Legionella-Polymorphonuclear Leukocytes Interaction

项目摘要 多形核白细胞(PMN)/中性粒细胞占中国人外周血白细胞的50-60% 人类，是抵御微生物感染的天然宿主防御系统的重要组成部分。静止的PMN 在体外通过上调促炎症反应对高水平的葡萄糖作出反应 有氧糖酵解。重要的是，中性粒细胞对糖尿病患者高血糖的反应是通过释放前体 炎性细胞因子。PMN抑制嗜肺军团菌胞内生长，其能力 肺炎的发生依赖于病原体在肺泡巨噬细胞内的增殖。Dot/ICM类型 LP的IV易位系统起到分子注射器的作用，将蛋白质效应器注入到 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介导的效应物移位到PMN胞浆中，细胞限制LP的增殖。这个 控制微生物感染的PMN的两个主要强大的抗菌机制是 活性氧物种(ROS)和抗菌剂在亲天蓝颗粒和特定颗粒中。它 目前尚不清楚两个主要的PMN抗菌机制中是否有任何一个参与了LP限制。 中性粒细胞在体内对LP具有促炎反应，但其机制尚不清楚。与之形成鲜明对比的是 巨噬细胞，我们的初步数据显示，PMN迅速降解LP的WT株，而Dot/ICM 易位缺陷突变体至少存活4小时。我们发现其中一个Dot/ICM-移位 Lp进入人PMN胞浆的效应物是一种军团菌淀粉酶(Legionella amylase，LAMA)，它能降解PMN 感染后30-60分钟内的糖原。有趣的是，PMN不能降解LAMA缺陷突变体，这是 不降解PMNS糖原，类似于Dot/ICM易位缺陷突变体。我们将测试 假设人多形核细胞对移位的LAMA效应器的作用是通过激活它们的 抗菌机制对LAMA介导的胞浆高糖的响应 糖原的降解。为了检验这一假设，我们的具体目标是：具体目标一：参与 针对LAMA效应的PMN抗菌机制；和特定目标II：促炎 中性粒细胞对LAMA通过糖原降解产生的胞浆高糖的反应。 当建议的研究完成后，我们将会了解先天免疫反应的机制 人中性粒细胞对胞浆葡萄糖异常高水平的反应。重要的是，我们的研究已经 广泛的意义，因为它们将与糖尿病条件下的先天性免疫机制相关， 调节失调的炎症是一种常见的持续性事件。我们提出的研究既是新颖的，也是 机制，并将在我们的PMN先天免疫反应的知识中产生一个新的范例 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.