Neutrophil Accumulation in Bacterial Pneumonia

描述（由申请人提供）：肺部感染是世界范围内发病率、死亡率和医疗费用的主要原因。病原体从呼吸道的快速清除是依赖于有效的先天免疫反应在肺。了解下呼吸道的先天防御机制对于开发新的治疗和/或预防策略以减轻这种疾病的负担至关重要。触发先天免疫反应的信号级联包括促进病原体清除的促炎反应和抑制控制过度全身炎症宿主反应的抗炎反应之间的微妙平衡。一般来说，人们对这些途径如何汇聚在一起调节宿主防御，同时最大限度地减少炎症组织损伤知之甚少。这项研究的长期目标是了解多种先天免疫事件如何整合到有效的抗菌素耐药性中。作为阐明宿主基本防御机制的模型，我们将重点放在了一种主要病原体肺炎克雷伯菌上，因为这种细胞外革兰氏阴性菌会导致严重的肺炎；以及多重耐药肺炎克雷伯菌菌株在世界范围内的广泛传播。尽管肺炎克雷伯菌通过toll样受体(TLR)-4和9发出信号，但一些研究也表明nod样受体（NLRs）作为细胞质免疫传感器参与其中。一些被称为“炎性小体”的nlr可以激活caspase-1以切割IL-1和IL-18。我们之前已经证明NLR家族CARD结构域包含4 （NLRC4； IPAF）作为人类和小鼠巨噬细胞中肺炎克雷伯菌的新传感器，调节炎性细胞因子，IL1¿和IL18依赖caspase-1的成熟。IL-1R1敲除（KO）小鼠比NLRC4 KO小鼠对肺内肺炎克雷伯菌感染的易感性更高，提示其他更突出的NLR蛋白参与其中。我们的初步数据表明：(1)细菌性肺炎患者肺部NLR家族pyrin结构域6 （NLRP6）炎性小体表达较高；(2)中性粒细胞在静息期和细菌感染时NLRP6表达最高；(3)与其他nlr （NOD1、NOD2、NLRP3和NLRC4）相比，NLRP6对肺炎克雷伯菌肺内细菌的清除作用最为突出；(4)细菌性肺炎时肺中性粒细胞产生IL-17A和IL-17F；(5) NLRP6敲低的人肺泡巨噬细胞在肺炎克雷伯菌LPS攻击后产生IL-1¿和IL-18蛋白水平降低。我们的主要发现支持了NLRP6在克雷伯菌感染期间的一个关键但尚未被认识的功能。该更新提案旨在解决核心假设，即NLRP6是革兰氏阴性细菌性肺炎期间通过诱导IL-17介导宿主防御的关键介质；因此，NLRP6是一个潜在的治疗靶点，可以增强宿主对急性呼吸道感染的防御。目的是：(1)确定NLRP6对肺炎克雷伯菌攻击后细菌清除率和中性粒细胞功能的影响；(2)确定nlrp6依赖性IL-17产生对宿主细菌性肺炎耐药性的影响；(3)鉴定细菌性肺炎患者NLRP6破坏引起的肺泡巨噬细胞功能改变；(4)探讨NLRP6信号的调控是否能增强肺炎克雷伯菌感染时宿主的耐药性。一个独特的体内和体外系统的组合，包括条件KO小鼠，慢病毒转导，过继转移和细胞因子恢复策略将被用来解决这些目标。证明特异性炎性小体在肺部炎症和宿主防御中起关键作用将导致重大的范式转变，并最终导致治疗细菌性肺炎ALI和ARDS的新治疗和预防策略，因为先前对NLRs和其他细胞质传感器作用的研究完全局限于体外研究和全身感染模型。

DESCRIPTION (provided by applicant): Lung infections are a leading cause of morbidity, mortality and health care costs worldwide. Rapid clearance of pathogens from the respiratory tract is dependent on effective innate immune responses in the lung. Understanding the innate defense mechanisms in the lower respiratory tract is critical for the development of novel treatment and/or prevention strategies to reduce this burden of disease. The signaling cascades triggering innate immune responses consist of a delicate balance between pro- inflammatory responses that facilitate pathogen clearance, and counteracting anti-inflammatory responses that control excessive systemic inflammatory host responses. In general, it is poorly understood how these pathways converge to regulate host defense while minimizing inflammatory tissue injury. The long-term goal of this research is to understand how multiple innate immune events are integrated into effective antimicrobial resistance. As a model to elucidate the basic host defense mechanisms, we have focused on a primary pathogen, Klebsiella pneumoniae because this extracellular Gram-negative bacterium causes severe pneumonia; and the extensive spread of multiple drug-resistant K. pneumoniae strains worldwide. Although K. pneumoniae signals via Toll-like receptor (TLR)-4 and 9, a few studies have also indicated the involvement of NOD-like receptors (NLRs) as cytosolic immune sensors. Some NLRs termed "inflammasomes" can activate caspase-1 in order to cleave IL-1¿ and IL-18. We have previously shown the NLR family CARD domain containing 4 (NLRC4; IPAF) as a new sensor to K. pneumoniae in human and mouse macrophages that regulates caspase-1 dependent maturation of the inflammatory cytokines, IL1¿ and IL18. IL-1R1 knockout (KO) mice show greater susceptibility than NLRC4 KO mice to intrapulmonary K. pneumoniae infection, suggesting the involvement of other more prominent NLR proteins. Our preliminary data demonstrate that (1) human lungs with bacterial pneumonia show higher expression of NLR family pyrin domain containing 6 (NLRP6) inflammasome; (2) neutrophils show the highest expression of NLRP6 during resting stage and upon bacterial infection; (3) as compared with other NLRs (NOD1, NOD2, NLRP3 and NLRC4), NLRP6 is most prominent for bacterial clearance in the lungs during Klebsiella pneumonia; (4) neutrophils produce IL-17A and IL-17F in the lungs during bacterial pneumonia; and (5) NLRP6 knockdown human alveolar macrophages produce attenuated levels of IL-1¿ and IL-18 proteins following K. pneumoniae LPS challenge. Our key findings support a critical yet unrecognized function for NLRP6 during Klebsiella infection. This renewal proposal seeks to address the central hypothesis that NLRP6 is a key mediator of host defense during gram-negative bacterial pneumonia via induction of IL-17; thus, NLRP6 is a potential therapeutic target that could augment host defenses to acute respiratory infections. The Aims are: (1) Determine the effects of NLRP6 on bacterial clearance and neutrophil function following K. pneumoniae challenge.; (2) Determine the effects of NLRP6-dependent IL-17 production on host resistance to bacterial pneumonia.; (3) Identify functional alterations in alveolar macrophages caused by NLRP6 disruption with bacterial pneumonia.; and (4) Explore if manipulation of NLRP6 signaling can augment host resistance during Klebsiella pneumonia. A unique combination of in vivo and in vitro systems, including conditional KO mice, lentiviral transduction, adoptive transfer, and cytokine restoration strategies will be employed to address these Aims. Proving that the specific inflammasome plays a critical role in lung inflammation and host defense will lead to a major paradigm shift and ultimately lead to new therapeutic and prevention strategies of the treatment of ALI and ARDS in bacterial pneumonia because prior studies of the role of NLRs and other cytosolic sensors have been exclusively confined to in vitro studies and systemic Infection models.