Pl3K Regulation of Microbicidal Pathways in Macrophages in Experimental Colitis

人类炎症性肠病(IBD)，克罗恩病和溃疡性结肠炎，是由于遗传易感宿主对肠道微生物区系的不适当定向免疫反应造成的。巨噬细胞对于识别、吞噬和清除肠道内的共生菌和病原菌是必不可少的。自噬和吞噬功能的改变已经成为巨噬细胞清除细胞内细菌能力的中心焦点，最近对相关基因的单核苷酸多态的描述突显了这些途径的重要性，这些基因与人类IBD的风险更高。在这里，我们描述了一种新的IBD小鼠模型中的自发性结肠炎，其中磷脂酰肌醇-3-激酶(PI3K)p110Delta亚单位包含一个点突变(p110Delta[D910A/D910A])。有趣的是，p110 Delta[D910A/D910A]巨噬细胞对共生K12大肠杆菌、肠粘附性NC101大肠杆菌和致病性鼠伤寒沙门氏菌显示出较低的细胞内杀菌活性。我已经获得了令人信服的初步数据，即p110Delta[D910A/D910A]巨噬细胞的吞噬溶酶体形成存在缺陷。 本项目旨在探索PI3K p110Delta[D910A/D910A]巨噬细胞的杀菌缺陷。在特定的目标1中，我们将表征巨噬细胞的吞噬酶体成熟和NADPH氧化酶活性，这是巨噬细胞杀菌活性的两个主要效应途径。在特定目标2中提出的实验将实现开发体内模型系统的重要目标，以确定杀菌活性缺陷对PI3K p110Delta[D910A/D910A]小鼠结肠炎发展的影响。我们将确定p110delta[D910A/D910A]小鼠是否增加了组织中的细菌负荷，包括结肠巨噬细胞。我们还将开发一个无菌的p110 Delta[D910A/D910A]小鼠群体。我们将监测无菌p110Delta[D910A/D910A]小鼠和那些重新定居的肠道微生物群小鼠的结肠炎发展情况。 这项工作的意义在于p110 delta[D910A/D910A]小鼠发展为自发发生的IBD。此外，最近在人类IBD中的遗传关联强调了了解粘膜天然免疫如何与肠道微生物区系相互作用的重要性。有趣的是，人类p110Delta基因定位于染色体1p36上的IBD7易感基因。我们将结合一系列基于细胞的研究和一种新的体内模型来实现这些目标。鉴于PI3K p110 Delta亚基在IBD发病机制中的先天免疫过程中的作用，诱导p110 Delta的表达和/或功能可能代表着人类IBD的新治疗策略。我的最终目标是从MSTP培训中走出来，拥有知识基础，开始作为一名内科科学家的富有成效的职业生涯。这个项目将让我在分子免疫学和微生物学方面有一个全面的背景，同时将这些技能应用于研究一组令人衰弱的人类疾病--IBDS。

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.