Pl3K Regulation of Microbicidal Pathways in Macrophages in Experimental Colitis

描述（由申请人提供）：人类炎症性肠病（IBD）、克罗恩病和溃疡性结肠炎是由遗传易感宿主对肠道微生物群的不适当定向免疫应答引起的。巨噬细胞对于肠道中细菌和致病菌的识别、吞噬和清除是必不可少的。自噬和吞噬体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，并且这些途径的重要性通过最近对与人类IBD风险较高相关的相关基因中的单核苷酸多态性的描述而突出。在此，我们描述了一种新型IBD小鼠模型中的自发性结肠炎，其中磷脂酰肌醇-3-激酶（PI3K）p110亚基含有点突变（p110d [D910A/D910A]）。有趣的是，p110d [D910A/D910A]巨噬细胞显示出对大肠杆菌K12 E的细胞内杀菌活性降低。大肠杆菌、肠粘附性大肠杆菌NC101。大肠杆菌和致病性鼠伤寒沙门氏菌。我已经获得了令人信服的初步数据，即p110d [D910A/D910A]巨噬细胞中吞噬溶酶体的形成是有缺陷的。 该项目旨在探索PI3K p110d [D910A/D910A]巨噬细胞中的杀菌缺陷。在特定目标1中，我们将描述p110 d [D910A/D910A]巨噬细胞中吞噬溶酶体成熟和NADPH氧化酶活性的特征，这是巨噬细胞杀菌活性的两个主要效应途径。具体目标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 p110 d [D910A/D910A]小鼠结肠炎发展的影响。我们将确定p110d [D910A/D910A]小鼠组织中的细菌负荷是否增加，包括结肠巨噬细胞。我们还将开发无菌p110d [D910A/D910A]小鼠群体。我们将监测无菌p110d [D910A/D910A]小鼠和肠道微生物群重新定植的小鼠结肠炎的发展。 这项工作的意义在于p110（D910A/D910A）小鼠自发发生IBD。此外，最近人类IBD的遗传关联强调了理解粘膜先天免疫如何与肠道微生物群相互作用的重要性。有趣的是，人类p110？基因定位于染色体1p36上的IBD 7易感性位点。我们将结合联合收割机一系列基于细胞的研究与一种新的体内模型来实现这些目标。鉴于PI3K p110的作用？由于p110是IBD发病机制的基础性先天免疫过程中的亚基，因此诱导p110的表达和/或功能可能代表人类IBD的新治疗策略。我的最终目标是从MSTP培训中脱颖而出，拥有知识基础，开始作为一名医生科学家的富有成效的职业生涯。这个项目将使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使人衰弱的人类疾病，IBD。

DESCRIPTION (provided by applicant): 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.