SKEWED TRYPTOPHAN METABOLISM CONTRIBUTES TO DISEASE PATHOGENESIS IN A LUPUS-PRONE MOUSE MODEL BY IMPACTING T CELL PHENOTYPES By JOSEPHINE MICHELLE BROWN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2020 © 2020 Josephine Michelle Brown To my father, Joseph Thomas Brown, who has unequivocally inspired me to spend my life working to improve the lives of others through scientific investigation ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Laurence Morel, who invested insurmountable time and energy for the past 4 years molding me into a skilled scientist by constantly challenging me. Most of all, I thank her for her immense patience and can only hope of having another mentor as thoughtful and skilled as her. I thank my committee for their guidance and constructive criticism on my work. Dr. Graciela Lorca gave me tremendous advice on our microbiology methodologies. Dr. Christian Jobin was an invaluable resource for advice and suggestions regarding our microbiota assessments and histological techniques. Dr. Todd Brusko provided guidance on the T cell portion of this study and has consistently encouraged me during my time as a PhD student. Without you all, I would not have such diverse technical expertise. I am forever grateful to my family for their unwavering support. I would absolutely not be where I am today without you. Thank you Haaris for always being my shoulder to cry on, for intelligent conversations about my research, and for constantly encouraging and believing in me, even in my greatest times of weakness. You are truly the most selfless person I know. Thank you Jess for being the most supportive sister, and for your encouragement and honest advice. Thank you to my mother and stepfather for always believing in me and for giving me advice and encouragement during the most difficult times. Thank you to my grandparents for thinking the world of me and for being proud. To my late grandmother, Sara – I am so thankful to have learned so many valuable life lessons from you. I am grateful to my friends and colleagues Dr. Williams, Dr. Shapiro, Dr. Lin, and Dr. Zuniga for your constant support and advice. 4 Finally, thank you to my fellow lab members, Nathalie Kanda, Tracoyia Roach, Dr. Elshikha, Dr. Abboud, and Dr. Choi, for your help with experiments and for your constructive criticism. I sincerely owe a portion of my success to all of you. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 14 CHAPTER 1 INTRODUCTION .................................................................................................... 16 Overview of Systemic Lupus Erythematosus .......................................................... 16 Overview of The TC Murine Model ......................................................................... 18 Endogenous Tryptophan Metabolism ..................................................................... 19 Microbial Tryptophan Metabolism ........................................................................... 20 Tryptophan Metabolites in Immunity ....................................................................... 21 Intestinal Dysbiosis in Human and Murine SLE ...................................................... 22 Potential Mechanisms for Microbiota Contributions to Autoimmunity ..................... 24 Evidence for Microbiota Contributions to SLE ......................................................... 25 Dysregulated Tryptophan Metabolism in SLE ......................................................... 27 Mammalian Target of Rapamycin (mTOR) and Relevance in SLE ......................... 28 2 MICROBIOTA-ASSOCIATED TRYPTOPHAN CATABOLISM INDUCES AUTOIMMUNE ACTIVATION IN THE TC MURINE MODEL .................................. 35 Background ............................................................................................................. 35 Methods .................................................................................................................. 36 Mice and Treatments ........................................................................................ 36 Assessment of Gut Leakage ............................................................................ 37 Gut Microbiota Analysis .................................................................................... 38 Metabolomic Analysis ....................................................................................... 39 Renal Pathology ............................................................................................... 41 Anti-dsDNA IgG Autoantibody ELISA ............................................................... 41 Statistical Analysis ............................................................................................ 42 Results .................................................................................................................... 42 Autoimmune TC mice Present Altered Gut Microbial Communities ................. 42 Intestinal Dysbiosis Contributes to Autoimmune Activation via a Mechanism other than Bacterial Translocation ................................................................. 43 Tryptophan Metabolites are Dysregulated in TC Mice ...................................... 45 6 Autoimmune Phenotype Severity is Dependent on the Amount of Dietary Tryptophan .................................................................................................... 45 Increased Dietary Tryptophan Percentage Further Disrupts Gut Microbial Communities in TC Mice ............................................................................... 46 Long-Term Antibiotic Treatment Restores Tryptophan Metabolites in TC Mice .............................................................................................................. 47 The TC Microbiota Mediates the Effects of Variations in Dietary Tryptophan on Autoimmune Activation............................................................................. 48 Discussion .............................................................................................................. 48 3 THE MICROBIOTA IS LARGELY RESPONSIBLE FOR SKEWED TRYPTOPHAN METABOLISM IN THE LUPUS-PRONE TC MODEL .................... 62 Background ............................................................................................................. 62 Methods .................................................................................................................. 63 Mice and Treatments ........................................................................................ 63 Analysis of Kynurenine and Tryptophan by HPLC ............................................ 64 Metabolomic Analysis ....................................................................................... 64 Gene Expression Analysis ................................................................................ 66 Flow Cytometry ................................................................................................ 67 Bone Marrow Chimeras .................................................................................... 67 Anti-dsDNA IgG Autoantibody ELISA ............................................................... 67 Statistical Analysis ............................................................................................ 68 Results .................................................................................................................... 68 IDO1 and TDO do not Contribute to Kynurenine Accumulation in TC Mice ..... 68 TC Hematopoietic and Non-hematopoietic Compartments Play a Role in Tryptophan Metabolite Alterations ................................................................. 70 The Microbiota Skews Tryptophan Metabolism in TC Mice .............................. 71 Discussion .............................................................................................................. 72 4 SKEWED TRYPTOPHAN METABOLISM IN THE LUPUS-PRONE TC MOUSE PROMOTES PRO-INFLAMMATORY T CELL PHENOTYPES .............................. 82 Background ............................................................................................................. 82 Methods .................................................................................................................. 83 Mice and Treatments ........................................................................................ 83 Flow Cytometry ................................................................................................ 84 Cellular Assays ................................................................................................. 85 Immunofluorescence Staining .......................................................................... 85 Western Blots ................................................................................................... 86