MACROPHAGE PHOSPHOINOSITIDE 3-KINASE P110δ REGULATES INTESTINAL HOMEOSTASIS by DIRECTING ADAPTIVE IMMUNITY and ENHANCING MICROBIAL CLEARANCE

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.

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.

To my parents, Jeanne and Chris

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. Y