P. falciparum are documented in the Greater Mekong subregion, including Cambodia, Lao People’s Democratic Republic, Myanmar, Thailand, and Vietnam. The chloroquine resistance of P. vivax malaria has been reported in a wider geographical area, including South America (Bolivia, Brazil, Peru), Africa (Ethiopia), Southeast Asia (Thailand, Indonesia, Malaysia, Myanmar, Papua New Guinea), and Oceania (the Solomon Islands). Both chloroquine and sulfadoxine-pyrimethamine are the former effective antimalarial drugs. The chloroquine resistance first emerged in Western Cambodia and Thailand– Myanmar border (White et al. 2014). Subsequently, the chloroquine resistance genes spread to Africa, leading to millions of deaths.

1.5.2

Intra-hepatic Parasite

There are two purposes of the treatment targeting liver-stage malaria: (1) prophylaxis of a clinical phase and (2) relapse prevention. Currently, four drug regimens are available for anti-hepatic malaria, e.g., atovaquone, proguanil, primaquine, and tafenoquine. Atovaquone and proguanil specifically target the hepatic schizont, preventing malaria for whom back from visiting the endemic area. As anti-relapse, primaquine and tafenoquine kill the hypnozoites (Lacerda et al. 2019; LlanosCuentas et al. 2019). A complete 14-day course of primaquine is required for effective treatment (Wells et al. 2010). However, primaquine induces hemolysis of erythrocytes in G6PD-deficient patients.

References Arrowsmith J, Miller P (2013) Trial watch: phase II and phase III attrition rates 2011–2012. Nat Rev Drug Discov 12:569. https://doi.org/10.1038/nrd4090 Gissen P, Arias IM (2015) Structural and functional hepatocyte polarity and liver disease. J Hepatol 63:1023–1037. https://doi.org/10.1016/j.jhep.2015.06.015 Haque A, Engwerda C (2014) Hepatocytes break the silence during liver-stage malaria. Nat Med 20:17–19. https://doi.org/10.1038/nm.3446

