Legionella-Polymorphonuclear Leukocytes Interaction

项目摘要 多形核白细胞（PMN）/中性粒细胞占外周血白细胞的50-60%， 人类，并且是先天宿主防御微生物感染的重要组成部分。静止PMN 在体外，通过上调葡萄糖受体介导的促炎反应， 有氧糖酵解更重要的是，糖尿病患者的中性粒细胞通过释放促中性粒细胞生成素来应对高血糖。 炎性细胞因子中性粒细胞限制嗜肺军团菌（Lp）的细胞内生长， 导致肺炎的病原体依赖于肺泡巨噬细胞内的病原体增殖。Dot/Icm类型 Lp的IV易位系统，其功能如同分子注射器，将蛋白质效应物注射到 巨噬细胞胞质溶胶以逃避溶酶体融合和巨噬细胞限制病原体增殖。尽管 Dot/Icm介导的效应子易位到PMNs胞质溶胶中，细胞限制Lp增殖。的 PMNs控制微生物感染的两个主要的强大的抗菌机制是产生 活性氧（ROS）和抗微生物剂在嗜天青和特定的颗粒。它 目前尚不清楚两种主要的PMNs抗微生物机制中是否有任何一种参与Lp限制。 PMN在体内对Lp产生促炎反应，但其机制尚不清楚。相比 我们的初步数据表明，PMN迅速降解Lp的WT株，而Dot/Icm 易位缺陷型突变体存活至少4 h。我们发现其中一个Dot/Icm易位 Lp进入人PMNs胞质溶胶的效应物是军团菌淀粉酶（LamA），其降解PMNs 感染后30-60分钟内的糖原。有趣的是，中性粒细胞不能降解lamA缺陷突变体， 不降解PMNs糖原，类似于Dot/Icm易位缺陷突变体。我们将测试 假设人PMNs通过与它们的细胞结合来响应易位的LamA效应物的作用， 抗微生物机制响应于由LamA介导的 糖原降解。为了检验这一假设，我们的具体目标是： PMNs抗微生物机制对LamA作用的响应;和特异性目标II：促炎性 响应于通过LamA的糖原降解产生的细胞溶质高葡萄糖，激活PMN。 在完成建议的研究后，我们将了解先天免疫反应的机制， 人中性粒细胞对脂蛋白的反应异常高水平的胞质葡萄糖。重要的是，我们的研究 广泛的意义，因为它们将与糖尿病条件下的先天免疫机制相关， 失调性炎症是常见的持续发生。我们提出的研究是新颖的， 机制，并将产生一个新的范例，在我们的知识中性粒细胞的先天免疫反应， 细菌病原体

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.