Novel Mechanisms and Therapies for Celiac Disease
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NOVEL MECHANISMS AND THERAPIES FOR CELIAC DISEASE EXPLORING NOVEL MECHANISMS AND THERAPIES FOR CELIAC DISEASE By HEATHER J GALIPEAU, B.H.Sc A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Heather J Galipeau, 2015 PhD Thesis- H.J. Galipeau; McMaster University- Medical Sciences DESCRIPTIVE NOTE Doctor of Philosophy (2015) McMaster University, Hamilton, Ontario (Medical Sciences) TITLE Exploring novel mechanisms and therapies for celiac disease AUTHOR Heather J. Galipeau, B.H.Sc SUPERVISOR Dr. Elena F. Verdu NUMBER OF PAGES xix, 314 ii PhD Thesis- H.J. Galipeau; McMaster University- Medical Sciences ABSTRACT The gastrointestinal tract forms the body’s largest interface with the external environment and is exposed to a vast amount of foreign material, including pathogenic and commensal bacteria, as well as food antigens. The gastrointestinal tract has multiple functions that are performed through complex interactions by its different components. It must be able to degrade food, absorb nutrients and eliminate waste, while at the same time maintain a balance between immune tolerance and protection against pathogenic and antigenic material. This concept of mucosally-induced tolerance is a key feature of the gut immune system, whereby a state of local and systemic unresponsiveness to food protein or systemic ignorance of commensal bacteria is maintained under homeostatic conditions through interactions between the host, dietary factors, and the intestinal microbiota. Dysfunctional interactions can lead to a breakdown in tolerance to otherwise innocuous antigens. One of the best characterized food sensitivities is celiac disease (CD). CD is a chronic immune-mediated disease triggered by the ingestion of gluten, the water insoluble protein fraction in wheat, rye and barley, in patients who are HLA/DQ2 or DQ8 positive. Celiac patients can experience a loss of oral tolerance to gluten any time throughout life. The clinical presentation of CD is variable and is often associated with extra-intestinal autoimmune diseases, such as type 1 diabetes (T1D). The increasing incidence of CD and the observation that only a small proportion of genetically susceptible individuals go on to develop active inflammation suggest a role for additional environmental factors in disease pathogenesis. The current treatment for CD is a strict, life-long adherence to a gluten-free diet (GFD), which is very demanding. Frequent gluten contamination can lead iii PhD Thesis- H.J. Galipeau; McMaster University- Medical Sciences to persistent mucosal damage and symptoms, which have a negative effect on quality of life. Understanding the environmental and host factors that contribute to gluten tolerance is critical for the development of adjuvant therapies to the GFD. Therefore, the overall aim of my thesis is to characterize a humanized mouse model of gluten sensitivity in order to study factors that influence host-responses to gluten and to investigate potential therapeutic strategies. In chapter 3 of this thesis I characterized host responses to gluten using transgenic non-obese diabetic (NOD)/DQ8 mice. I found that gluten sensitization in NOD/DQ8 mice induced barrier dysfunction with a moderate degree of enteropathy and the development of anti-gliadin and anti-tissue-transglutaminase antibodies. I also explored the potential role of gluten in the development of T1D and found that gluten- induced barrier dysfunction was not sufficient to induce insulitis; a partial depletion of regulatory T cells (Tregs) plus gluten sensitization was required. In chapter 4, I utilized this model to demonstrate that the microbiota can modulate host responses to gluten. I found that both the presence and absence of a microbiota, as well as the composition of the microbiota influenced host responses to gluten in NOD/DQ8 mice. Finally, Chapters 5 and 6 of this thesis utilized transgenic DQ8 mice to explore two different adjuvant therapies for CD. In chapter 5, I showed that administration of a gluten binding polymer, P(HEMA-co-SS, to gluten-sensitive HLA/DQ8 mice reduced short-term and long-term gluten-induced barrier dysfunction and inflammation. In chapter 6, I discovered that elafin, a human anti-protease, was decreased in patients with active CD and in vitro, it inhibited the deamidation of gliadin peptides, a key step in the pathogenesis of CD. I iv PhD Thesis- H.J. Galipeau; McMaster University- Medical Sciences showed that administration of elafin prevented gluten-induced barrier dysfunction and intraepithelial lymphocytosis. Together these results, (a) provide an in depth characterization of a humanized animal model for studying gluten-induced intestinal and extra-intestinal immune responses, (b) demonstrate the role of the microbiota as an environmental modulator of gluten-induced immune responses, (c) support the preclinical potential of two novel adjuvant therapies to the GFD. These findings emphasize the translational value of using relevant animal models to study the complex interactions between environmental and host factors that contribute to intestinal health and disease. v PhD Thesis- H.J. Galipeau; McMaster University- Medical Sciences ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Dr. Elena Verdu. She has taught me so much over the past six years, and I would not be where I am today without her support and guidance. Her enthusiasm and love for science are inspiring. Since starting in Elena’s lab as a shy, undergraduate student, I have gained so much more confidence and have grown as a person, and as a scientist as a result of Elena’s encouragement and guidance. She is truly a great teacher, mentor and the perfect example of a role model. I cannot thank her enough for her mentorship and the opportunities she has given me. I also need to thank Jennifer Jury. Without the guidance and help from Jen, I would have been totally lost in the lab. Her constant willingness to help and ability to always make the chambers work were invaluable. We have had many great memories over the past years, both in the lab and outside the lab, and I am truly grateful for your friendship. To all other members of the lab, both past and present, I am so thankful for all the help you have given me over the years. To Jane Natividad, your work ethic and passion for science was truly inspiring. You were always willing to help me with my experiments and provide advice when I needed it, but more importantly, you were always able to make me laugh. To the members of the AGU, thank you for all your help with my gnotobiotic experiments. Without your assistance, much of this work would not have been possible. I also would like to thank my supervisory committee, Dr. Stephen Collins and Dr. Michael Surette, for always pushing me to think critically. The insight and guidance they provided me was invaluable. I would also like to thank our collaborators Dr. Manel Jordana and Dr. Fernando Chirdo for their insight, suggestions, and advice over the years. You have provided me with valuable input and constructive criticism for which I am very grateful. Finally, I would like to thank my family and friends for their love and encouragement. You are always interested and excited to hear about my research and you continue to support me as I continue my scientific career. I really cannot thank you enough for always being there for me when I was frustrated and helping me forget my graduate worries and enjoy life. vi PhD Thesis- H.J. Galipeau; McMaster University- Medical Sciences TABLE OF CONTENTS EXPLORING NOVEL MECHANISMS AND THERAPIES FOR CELIAC DISEASE ............................................................................................................................ i DESCRIPTIVE NOTE ..................................................................................................... ii ABSTRACT ...................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................ vi TABLE OF CONTENTS ............................................................................................... vii LIST OF FIGURES .......................................................................................................... xi LIST OF TABLES ........................................................................................................... xv LIST OF ABBREVIATIONS AND SYMBOLS .......................................................... xvi CHAPTER 1 INTRODUCTION .............................................................................................................. 1 1.1 Regulation of intestinal homeostasis ......................................................................... 2 1.1.1 Small intestinal barrier function .................................................................. 2 1.1.2 Intestinal immune system ............................................................................. 6 1.1.3 Protease and anti-protease balance ............................................................ 12 1.1.4 Intestinal microbiota and dietary antigens ................................................ 13 1.1.4.1 The intestinal microbiota .............................................................. 13 1.1.4.2 Dietary antigens ............................................................................ 16 1.1.4.3