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Kulkeaw K, Pengsart W (2021) Progress and challenges in the use of a liver-on-a-chip for hepatotropic infectious diseases. Micromachines (Basel) 12(7):842 Lacerda MVG, Llanos-Cuentas A, Krudsood S, Lon C, Saunders DL, Mohammed R, Yilma D, Batista Pereira D, Espino FEJ, Mia RZ, Chuquiyauri R, Val F, Casapia M, Monteiro WM, Brito MAM, Costa MRF, Buathong N, Noedl H, Diro E, Getie S, Wubie KM, Abdissa A, Zeynudin A, Abebe C, Tada MS, Brand F, Beck HP, Angus B, Duparc S, Kleim JP, Kellam LM, Rousell VM, Jones SW, Hardaker E, Mohamed K, Clover DD, Fletcher K, Breton JJ, Ugwuegbulam CO, Green JA, Koh G (2019) Single-dose tafenoquine to prevent relapse of Plasmodium vivax malaria. N Engl J Med 380:215–228. https://doi.org/10.1056/ NEJMoa1710775 Lalloo DG, Olukoya P, Olliaro P (2006) Malaria in adolescence: burden of disease, consequences, and opportunities for intervention. Lancet Infect Dis 6:780–793. https://doi.org/10.1016/S14733099(06)70655-7 Lautt W (2009) Hepatic circulation: physiology and pathophysiology. Morgan & Claypool Life Sciences, San Rafael Llanos-Cuentas A, Lacerda MVG, Hien TT, Velez ID, Namaik-Larp C, Chu CS, Villegas MF, Val F, Monteiro WM, Brito MAM, Costa MRF, Chuquiyauri R, Casapia M, Nguyen CH, Aruachan S, Papwijitsil R, Nosten FH, Bancone G, Angus B, Duparc S, Craig G, Rousell VM, Jones SW, Hardaker E, Clover DD, Kendall L, Mohamed K, Koh G, Wilches VM, Breton JJ, Green JA (2019) Tafenoquine versus primaquine to prevent relapse of Plasmodium vivax malaria. N Engl J Med 380:229–241. https://doi.org/10.1056/NEJMoa1802537 Markus MB (2011) Malaria: origin of the term “hypnozoite”. J Hist Biol 44:781–786. https://doi. org/10.1007/s10739-010-9239-3 Meis JF, Verhave JP, Brouwer A, Meuwissen JH (1985) Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites. Z Parasitenkd 71:473– 483. https://doi.org/10.1007/BF00928350 Meis JF, Verhave JP, Jap PH, Meuwissen JH (1983) An ultrastructural study on the role of Kupffer cells in the process of infection by Plasmodium berghei sporozoites in rats. Parasitology 86 (Pt 2):231–242. https://doi.org/10.1017/s003118200005040x Menard R (2001) Gliding motility and cell invasion by Apicomplexa: insights from the Plasmodium sporozoite. Cell Microbiol 3:63–73. https://doi.org/10.1046/j.1462-5822.2001.00097.x Mota MM, Hafalla JC, Rodriguez A (2002) Migration through host cells activates Plasmodium sporozoites for infection. Nat Med 8:1318–1322. https://doi.org/10.1038/nm785 Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Frevert U, Nussenzweig RS, Nussenzweig V, Rodriguez A (2001) Migration of Plasmodium sporozoites through cells before infection. Science 291:141–144. https://doi.org/10.1126/science.291.5501.141 Mota MM, Rodriguez A (2002) Invasion of mammalian host cells by Plasmodium sporozoites. Bio Essays 24:149–156. https://doi.org/10.1002/bies.10050 Munter S, Sabass B, Selhuber-Unkel C, Kudryashev M, Hegge S, Engel U, Spatz JP, Matuschewski K, Schwarz US, Frischknecht F (2009) Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites. Cell Host Microbe 6:551–562. https:// doi.org/10.1016/j.chom.2009.11.007 Portugal S, Drakesmith H, Mota MM (2011) Superinfection in malaria: Plasmodium shows its iron will. EMBO Rep 12:1233–1242. https://doi.org/10.1038/embor.2011.213 Pradel G, Frevert U (2001) Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion. Hepatology 33:1154–1165. https://doi.org/10.1053/jhep.2001. 24237 Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM (2007) Vivax malaria: neglected and not benign. Am J Trop Med Hyg 77:79–87 Shin SC, Vanderberg JP, Terzakis JA (1982) Direct infection of hepatocytes by sporozoites of Plasmodium berghei. J Protozool 29:448–454. https://doi.org/10.1111/j.1550-7408.1982. tb05431.x Stanger BZ (2015) Cellular homeostasis and repair in the mammalian liver. Annu Rev Physiol 77: 179–200. https://doi.org/10.1146/annurev-physiol-021113-170255

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Steffan AM, Gendrault JL, Kirn A (1987) Increase in the number of fenestrae in mouse endothelial liver cells by altering the cytoskeleton with cytochalasin B. Hepatology 7:1230–1238. https:// doi.org/10.1002/hep.1840070610 Wells TN, Burrows JN, Baird JK (2010) Targeting the hypnozoite reservoir of Plasmodium vivax: the hidden obstacle to malaria elimination. Trends Parasitol 26:145–151. https://doi.org/10. 1016/j.pt.2009.12.005 White NJ (2011) Determinants of relapse periodicity in Plasmodium vivax malaria. Malar J 10:297. https://doi.org/10.1186/1475-2875-10-297 White NJ, Imwong M (2012) Relapse. Adv Parasitol 80:113–150. https://doi.org/10.1016/B978-012-397900-1.00002-5 White NJ, Pukrittayakamee S, Hien TT, Faiz MA, Mokuolu OA, Dondorp AM (2014) Malaria. Lancet 383:723–735. https://doi.org/10.1016/S0140-6736(13)60024-0 Wojciech Pawlina MHR (2006) Histology: a text and atlas. Lippincott Wiliams & Wikins, Baltimore, MD

2

Modeling a Liver-Stage Malaria

This chapter starts with problems in the use of the disease model in drug discovery and pre-clinical study. Then, it provides information on conventional cell cultures and animals for modeling liver-stage malaria.

