Legionella-Polymorphonuclear Leukocytes Interaction

军团菌-多形核白细胞相互作用

• 批准号: 10057609
• 负责人: Yousef A Abu Kwaik
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-17 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057609
• 关键词: Alveolar Macrophages; Amylases; Animals; Bacteria; Bacterial Infections; Biopsy; Cells; Cytoplasmic Granules; Cytosol; Data; Exposure to; Generations; Glucose; Glycogen; Growth; Host Defense; Human; Hyperglycemia; Immune; In Vitro; Infection; Infection Control; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Knowledge; Learning; Legionella; Legionella pneumophila; Leukocytes; Mediating; Metabolic; Metabolism; Molecular; NADPH Oxidase; Natural Immunity; Patients; Phagolysosome; Phagosomes; Pneumonia; Production; Proliferating; Proteins; Pulmonary alveolar structure; Reactive Oxygen Species; Reporting; Syringes; System; Testing; Up-Regulation; aerobic glycolysis; antimicrobial; antimicrobial drug; cytokine; diabetic; diabetic patient; glycogenolysis; in vivo; innate immune mechanisms; macrophage; microbial; monocyte; mutant; neutrophil; novel; pathogen; pathogenic bacteria; peripheral blood; response

Project Summary Polymorphonuclear leukocytes (PMNs)/Neutrophils account for 50-60% of peripheral blood leukocytes in humans, and are an essential part of the innate host defense against microbial infections. Quiescent PMNs respond to high levels of glucose in vitro through a pro-inflammatory response mediated by up-regulation of aerobic glycolysis. Importantly, PMNs respond to hyperglycemia in diabetic patients by releasing pro- inflammatory cytokines. PMNs restrict intracellular growth of Legionella pneumophila (Lp), whose ability to cause pneumonia is dependent on pathogen proliferation within alveolar macrophages. The Dot/Icm type IV translocation system of Lp, which functions as a molecular syringe, injects protein effectors into the macrophage cytosol to evade lysosomal fusion and macrophage restriction of pathogen proliferation. Despite the Dot/Icm-mediated translocation of effectors into the PMNs cytosol, the cells restrict Lp proliferation. The two main powerful antimicrobial machineries of PMNs to control microbial infections are the generation of reactive oxygen species (ROS) and the antimicrobial agents within the azurophilic and specific granules. It is not known whether any of the two major PMNs antimicrobial machineries are involved in Lp restriction. The PMNs mount a pro-inflammatory response to Lp in vivo, but the mechanism is not known. In contrast to macrophages, our preliminary data show that PMNs rapidly degrade the WT strain of Lp, while the Dot/Icm translocation-defective mutant survives for at least 4h. We discovered that one of the Dot/Icm-translocated effectors of Lp into the cytosol of human PMNs is a Legionella amylase (LamA), which degrades the PMNs glycogen within 30-60 min of infection. Interestingly, PMNs fail to degrade the lamA-deficient mutant, which does not degrade PMNs glycogen, similar to the Dot/Icm translocation-defective mutant. We will test the hypothesis that human PMNs respond to the effect of the translocated LamA effector by engaging their antimicrobial machineries in response to the cytosolic hyper-glucose generated by the LamA-mediated degradation of glycogen. To test the hypothesis, our specific aims are: Specific Aim I: Engagement of the PMNs antimicrobial machineries in response to the effect of LamA; and Specific Aim II: Pro-inflammatory activation of PMNs in response to cytosolic hyper-glucose generated through glycogen degradation by LamA. Upon completion of the proposed studies, we will learn the mechanism of the innate immune response of human PMNs to Lp in response to abnormally high levels of cytosolic glucose. Importantly, our studies have broad significance as they will be relevant to mechanisms of innate immunity under diabetic conditions where dysregulated inflammation is a common persistent occurrence. Our proposed studies are novel as well as mechanistic, and would generate a new paradigm in our knowledge of PMNs innate immune responses to bacterial pathogens.

项目概要 多形核白细胞 (PMN)/中性粒细胞占外周血白细胞的 50-60% 人类，是宿主抵抗微生物感染的先天防御的重要组成部分。静态中性粒细胞 在体外通过上调介导的促炎症反应对高水平葡萄糖做出反应 有氧糖酵解。重要的是，中性粒细胞通过释放促胰岛素来应对糖尿病患者的高血糖。 炎症细胞因子。 PMN 限制嗜肺军团菌 (Lp) 的细胞内生长，其能力 引起肺炎取决于肺泡巨噬细胞内病原体的增殖。点/Icm 型 Lp 的 IV 易位系统起到分子注射器的作用，将蛋白质效应子注入 巨噬细胞胞浆逃避溶酶体融合和巨噬细胞对病原体增殖的限制。尽管 Dot/Icm 介导的效应器易位至 PMN 胞质中，细胞限制 Lp 增殖。这 PMN 控制微生物感染的两个主要强大抗菌机制是产生 活性氧 (ROS) 和亲天青颗粒和特定颗粒内的抗菌剂。它 目前尚不清楚两种主要的 PMN 抗菌机制是否参与 Lp 限制。 PMN 在体内对 Lp 产生促炎症反应，但其机制尚不清楚。相比之下 巨噬细胞，我们的初步数据表明 PMN 快速降解 Lp 的 WT 菌株，而 Dot/Icm 易位缺陷突变体存活至少 4 小时。我们发现 Dot/Icm 易位之一 Lp 进入人中性粒细胞胞浆的效应器是军团菌淀粉酶 (LamA)，它能降解中性粒细胞 感染后 30-60 分钟内的糖原。有趣的是，PMN 无法降解 lamA 缺陷突变体，这 与 Dot/Icm 易位缺陷突变体类似，不会降解 PMN 糖原。我们将测试 假设人类 PMN 通过参与其自身来响应易位的 LamA 效应子的作用 抗菌机制响应 LamA 介导的胞质高葡萄糖 糖原的降解。为了检验这一假设，我们的具体目标是： 具体目标 I： PMNs 抗菌机制响应 LamA 的作用；具体目标 II：促炎 PMN 响应 LamA 糖原降解产生的胞质高葡萄糖而激活。 完成拟议的研究后，我们将了解先天免疫反应的机制 人类中性粒细胞（PMN）对脂蛋白（Lp）的反应是对异常高水平的胞浆葡萄糖的反应。重要的是，我们的研究 具有广泛的意义，因为它们与糖尿病条件下的先天免疫机制有关，其中 炎症失调是一种常见的持续现象。我们提出的研究既新颖又 机制，并将在我们对 PMN 先天免疫反应的认识中产生一个新的范式 细菌病原体。