Pulmonary Defenses Against Intracellular Infection

描述（由申请人提供）：肺炎是发病率和死亡率的主要原因。了解宿主在下呼吸道的防御机制对于开发新的策略来减轻这种疾病的负担是必不可少的。嗜肺军团菌（Legionella pneumophila, Lp）是细菌性肺炎的重要病因，也是肺泡巨噬细胞（alveolar macrophages， AM）的胞内病原体。宿主对Lp的防御包括通过膜结合的Toll样受体（TLRs）和细胞质核苷酸结合的富亮氨酸重复蛋白（NLRs）进行微生物检测，前者可诱导促炎细胞因子，后者可组装激活半胱天冬酶-1的炎性小体。Caspase-1将白细胞介素-1 （IL-1）和IL-18切割成活性形式，促进吞噬体成熟，并诱导溶解性细胞死亡（焦亡）。最近的研究强调了caspase-1对小鼠骨髓源性巨噬细胞（BMM）抵抗细胞内Lp感染的重要性，但炎症体激活对体内综合宿主防御的贡献尚未得到研究。此外，caspase-1在人类AM对Lp的抗性中的作用无法从其他细胞群的研究中预测，因为人类AM的炎性体功能是唯一的沉默。该项目的总体目标是了解关键的细菌识别事件如何整合到体内有效的免疫反应中，并确定这些发现与人类疾病的相关性。为实现这一目标，我们制定了以下具体目标：确定炎症小体活化在人AM对Lp的抵抗中的作用。这一目的将验证NLRC4炎性小体的静音功能是人类AM对Lp寄生的易感性的基础。本研究旨在确定与单核细胞和小鼠AM相比，人AM NLRC4炎症小体的配体敏感性，确定Lp是否诱导或抑制AM中的炎症小体激活，并确定NLRC4炎症小体的刺激是否诱导人AM对Lp的抗性。具体目标2。确定NLRC4炎性体在肺炎军团菌病抵抗中的作用。该目标将使用敲除小鼠，空气传播感染模型和组织培养研究来验证nlr介导的Lp体内抗性涉及细胞特异性IL-1 / il -18依赖和独立机制的假设。缺乏一种或多种炎症小体成分的小鼠和野生型小鼠将暴露于雾化Lp中，并比较细菌清除率、细胞死亡和免疫反应。具体目标#3。确定IL-1¿和IL-18在Lp肺炎耐药中的作用。MyD88是一种介导tlr和IL-1/IL-18受体信号传导的接头，是肺炎军团菌病存活所必需的，但tlr缺乏不会重现MyD88-/-小鼠的表型。该目标将使用敲除小鼠和阻断抗体，空气传播感染模型和组织培养研究来验证IL-1和IL-18通过刺激干扰素-18在介导肺炎军团菌病抗性中发挥关键作用的假设。产生，IL-17反应，并直接增加巨噬细胞对Lp的抗性。

DESCRIPTION (provided by applicant): Pneumonia is a leading cause of morbidity and mortality. Understanding mechanisms of host defense in the lower respiratory tract is essential for the development of novel strategies to reduce this burden of disease. Legionella pneumophila (Lp) is an important cause of bacterial pneumonia and an intracellular pathogen of alveolar macrophages (AM). Host defense against Lp involves microbial detection by membrane-bound Toll- like receptors (TLRs), which induce pro-inflammatory cytokines, and by cytosolic nucleotide-binding leucine rich repeat proteins (NLRs), which assemble inflammasomes that activates caspase-1. Caspase-1 cleaves interleukin-1¿ (IL-1¿) and IL-18 to the active forms, favors phagosome maturation, and induces lytic cell death (pyroptosis). Recent studies have highlighted the importance of caspase-1 to the resistance of mouse bone marrow-derived macrophages (BMM) to intracellular infection with Lp, but the contribution of inflammasome activation to integrated host defense in vivo has not been studied. Furthermore, the role of caspase-1 in the resistance of human AM to Lp cannot be predicted from studies with other cell populations, as inflammasome function in human AM is uniquely muted. The overall goal of this project is to understand how key bacterial recognition events are integrated into effective immune responses in vivo, and to determine the relevance of these findings for human disease. To achieve this end we have formulated the following specific aims: Specific aim #1. Determine role of inflammasome activation in the resistance of human AM to Lp. This aim will test the hypothesis that muted function of the NLRC4 inflammasome underlies the susceptibility of human AM to parasitism by Lp. This aim will determine the ligand sensitivity of the NLRC4 inflammasome of human AM in comparison with monocytes and murine AM, determine if Lp induces or suppresses inflammasome activation in AM, and determine if stimulation of the NLRC4 inflammasome induces resistance of human AM to Lp. Specific aim #2. Determine the role of the NLRC4 inflammasome in resistance to pneumonic legionellosis. This aim will use knockout mice, a model of airborne infection, and tissue culture studies to test the hypotheses that NLR-mediated resistance to Lp vivo involves both IL-1¿/IL-18-dependent and -independent mechanisms that are cell-specific. Mice with lacking one or more inflammasome component and wild type mice will be exposed to aerosolized Lp and compared for bacterial clearance, cell death, and immune responses. Specific aim #3. Determine the roles of IL-1¿ and IL-18 in resistance to Lp pneumonia. MyD88, an adaptor that mediates signaling from TLRs and IL-1/IL-18 receptors, is required for survival from pneumonic legionellosis, but deficiencies of TLRs do not reproduce the phenotype of MyD88-/- mice. This aim will use knockout mice and blocking antibodies, a model of airborne infection, and tissue culture studies to test the hypothesis that IL-1¿ and IL-18 play key roles in mediating resistance to pneumonic legionellosis by stimulating interferon-? production, IL-17 responses, and directly augmenting macrophage resistance to Lp.