2.1

Necessity of the In Vitro Models for Drug Discovery and Pre-clinical Study

Drug development consists of three major phases, e.g., drug discovery, pre-clinical study, and clinical study. In vitro cell culture systems are the most common tools for understanding the disease mechanism, identifying drug targets, and screening potential drugs. Before the drug is tested in human subjects, a pre-clinical study is inevitable. The pre-clinical study ensures the toxicity and gains information on pharmacokinetics, which determine the first dose in humans. Then, the selected drug will enter the long-term process of a three-phase clinical trial. Hence, the drug discovery and the pre-clinical study are essential as bottlenecks prior to the clinical study, which is a high-cost and long-time phase, taking about 10–15 years. Pre-clinical study involves the in vitro culture of cells and animal experiments. Given the high proliferation and ease to use, cancer cell lines and immortalized cells are widely used in in vitro culture systems. Most conventional cell culture platforms rely on a monolayer of the cells on a stationary plate made of glass or plastic polystyrene. As a monolayer, the individual cells interact laterally with the adjacent cells and basally with extracellular matrix protein, the two-dimensional interaction. The 2D cell cultures are easy, time saving, and cost-effective. Given the versatility and reproducibility, the 2D cell culture platform allows drug target identification, therapeutic compound screening, and toxicity and drug metabolism assays. However, long-term culture of the proliferative cell cultures may induce mutations and leads to a change in cell physiology and functions. The culture-induced mutations could limit the accuracy of disease modeling or drug screening. The dependence on # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 K. Kulkeaw, Emergence of In Vitro 3D Systems to Model Human Malaria, https://doi.org/10.1007/978-981-99-0691-8_2

15

16

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Modeling a Liver-Stage Malaria

such non-physiological systems may contribute to unsuccessful clinical trials. In phase II clinical trials of infectious diseases, approximately 10% of the drug failed. In phases II and III of clinical trials, more than half lack efficacy and safety (Arrowsmith and Miller 2013; Hwang et al. 2016). Authority withdraws some drugs from the market due to toxicity to the human body (Stevens and Baker 2009). In 2D cell culture, all cells are exposed to nutrients and gas linearly, which differs from the microenvironments in tissues. In a given tissue, many cell types polarize in the apical or basolateral direction. The basal membrane of some cells adheres to the matrix proteins, while the lateral membranes interact with the surrounding cells. The way of cell interaction is referred to as 3D, in which the cells are exposed to nutrients and gas in a gradient manner. As a result, cell functions and metabolism in the 3D culture mimic those in vivo (Rubashkin et al. 2014). Therefore, tissue microenvironment is a central issue in modeling human diseases in a near-physiology setting. A breakthrough revolution of the in vitro cell culture relies on understanding stem cell biology and the advancement of materials and tissue engineering. An organoid is one technological breakthrough applicable to a broad range of basic biomedical science to clinical applications. Several 3D culture models provide a body-relevant microenvironment suitable for elucidating tissue renewal (regeneration), dissecting stem cell/niche functions, and investigating tissue responses to disease-underlying mutations or drug-induced damage. Thus, the use of organoids has emerged in translation medicine, in which the drug efficacy and safety obtained from the organoid can predict the clinical use in an individual.

2.2

In Vitro 2D Models

In pre-clinical studies, primary cells and cell lines are used for testing drug efficacy against hepatic schizont or hypnozoites and vaccines (Table 2.1). This part provides an overview of the 2D culture systems for liver-stage malaria.

2.2.1

Human Hepatoma Lines

A few hepatoma cell lines are deployed for liver-stage malaria studies: HepG2-A16, Huh7, and HHS-102. Since CD81 (known as the target of the antiproliferative antibody-1 or tetraspanin-28) is essential for the sporozoite invasion. Each hepatic cell line expresses CD81 at a different level, resulting in variation in infectivity or resistance to cell entry. P. vivax sporozoites could develop in the HepG2 cells, resulting in the release of merozoites infectious to human erythrocytes. By contrast, P. falciparum sporozoites failed to develop in the HepG2 cells (Hollingdale et al. 1985) that lack CD81 on the surface (Flint et al. 1999). However, P. vivax sporozoites could invade HepG2-A16 cells (Manzoni et al. 2017) and develop into schizonts capable of releasing merozoites (Hollingdale et al. 1985). A Thai isolate of P. falciparum sporozoites could undergo schizogony in the HHS-102 hepatoma cell

• Rodent

• Organoid

• Spheroid

• iPSCs • Co-culture

imHC

• Immortalized cells • Liver

Adult hepatocyte Hepatocytes Adult hepatocytes + fibroblastsa HepG2 Adult hepatocyte iPSC-derived cells Mice with chimeric liver or humanized mice

