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 polymorphisms 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) p110( subunit contains a point mutation (p110d[D910A/D910A]). Interestingly, p110d[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 p110d[D910A/D910A] macrophages is defective. This project seeks to explore bactericidal defects in PI3K p110d[D910A/D910A] macrophages.
In Specific Aim 1, we will characterize phagolysosome maturation and NADPH oxidase activity in p110d[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 p110d[D910A/D910A] mice. We will determine if p110d[D910A/D910A] mice have increased bacterial load in tissues, including colonic macrophages. We will also develop a germ-free p110d[D910A/D910A] mouse colony. We will monitor germ-free p110d[D910A/D910A] mice and those re-colonized with enteric microbiota for the development of colitis. The significance of this work is that p110(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 p110??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 p110? subunit in innate immune processes fundamental to the pathogenesis of IBD, induction of p110( 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.
Interactions between macrophages and enteric microbiota have emerged as early and important events in inflammatory bowel disease (IBD) pathogenesis. Decreased clearance of otherwise non-pathogenic bacteria in PI3K p110d-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 p110d-mutant macrophages and relate this to the pathogenesis of colitis in this novel experimental model.
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|Uno, Jennifer K; Rao, Kavitha N; Matsuoka, Katsuyoshi et al. (2010) Altered macrophage function contributes to colitis in mice defective in the phosphoinositide-3 kinase subunit p110?. Gastroenterology 139:1642-53, 1653.e1-6|