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: 8471701
• 负责人: Erin C. Steinbach
• 金额: $ 3.42万
• 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/8471701
• 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; Interleukin-12; Intestines; Label; Laboratories; Lead; Leukocytes; Mannose; Measures; Mediating; Mentors; Microbiology; Modeling; Molecular; Molecular Immunology; Monitor; Mus; Mutant Strains Mice; NADPH Oxidase; Natural Immunity; Nucleotides; Nutrient; Pathogenesis; Pathway interactions; Phagocytosis; Phagolysosome; Phosphatidylinositols; 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; Specific Pathogen Frees; Staining method; Stains; Tissues; Toll-like receptors; Training; Ulcerative Colitis; Virginia; Work; bactericide; base; career; cohort; commensal microbes; cytokine; genetic association; germ free condition; high risk; human disease; in vivo; in vivo Model; killings; knowledge base; macrophage; microbicide; mouse model; mutant; neutrophil; novel; novel therapeutics; pathogenic Escherichia coli; pathogenic bacteria; research study; response; skills

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.

人类炎症性肠病（IBD），克罗恩氏病和溃疡性结肠炎是由对遗传易感宿主的肠性微生物群的不当定向的免疫反应引起的。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，并且这些途径的重要性通过最近描述与人IBD较高风险相关的相关基因中的单核苷酸多晶型ISMS的最新描述强调了。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）p110delta subunit包含一个点突变（p110delta [d910a/d910a]）。有趣的是，p110delta [D910A/D910A]巨噬细胞显示出针对Cencensal K12大肠杆菌，肠疗养NC101大肠杆菌和发病型沙门氏菌的细胞内杀菌活性降低。我获得了令人信服的初步数据，即p110delta [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 p110delta [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p110delta [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种主要效应途径。特定目标2中提出的实验将实现开发一个体内模型系统，以确定有缺陷的杀菌活性对PI3K P110DELTA [D910A/D910A]小鼠的结肠炎的影响。我们将确定P110DELTA [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P110delta [D910A/D910A]小鼠菌落。我们将监测无细菌P110DELTA [D910A/D910A]小鼠，并用肠子微生物殖民地重新殖民以开发结肠炎。 这项工作的重要性是p110delta [D910a/d910a]小鼠自发发生IBD。此外，人类IBD的最新遗传关联强调了了解粘膜先天免疫如何与肠性微生物群相互作用的重要性。有趣的是，人类p110delta基因映射到1p36染色体上的IBD7易感基因座。我们将将一系列基于细胞的研究与新型的体内模型相结合，以实现这些目标。鉴于PI3K p110delta亚基在IBD发病机理的先天免疫过程中的作用，p110delta表达和/或功能的诱导可能代表人IBD中的新型治疗策略。我的最终目标是从知识库的MSTP培训中脱颖而出，以开始作为医生科学家的富有成效的职业。该项目将使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。