Cell types HepG2-A16 HC-04

Sources • Cancer cell lines

Derived from a mouse embryo • = applicable

a

Animals

3D

Types 2D

•

• •

•

Liver-stage development

Applications

•

•

Sporozoite invasion •

• •

• •

•

Antimalarial drug test

Table 2.1 Current pre-clinical models and applications for study of the liver-stage malaria

•

vaccine test

•

Host immune responses

Vaughan et al. (2012), Kaushansky et al. (2015a), Soulard et al. (2015), Wijayalath et al. (2014)

Kulkeaw (2021)

Arez et al. (2019) Chua et al. (2019)

Silvie et al. (2003), Rodrigues et al. (2008) Ng et al. (2015) March et al. (2013)

References Manzoni et al. (2017) Sattabongkot et al. (2006) Kaushansky et al. (2015b) Pewkliang et al. (2018)

2.2 In Vitro 2D Models 17

18

2

Modeling a Liver-Stage Malaria

line. On day 12 post sporozoite inoculation, merozoite-infected erythrocytes were observed under a light microscope (Table 2.2, Karnasuta et al. 1995). Given the cancer origin, the hepatoma cell lines are immature and highly proliferative relative to the hepatocytes. By contrast, the hepatocytes are dormant in the adult human liver. Overgrowth of the hepatoma cells leads to cell detachment from a plastic plate. Long-term culture of hepatoma cells is necessary for hypnozoite formation. Chemicals or radiation are used to inhibit cell proliferation, allowing a long-term culture (Hollingdale et al. 1985). However, these methods may affect the intrahepatic development of Plasmodium spp. Moreover, the sporozoite’s poor invasion rate results from an insufficiency of sporozoite-binding receptor on the cell membrane. Key features of the hepatoma cells are rapid proliferation and simple culture protocol. However, overgrowth of the hepatoma cells lead to cell detachment from the plastic plate, especially in a long-time culture of sporozoite development. The use of chemicals or radiation could stop cell proliferation but consequently, alter intracellular activity of hepatoma cells required for the parasite development.

2.2.2

Primary Human Hepatocytes (PHH)

The PHHs can be isolated from a donated liver, and cultured under an optimal condition. The frozen stock of the PHH is also commercially available. A 2D culture of the PHHs requires collagen type IV as adhesive matrix protein. The optimal medium primarily consists of growth factors (e.g., hepatocyte growth factor and oncostatin-M) and essential supplements. Examples of common supplements used for the PHH culture are transferrin, ascorbic acid, human epidermal growth factor, insulin, hydrocortisone, and fatty acid-free BSA. A pioneering study shows the use of PHHs to model falciparum malaria in the liver. The P. falciparum sporozoites undergo the schizogony, releasing the merozoites infectious to the erythrocytes (Mazier et al. 1985). Moreover, the PHH culture allowed studies of the invasion mechanism, including the surface receptors (CD81 (Silvie et al. 2003) and SR-BI (Rodrigues et al. 2008)) for the sporozoite, and the molecular mechanism, in which the sporozoite invade into hepatocytes (Manzoni et al. 2017; Silvie et al. 2006; Yalaoui et al. 2008). Nevertheless, the invasion rate of a given sporozoite varies depending on the PHH origin (March et al. 2013; Roth et al. 2018) and on the Plasmodium parasite strain (McCall et al. 2017). Co-culture of the PHHs with murine fibroblasts reportedly supports the development of sporozoites. This chimeric cell culture allows the sporozoites of the P. falciparum and P. vivax sporozoites to complete their development, resulting in the generation of the erythrocyte-infective merozoites (March et al. 2013). Given the PHH-fibroblast culture for 21 days, presumptive hypnozoites of P. vivax could be observed based on morphology. Moreover, an attempt to develop a platform for high-throughput screening at a lower cost was a success. The PHHs are cultured as a scale of 500 um on 384-well collagen-coated plates. This study selects a batch of the PHHs that are highly susceptible to the sporozoite. The cryopreserved hepatocytes of the patient donors are screened for the batch that meets the following criteria:

N/A

0.066 (P.f.) 0.041 (P.v.) N/A 6 (P.f.) 8 (P.v.)

N/A

Yes Yes N/A

b

N/A

Yes (14)

Yes (28)

Yes (6–8)

Yes

Yes Yes (P.f.)

