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Novel Mechanisms of Antihelminth Immunity

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Novel Mechanisms of Antihelminth Immunity Novel Mechanisms of Antihelminth Immunity Lewis James Entwistle The Francis Crick Institute, London University College London PhD Supervisor: Mark S. Wilson A thesis submitted for the degree of Doctor of Philosophy 2018 1 Declaration I, Lewis James Entwistle, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Intestinal helminths are highly prevalent worldwide, infecting approximately a third of the world’s population, causing significant host morbidity. With no current vaccines, a limited number of effective chemotherapeutic drugs available and the emergence of drug-resistant helminths, it is essential to further our understanding of the mechanisms of antihelminth immunity. Our current understanding of antihelminth immunity places the type 2 immune response at the forefront of protection, with type 2 cytokines orchestrating and activating a plethora of immune and non-immune cells to mediate parasite expulsion. The naturally occurring intestinal helminth Heligmosomoides polygyrus establishes a chronic infection in many inbred naïve mice, with resistance to a challenge infection established following drug-cure. This experimental model allows us to identify novel mechanisms of drug-induced resistance, relative to susceptibility. In this thesis, we utilised next generation sequencing technology to identify two novel mechanisms of antihelminth immunity. Firstly, we determined that the enzyme phospholipase A2 group 1B (PLA2g1B) is an endogenous anthelmintic, upregulated in intestinal epithelial cells of resistant mice. We demonstrated that PLA2g1B was essential for resistance to H. polygyrus and that PLA2g1B directly cleaves phospholipids off infective H. polygyrus larvae. Secondly, we identified that the microRNAs miR-99a-5p, miR-148a-3p and miR-155-5p were upregulated in mice resistant to H. polygyrus during infection and were also essential for functional immunity. In summary, we have identified and characterised two novel mechanisms of antihelminth immunity and propose a model of tissue memory, essential for acquired resistance to H. polygyrus. 3 Impact Statement The analysis, discovery and insight presented in this thesis further our understanding of antihelminth immunity. Utilising RNA sequencing, we identified and describe two novel mechanisms of immunity to intestinal helminth infection. These findings have the potential to benefit other basic, translational and clinical researchers in the fields of parasitology and immunology. Furthermore, these findings may also impact the pharmaceutical industry and improve human health by providing new therapeutic opportunities in the rational design of anthelmintic drugs and/or the manipulation of microRNA (miRNA) expression. Specifically, the vast RNA sequencing datasets created and analysed in this thesis provide an abundance of high quality, high resolution data from mice susceptible and resistant to an experimental intestinal helminth infection. These datasets are publically available and knowledge of this will be disseminated upon publishing of these results in scientific journals. Further mining of these transcriptomic datasets has the potential for the discovery of further novel mechanisms of antihelminth immunity. This will therefore benefit basic and translational researchers in the field of antihelminth immunity. The discovery that phospholipase A2 group 1B (PLA2g1B) acts as an endogenous anthelmintic and is critical for protective immunity to intestinal helminth infection furthers our understanding of important antihelminth responses. Furthermore, the demonstration that PLA2g1B cleaves phospholipids from infective larvae has revealed a completely new mechanism of antihelminth immunity. This discovery should spur interest in identifying the specific roles of larval phospholipids in infection, host-parasite interaction and larval development amongst the basic parasitology and immunoparasitology research communities. Moreover, further elucidation of PLA2g1B’s effect on intestinal helminths and the role of parasite-phospholipids in helminth infection and health would be of interest in the pharmaceutical industry. Specifically, in the design of synthetic helminth-specific molecules to recapitulate the effects of endogenous PLA2g1B, leading to the generation of new anthelmintics. The discovery of PLA2g1B as an endogenous anthelmintic could therefore impact treatment strategies of intestinal helminth infection, thus improving human health. 4 As part of this thesis, we may have identified a discrepancy between pharmacological inhibition of miRNAs and genetic deletion studies of miRNAs. This discrepancy is particularly important in the translation of basic research using genetic miRNA- deficient models into pharmacological inhibition for potential use in the clinic. We also demonstrate that inhibition of a suite of miRNAs abrogated protective immunity to intestinal helminth infection, whereas individual miRNA inhibition did not. This highlights the complex nature of miRNAs in vivo cooperating to regulate specific biological functions and pathways. These insights into miRNA-regulated antihelminth immunity will benefit basic researchers with interests in immunoparasitology, immunology and miRNA biology. Finally, our data provides insights into the phenomenon of ‘tissue memory’ being both present and essential in protection against intestinal helminth infection. Our findings should stimulate important questions in the realm of ‘tissue memory’ that are of interest to the wider community of basic, translational and clinical researches. 5 Acknowledgement “A scientist in his laboratory is not a mere technician: he is also a child confronting natural phenomena that impress him as though they were fairy tales” – Marie Curie I firstly would like to thank my supervisor, Mark Wilson, for his endless guidance, support and encouragement, inside and outside the lab, from San Francisco to London. I will be forever grateful for his kindness, generosity and friendship, as well as helping me to develop as a scientist. I am also forever indebted to my friends and colleagues Yashaswini Kannan, Victoria Pelly, Stephanie Coomes, Riccardo Guidi and Jimena Perez Lloret for their teaching, guidance and thoughtful advice throughout my time in the lab. I would also like to thank all past members of the Wilson lab for producing the most wonderful atmosphere to learn and develop in. I am also extremely grateful to the Stockinger lab for their kindness in incorporating me into their lab following Mark’s move to San Francisco and our move to the Crick. I am also thankful to my Thesis Committee, Gitta Stockinger, Abdul Sesay and Mike Gilchrist, for their constructive scientific input and guidance. My research would have not been possible without the fantastic facilities and staff at both the National Institute for Medical Research and The Francis Crick Institute. Firstly, I would like to thank Abdul Sesay, Leena Bhaw, Harsha Jani and Deb Jackson in the Advanced Sequencing Facility for all of their help and efforts with my RNA sequencing, as well as Nikolay Nikolov and Probir Chakravarty for their bioinformatic expertise. I would also like to thank Ade Adekoya, Jamie Barratt and all of the BRF staff for all of their efforts in the breeding and maintenance of our mice. I am grateful to James MacRae and Mariana Silva dos Santos in Metabolomics for their help and expertise with the lipidomic experiments. I would also like to thank the Flow Cytometry staff for all their help and hard work with cell sorting. I am also thankful to Lucy Collinson in the Electron Microscopy department for her help and expertise. I would also like to thank the infection and immunology community at both the NIMR and the Crick for generating a friendly atmosphere to share ideas, provide feedback and form collaborations. I am also extremely fortunate to have some amazing friends whom I thank for their support and keeping me sane, especially to Helena at the NIMR/Crick in the sharing of the trials and tribulations of being a PhD student. 6 I would finally like to thank my family for their unwavering love and support. Thanks to my siblings, Jake and Ella, for their encouragement. Thank you to my late father, Mark, for instilling an early drive to be the best that I can possibly be. Thank you to my fiancée, Becky, I will be forever grateful for her sacrifice, patience and day to day support over the past four years. Finally, thank you to my Mum, Amanda. I will be forever grateful and indebted to her for making me the man I am today, placing my feet on the ground and eyes toward the sky. I am humbled by your faith in me, I hope I can continue to make you proud. 7 Table of Contents Abstract ............................................................................................................. 3 Impact Statement .............................................................................................. 4 Acknowledgement ............................................................................................ 6 Table of Contents .............................................................................................. 8 Table of Figures .............................................................................................. 11 List of Tables ................................................................................................... 14 Abbreviations .................................................................................................
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