Innate immunity and inflammatory response of macrophages to Legionella infection

项目摘要 根据体外的信号，巨噬细胞可以进行瞬时和可逆的分化 分为两个主要子集：促炎“M1”(经典激活)或抗炎“M2” (交替激活的)表型。巨噬细胞暴露于高水平葡萄糖的体外实验 或在体内的糖尿病患者中，它们经历分化和激活为促炎症的M1 通过细胞代谢重编程产生促炎细胞因子的状态 通过上调有氧糖酵解。巨噬细胞的M1促炎分化是 先天免疫反应的一个重要分支，用于控制细菌感染。我们的初步数据 显示当巨噬细胞吞噬细胞内细菌嗜肺军团菌时 (LP)，它们通过分化为活化的M1样促炎表型而迅速反应。 尽管巨噬细胞有快速的促炎活性，但含有军团菌的空泡 (LCV)通过限制其与溶酶体的融合来绕过巨噬细胞的激活。我们证明了早期的 人单核细胞来源的巨噬细胞(HMDM)向M1样促炎分化 LP是一种对胞浆高血糖的先天性免疫反应，通过快速降解 注射军团菌淀粉酶(LAMA)效应器的糖原。我们发现LP已经进化成 注射MAVE在促炎性hMDM内的溶酶体降解旁路途径 效应器，定位于LCV膜胞质侧的微区。 Hmdms将MAVE缺陷突变体定位于溶酶体进行降解，突变体 在体外和体内都是完全衰减的。我们解析出了新颖的三维晶体结构 MAVE，它显示了一个胞浆表面暴露的结构域，涉及一个NPXY真核基序 与磷酸酪氨酸结合接头蛋白结合。我们的中心假设是：类似M1的PRO- HMDM对LP感染的炎症激活是对胞浆的一种先天免疫反应 LAMA诱导高血糖，病原菌利用MAVE绕过先天免疫 溶酶体降解的巨噬细胞途径。为了检验这一假设，我们的具体目标是： 特异性目标I：HMDM的M1样促炎分化机制 对喇嘛效应的反应；和特定目标II：先天的逃避机制 巨噬细胞利用溶酶体旁路降解溶酶体的功能 MAVE介导的途径。当我们完成建议的研究后，我们会发现 巨噬细胞对脂蛋白和溶酶体的先天促炎反应机制 由病原体操纵的降解旁路以避免致命的命运。

Project Summary Depending on the signals in vitro, macrophages can undergo transient and reversible differentiation into two main subsets: pro-inflammatory “M1” (classically activated) or anti-inflammatory “M2” (alternatively activated) phenotype. Upon exposure of macrophages to high levels of glucose in vitro or in diabetic patients in vivo, they undergo differentiation and activation into a M1 pro-inflammatory state, which produces pro-inflammatory cytokines mediated by cellular reprogramming of metabolism through upregulating aerobic glycolysis. The M1 pro-inflammatory differentiation of macrophages is an import arm of the innate immune response to control bacterial infections. Our preliminary data show that when macrophages engulf the intracellular bacterial pathogen Legionella pneumophila (Lp), they rapidly respond by differentiation into an activated M1-like pro-inflammatory phenotype. Despite this rapid macrophage pro-inflammatory activation, the Legionella-containing vacuole (LCV) bypasses macrophage activation by limiting its fusion to lysosomes. We show that the early M1-like pro-inflammatory differentiation of human monocytes-derived macrophages (hMDMs) to Lp is an innate immune response to cytosolic hyper-glucose generated through rapid degradation of glycogen by the injected Legionella amylase (LamA) effector. We discovered that Lp has evolved a lysosomal degradation bypass pathway within the pro-inflammatory hMDMs by injecting the MavE effector, which becomes localized in a micro-domain on the cytosolic side of the LCV membrane. The mavE-deficient mutant is targeted by hMDMs to the lysosomes for degradation, and the mutant is totally attenuated in vitro and in vivo. We have resolved the novel three dimensional crystal structure of MavE, which shows a cytosolic surface-exposed domain with an NPxY eukaryotic motif involved in binding to phosphotyrosine-binding adaptor proteins. Our central hypothesis is: the M1-like pro- inflammatory activation of hMDMs to Lp infection is an innate immune response to the cytosolic hyper-glucose elicited by LamA, and the pathogen utilizes MavE to bypass the innate immune macrophage pathway of lysosomal degradation. To test the hypothesis, our specific aims are: Specific Aim I: The mechanisms of M1-like pro-inflammatory differentiation of hMDMs in response to the effect of LamA; and Specific Aim II: The mechanisms of evasion of the innate macrophage function of lysosomal degradation through utilization of a lysosomes bypass pathway mediated by MavE. Upon completion of our proposed studies, we will discover the mechanism of the innate pro-inflammatory response of macrophages to Lp and the lysosomal degradation bypass pathway maneuvered by the pathogen to avoid a fatal fate.