The human inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, result from an inappropriately directed immune response to enteric microbiota in a genetically susceptible host. Macrophages are essential for the recognition, phagocytosis and clearance of commensal and pathogenic bacteria in the intestine. Alterations in autophagy and phagosomal function have emerged as a central focus in macrophage ability to eradicate intracellular bacteria, and the importance of these pathways is highlighted by recent descriptions of single nucleotide polymorph isms in related genes that are associated with a higher risk for human IBD. Here, we describe spontaneous colitis in a novel mouse model of IBD where the phosphatidylinositol-3-kinase (PI3K) p110delta subunit contains a point mutation (p110delta[D910A/D910A]). Interestingly, p110delta[D910A/D910A] macrophages show decreased intracellular bactericidal activity against commensal K12 E. coli, enteroadherent NC101 E. coli, and pathogenic Salmonella typhimurium. I have obtained compelling preliminary data that phagolysosome formation in p110delta[D910A/D910A] macrophages is defective. This project seeks to explore bactericidal defects in PI3K p110delta[D910A/D910A] macrophages. In Specific Aim 1, we will characterize phagolysosome maturation and NADPH oxidase activity in p110delta[D910A/D910A] macrophages, two major effector pathways of macrophage bactericidal activity. Experiments proposed in Specific Aim 2 will achieve the important goal of developing an in vivo model system for determining the impact of defective bactericidal activity on the development of colitis in PI3K p110delta[D910A/D910A] mice. We will determine if p110delta[D910A/D910A] mice have increased bacterial load in tissues, including colonic macrophages. We will also develop a germ-free p110delta[D910A/D910A] mouse colony. We will monitor germ-free p110delta[D910A/D910A] mice and those re-colonized with enteric micro biota for the development of colitis. The significance of this work is that p110delta[D910A/D910A] mice develop spontaneously occurring IBD. Furthermore, recent genetic associations in human IBD emphasize the importance of understanding how mucosal innate immunity interacts with the enteric microbiota. Interestingly, the human p110delta gene maps to the IBD7 susceptibility locus on chromosome 1p36. We will combine a series of cell-based studies with a novel in vivo model to accomplish these goals. Given the role of the PI3K p110delta subunit in innate immune processes fundamental to the pathogenesis of IBD, induction of p110delta expression and/or function may represent novel therapeutic strategies in human IBD. My ultimate goal is to emerge from MSTP training with the knowledge base to begin a productive career as a physician-scientist. This project will give me a comprehensive background in molecular immunology and microbiology, while applying these skills to the study of a debilitating group of human diseases, the IBDs.

PROJECT NARRATIVE STATEMENT Interactions between macrophages and enteric micro biota have emerged as early and important events in inflammatory bowel disease (IBD) pathogenesis. Decreased clearance of otherwise non-pathogenic bacteria in PI3K p110delta-mutant enteric macrophages may potentiate chronic immune signaling and lead to IBD. In this proposal, we aim to determine the molecular basis of bactericidal defects that we describe in p110delta-mutant macrophages and relate this to the pathogenesis of colitis in this novel experimental model.