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。了解下呼吸道中宿主防御机制对于开发新策略以减少这种疾病负担至关重要。嗜肺军团菌（Legionella pneumophila，Lp）是细菌性肺炎的重要病原体，也是肺泡巨噬细胞（alveolar macrophages，AM）的胞内病原体。宿主对Lp的防御涉及通过膜结合的Toll样受体（TLR）和细胞溶质核苷酸结合的富含亮氨酸的重复蛋白（NLR）进行微生物检测，Toll样受体（TLR）诱导促炎细胞因子，NLR组装激活半胱天冬酶-1的炎性体。半胱天冬酶-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（细胞凋亡）。最近的研究强调了caspase-1对小鼠骨髓源性巨噬细胞（BMM）抵抗Lp细胞内感染的重要性，但尚未研究炎性小体激活对体内整合宿主防御的贡献。此外，胱天蛋白酶-1在人AM对Lp的抗性中的作用不能从其他细胞群体的研究中预测，因为人AM中的炎性小体功能是独特的沉默。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。为了实现这一目标，我们制定了以下具体目标：具体目标1。确定炎性小体活化在人AM对Lp抵抗中的作用。这一目的将检验NLRC 4炎性小体的沉默功能是人AM对Lp寄生的易感性的基础的假设。该目的将确定与单核细胞和鼠AM相比，人AM的NLRC 4炎性体的配体敏感性，确定Lp是否诱导或抑制AM中的炎性体活化，并确定NLRC 4炎性体的刺激是否诱导人AM对Lp的抗性。具体目标#2确定NLRC 4炎性小体在抵抗肺炎性军团菌病中的作用。该目标将使用基因敲除小鼠（空气传播感染模型）和组织培养研究来测试NLR介导的对Lp体内抗性涉及细胞特异性的IL-1 β/IL-18依赖性和非依赖性机制的假设。将缺乏一种或多种炎性小体组分的小鼠和野生型小鼠暴露于雾化的Lp，并比较细菌清除、细胞死亡和免疫应答。具体目标#3确定IL-1和IL-18在抵抗Lp肺炎中的作用。MyD88是一种介导TLR和IL-1/IL-18受体信号传导的衔接子，是肺炎性军团菌病生存所必需的，但TLR的缺陷不会复制MyD88-/-小鼠的表型。这个目标将使用基因敲除小鼠和封闭抗体，空气传播感染的模型，和组织培养研究，以测试假设，IL-1和IL-18发挥关键作用，通过刺激干扰素介导的耐药性肺军团菌病？产生、IL-17应答和直接增强巨噬细胞对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.