MACROPHAGE PHOSPHOINOSITIDE 3-KINASE p110δ REGULATES INTESTINAL HOMEOSTASIS BY DIRECTING ADAPTIVE IMMUNITY AND ENHANCING MICROBIAL CLEARANCE ERIN CATHLEEN STEINBACH A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology. Chapel Hill 2013 Approved by: Scott E. Plevy, MD. R. Balfour Sartor, MD. Virginia L. Miller, PhD. Miriam Braunstein, PhD. William E. Goldman, PhD. © 2013 Erin Cathleen Steinbach ALL RIGHTS RESERVED ii ABSTRACT ERIN CATHLEEN STEINBACH: Macrophage Phosphoinositide 3-kinase p110δ Regulates Intestinal Homeostasis by Directing Adaptive Immunity and Enhancing Microbial Clearance (Under the direction of Scott E. Plevy, MD) The human inflammatory bowel diseases (IBDs), Crohn’s disease (CD) and ulcerative colitis, result from an inappropriately directed immune response to enteric microbiota in a genetically susceptible host. IBDs represent an increasing burden on the global health care system, as incidence is increasing and effective therapies remain elusive. Genome-wide association studies highlight the importance of host innate immune cell-microbial interactions in the pathogenesis of IBDs. PI3K signaling regulates diverse functions, including cell growth, differentiation, proliferation and survival. The Class IA PI3K catalytic subunit p110δ negatively regulates toll-like receptor signaling in innate immune cells. The importance of p110δ in intestinal homeostasis is shown in a mouse harboring a kinase-dead p110δ (p110δKD) that develops spontaneous Th1/Th17-skewed colitis. We describe a requirement for the enteric microbiota to drive intestinal inflammation in p110δKD mice. Microbial-innate immune interactions maintain homeostasis through regulation of both protective (IL-10) and inflammatory (IL-12p40) cytokines, and p110δ is a central regulator of this balance. Additionally, p110δ positively regulates eradication of intracellular bacteria in macrophages. Persistence of intracellular bacteria and chronic stimulation in intestinal p110δKD macrophages propagates the cytokine imbalance. Furthermore, p110δ iii orchestrates innate immune cell regulation of pathogenic adaptive immune responses. Importantly, in human CD, decreased intestinal PIK3CD gene expression and an inverse correlation with intestinal IL12B:IL10 ratios are demonstrated. Thus, p110δ appears to be a central homeostatic switch in the intestine, governing the critical balance between IL- 12/23 and IL-10 induced by the microbiota that determines the subsequent T cell response. Counter to prevailing paradigms where p110δ inhibition is a strategic approach in inflammatory diseases, strategies to induce p110δ gene expression could be a potential therapeutic approach in human IBDs. iv To my parents, Jeanne and Chris v ACKNOWLEDGEMENTS To my mentor, Dr. Scott Plevy, I extend heartfelt gratitude for guiding me through the harrowing journey of the PhD. Some parts were messy! But you never gave up on me. Your advice went beyond the science and taught me how to maneuver through the physician scientist’s career while maintaining the drive to help your patients – always your patients were kept at the forefront. For the unending guidance and support during my PhD education, I thank my thesis committee members: Drs. Balfour Sartor, Virginia Miller, Miriam Braunstein, William Goldman, and Christian Jobin who recently moved to another institution. Thank you for your wise advice at all stages of my dissertation. A special thank you to the National Institutes of Health for funding my education (F30 DK089692). To Drs. Taku Kobyashi and Shehzad Sheikh, I owe you everything. You kept me on the right track day to day and were always willing (and loved!) to talk science. You were there for me during some of my lowest moments to pick me up, and you were always there to celebrate the highs. You are not only physician scientists I admire and hope to emulate someday, but you are dear friends. Thank you for investing so much in me – because you did, I know I will be successful in whatever the future brings. To my colleagues who journeyed with me, thank you for your support and friendship: Melissa Ellermann, Steve Russo, the entire 2007 matriculating MD/PhD class (and those who joined later), my dear friends from medical school, and the entire vi MD/PhD program. I am really excited to follow each of your careers, which I know will be as amazing as each of you are. Here’s to OUR future! To both my sets of parents, who I am incredibly lucky to have: thank you! Thank you, Mom and Dad Klein, for your unconditional love and support. I think you may have always known I was destined to be a lifelong student, but you have taken it in stride with a grace for which I am so grateful. Thank you for raising me to become who I am; it hasn’t always been easy, but where’s the fun in an easy ride? I love you more than I can express! Thank you, Mom and Dad Steinbach. You accepted me into your family without question and have been incredibly supportive, even when I dragged your only son to live far away in North Carolina. Thank you for making me feel like one of your own. Most importantly, thank you for Jeff. I suppose you know how special he is, but it’s because of your strong values and love that he is such a wonderful person. To my love, Jeff: even as I write this I know it will never fully express what you have been to me during this time. But allow me to attempt to convey how grateful I am for you, and know that whatever I write, I mean it a thousands times more strongly than it comes across. You are my everything. Thank you for never giving up on me or letting me give up on myself. Thank you for spending endless nights alone while I worked in the lab. Thank you for making dinner without complaining so many nights when I came home physically and mentally exhausted. Thank you for listening to my hopes and fears and for truly sharing them with me so that I didn’t have to feel them alone. This PhD is yours, too, for you shared in all my struggles and triumphs. I love you more than words could ever express! Thank you. Thank you a million times. vii TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................... xiii LIST OF ABBREVIATIONS ....................................................................................... xvi CHAPTER 1 INTRODUCTION ............................................................................................................ 1 1.1 Inflammatory Bowel Diseases ................................................................................ 1 1.2 Macrophages and Dendritic Cells in Innate Immunity ....................................... 3 1.2.1 Macrophages ...................................................................................................... 3 1.2.2 Dendritic Cells ................................................................................................... 5 1.3 Recognition of Pathogen-associated Molecular Patterns .................................... 6 1.3.1 Toll-like Receptors (TLRs) ................................................................................. 7 1.3.2 Nucleotide-binding Oligomerization Domain (NOD), Leucine-rich Repeat (LRR) Receptors (NLRs) ................................................... 8 1.3.3 C-type Lectin Receptors (CLRs) ........................................................................ 9 1.3.4 Retinoic Acid-inducible Gene-1 (RIG-I)-like Receptors (RLRs) ................................................................................................................. 9 1.3.5 Functional Integration of Pathogen Recognition Receptor Signaling ........................................................................................... 10 1.4 Macrophage Intracellular Bactericidal Functions ............................................. 11 1.5 Lamina Propria Mononuclear Cells in the Healthy Gastrointestinal Tract .......................................................................................... 13 1.5.1 Lamina Propria Mononuclear Cells ................................................................ 13 1.5.2 Lamina Propria Macrophages ......................................................................... 15 viii 1.5.3 Lamina Propria Dendritic Cells ...................................................................... 17 1.6 Lamina Propria Mononuclear Cells in IBD ....................................................... 20 1.6.1 Murine Experimental IBD ................................................................................ 20 1.6.2 Human IBDs ..................................................................................................... 27 1.7 Phosphoinositide 3-kinases in Immune Responses ............................................ 29 1.7.1 Structure and Signaling Downstream of the Class IA PI3Ks ................................................................................................................ 31 1.7.2 PI3K p110δ in Innate Immune Cells ................................................................ 32 1.7.3 PI3K p110δ in Adaptive Immune Cells ............................................................ 33 1.8 PI3K p110δ in Intestinal Homeostasis ................................................................ 34 1.9 Figures ...................................................................................................................