Pl3K Regulation of Microbicidal Pathways in Macrophages in Experimental Colitis

实验性结肠炎巨噬细胞中 Pl3K 杀菌途径的调节

• 批准号: 8000525
• 负责人: Erin C. Steinbach
• 金额: $ 2.97万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000525
• 关键词: 1-Phosphatidylinositol 3-Kinase; 1p36; Agar; Animal Model; Attenuated; Autophagocytosis; Bacteria; Bacteriophages; Biological Assay; Biological Models; Biota; Cathepsins; Cells; Chromosome Mapping; Chromosomes; Chronic; Colitis; Crohn' s disease; Data; Defect; Development; Enteral; Enterobacteriaceae; Epithelial; Escherichia coli K12; Event; Experimental Models; Genes; Genetically Engineered Mouse; Gentamicins; Germ-Free; Goals; Human; IGF Type 2 Receptor; Immune; Immune response; Immune system; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Intestines; Label; Laboratories; Lead; Leukocytes; Mannose; Measures; Mediating; Mentors; Microbiology; Modeling; Molecular; Molecular Immunology; Monitor; Mus; Mutant Strains Mice; NADPH Oxidase; Natural Immunity; Nutrient; Pathogenesis; Pathway interactions; Phagocytosis; Phagolysosome; Phosphatidylinositols; Phosphotransferases; Physicians; Point Mutation; Population; Positioning Attribute; Predisposition; Process; Production; Proteins; Reactive Oxygen Species; Receptor Signaling; Regulation; Risk; Role; Salmonella typhimurium; Scientist; Series; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Specific Pathogen Frees; Staining method; Stains; Tissues; Toll-like receptors; Training; Ulcerative Colitis; Virginia; Work; bactericide; base; career; cohort; cytokine; genetic association; germ free condition; high risk; human disease; in vivo; in vivo Model; killings; knowledge base; macrophage; microbicide; mouse model; mutant; novel; novel therapeutics; pathogenic Escherichia coli; pathogenic bacteria; public health relevance; research study; response; skills

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.

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，是由于对遗传易感宿主中对肠性微生物群的免疫反应不当引起的。巨噬细胞对于肠道中的共生和致病细菌的识别，吞噬作用以及清除率至关重要。自噬和吞噬功能的改变已成为巨噬细胞消除细胞内细菌能力的中心重点，这些途径的重要性突显了相关基因中单个核苷酸多态性与人类IBD较高风险相关的相关基因中的单个核苷酸多态性的重要性。在这里，我们在IBD的新型小鼠模型中描述了自发性结肠炎，其中磷脂酰肌醇-3-激酶（PI3K）p110（亚基包含一个点突变（p110d [d910a/d910a]）。肠巢NC101大肠杆菌和致病性沙门氏菌i已获得令人信服的初步数据，即p110d [D910A/D910A]巨噬细胞中的吞噬溶血体形成是有缺陷的。 该项目旨在探索PI3K P110D [D910A/D910A]巨噬细胞中的杀菌缺陷。在特定的目标1中，我们将表征p110d [D910A/D910A]巨噬细胞中的吞噬酶体成熟和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。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，以确定有缺陷的杀菌活性对PI3K P110D [D910A/D910A]小鼠中结肠炎发展的影响。我们将确定P110D [D910A/D910A]小鼠在包括结肠巨噬细胞在内的组织中的细菌负荷增加。我们还将开发无菌的P110D [D910A/D910A]小鼠菌落。我们将监测无细菌P110D [D910A/D910A]小鼠，并用肠菌群重新殖民以开发结肠炎的小鼠。 这项工作的重要性是p110（D910a/d910a小鼠出现自发发生。通过新的体内模型的基于细胞的研究，可以实现PI3K P110的作用？我在分子免疫学和微生物学方面具有全面的背景，同时将这些技能应用于IBD的一个使人衰弱的人类疾病组。