Pulmonary Defenses Against Intracellular Infection

描述(申请人提供)：肺炎是发病率和死亡率的主要原因。了解下呼吸道的宿主防御机制对于开发减少这种疾病负担的新策略至关重要。嗜肺军团菌是细菌性肺炎的重要病原，也是肺泡巨噬细胞的胞内致病菌。宿主对LP的防御包括通过膜结合的Toll样受体(TLRs)和胞浆核苷酸结合的亮氨酸富含重复蛋白(NLRs)进行微生物检测，TLRs诱导促炎细胞因子，NLRs组装炎性小体激活caspase-1。Caspase-1将IL-1和IL-18分解为活性形式，促进吞噬小体成熟，并诱导溶血性细胞死亡(下垂)。最近的研究强调了caspase-1在小鼠骨髓来源的巨噬细胞(BMM)抵抗LP细胞内感染中的重要性，但体内炎性小体激活在整合宿主防御中的作用尚未被研究。此外，caspase-1在人AM对LP的抗性中的作用不能通过对其他细胞群体的研究来预测，因为人AM中的炎症体功能是唯一沉默的。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。为了达到这一目的，我们制定了以下具体目标：具体目标#1.确定炎性小体激活在人AM对脂蛋白抵抗中的作用。这一目的将验证NLRC4炎症体的沉默功能是人AM对LP寄生的易感性的假设。本研究的目的是通过与单核细胞和小鼠AM比较，确定人AM的NLRC4炎性小体的配体敏感性，确定LP是否诱导或抑制AM的炎性小体激活，以及刺激NLRC4炎性小体是否诱导人AM对LP产生抵抗。特定目标#2.确定NLRC4炎症体在抵抗肺炎军团菌病中的作用。这一目标将使用空气传播感染的模型基因敲除小鼠和组织培养研究来检验NLR介导的对LP vivo的抵抗涉及细胞特异性的IL-1/IL-18依赖和非独立机制的假设。缺乏一个或多个炎症体成分的小鼠和野生型小鼠将暴露在雾化LP中，并比较细菌清除、细胞死亡和免疫反应。特定目的#3.确定IL-1β和IL-18在LP肺炎耐药中的作用。MyD88是一种介导TLRs和IL-1/IL-18受体信号的适配器，是肺炎军团菌病生存所必需的，但TLRs的缺失不能复制MyD88-/-小鼠的表型。这一目标将使用基因敲除小鼠和阻断抗体、空气传播感染的模型和组织培养研究来检验IL-1和IL-18通过刺激干扰素-？在介导对肺炎军团菌病的耐药性中起关键作用的假设。产生，IL-17反应，并直接增强巨噬细胞对LP的抵抗力。

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.