N/A

Hypnozoitesc (dpi) Yes (5–15)

Not applicable Not applicable Yes (up to 21)

Yes

N/A

Infectivity N/A

Nondividing, small-form parasites presumably recognized as hypnozoites Number of infected hepatocytes/total number of hepatocytes c Number of sporozoite-infected hepatocytes/total number of viable, inoculated sporozoites Dpi day post inoculation, N/A not determined, P.f. P. falciparum, P.v. P. vivax

a

Hepatocytes

0.14 ± 0.16 (P.v.) N/A

imHC

Pluripotent stem cells

N/A

HC-04

7–8 (P.f.) 9–11 (P.v.) 7 (P.f.) 10 (P.v.) 10 (P.v.)

N/A

Immortalized cells

6–10

0.18 (P.f.)

0.03 (P.f.) 0.013 (P.v.) 0.6–2 (P.f.) 2–8.3 (P.v.)

12-13

12–13

N/A

N/A

0.009 (P.f.)

N/A

Hepatocytes N/A

b

Merozoites Detection (dpi) 9

N/A

Sporozoites 0.0001 (P.v.) 0.4–2.5 (P.f.) N/A

Mazier et al. (1985) March et al. (2013) Roth et al. (2018)

HHS-102

HepG2-A16

Primary human hepatocytes

Models Hepatocellular Carcinoma cell Lines

a

Infection rate (%) based on

Ng et al. (2015)

Sattabongkot et al. (2006), Kaushansky et al. (2015b) Pewkliang et al. (2018)

Roth et al. (2018)

March et al. (2013)

Mazier et al. (1985)

Karnasuta et al. (1995)

Hollingdale et al. (1984)

References Hollingdale et al. (1985)

Table 2.2 Infectivity of the Plasmodium sporozoites and the presumptive hypnozoites in the models of the liver-stage malaria

2.2 In Vitro 2D Models 19

20

2

Modeling a Liver-Stage Malaria

1. Adhering to collagen type I. 2. Functioning in albumin expression, urea production, and CYP450 activity for a maximum of 3 weeks. 3. Displaying a sporozoite entry receptor CD81. The variation in hepatocyte functions and CD81 expression was noticed among eight donors. Amount of the secreted albumin was stable, while the level of urea increased in half of the donors during 2 weeks (day 7–21) of the culture. CYP450 activity remains unchanged during day 18–24. Intracellular sporozoite was detected using anti-HSP70. Based on the HSP70-expressing exoerythrocytic form on day 3 post-inoculation, the highest percentage of the sporozoite-infected hepatocytes was only 0.2%, and the percentage of the exoerythrocytic form did not correlate with the level of CD81 expression. About one-third of the intracellular sporozoite (day 3 post-inoculation) was capable of developing into the hepatic schizont on day 6 post-inoculation. The progression rate of the sporozoites to schizonts was higher than that in the immortalized hepatic HC-04 cell line (Table 2.2). Moreover, not only the donors of the PHHs, a batch of the cryopreserved sporozoites yielded different infectivity. The batch-to-batch difference in the formation of exoerythrocytic stage of the cryopreserved sporozoite was documented as well. Inoculation with the fresh sporozoite allows a higher rate of hepatocyte infection (March et al. 2013). Then, the PHHs were cultured on the collagen-coated 384-well format on the microscale. Together with high-content imaging, this way allow the screening of potential drug and testing vaccine efficacy in a high-throughput manner. Given the microscale of the PHHs, it allow phenotypic screening in the high-throughput settings, the complete schizogony, hypnozoite formation, inhibition of sporozoite invasion, and prophylactic activity (Roth et al. 2018). Despite the broad applications of the PHHs, the limitations in the use of primary cell remain challenging, e.g., a lotto-lot variation in the Plasmodium susceptibility (March et al. 2013), a shortage of donors, and the complex culture protocol steps. Apart from the nature of the PHHs, the intrahepatic development rates, based on the number of liver-stage parasites divided by the number of inoculated sporozoite, are less than 1%.

2.2.3

Human Immortalized Hepatic Cells

To minimize quality variation, and allow a constant supply with scalability, the PHHs were genetically transformed into immortalized hepatic cells. The immortalized cells proliferate unlimitedly, while remaining hepatocyte functions. The human immortalized HC-04 cells allow development of P. falciparum and P. vivax sporozoites into the exoerythrocytic merozoites (Sattabongkot et al. 2006). Although this way addresses the issue regarding cell proliferation; however, infection rate remains low (