Neutrophil Accumulation in Bacterial Pneumonia

描述（由申请人提供）：肺部感染是全球发病率、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依赖于肺中有效的先天免疫应答。了解下呼吸道的先天防御机制对于开发新的治疗和/或预防策略以减少这种疾病负担至关重要。触发先天性免疫应答的信号传导级联由促进病原体清除的促炎应答和控制过度全身性炎症宿主应答的抵消抗炎应答之间的微妙平衡组成。一般来说，人们对这些途径如何收敛以调节宿主防御，同时最大限度地减少炎性组织损伤知之甚少。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。作为阐明基本宿主防御机制的模型，我们集中在主要病原体，肺炎克雷伯氏菌，因为这种细胞外革兰氏阴性菌引起严重肺炎;和广泛传播的多重耐药K。全世界的肺炎菌株。虽然K.尽管N 0 D样受体（NLR）通过Toll样受体（TLR）-4和9参与肺炎链球菌的信号转导，但一些研究也表明N 0 D样受体（NLR）作为细胞溶质免疫传感器参与。一些称为“炎性小体”的NLR可以激活半胱天冬酶-1以裂解IL-1和IL-18。我们先前已经显示了NLR家族CARD结构域包含4（NLRC 4; IPAF）作为K的新传感器。在人类和小鼠巨噬细胞中的肺炎链球菌，调节炎症细胞因子IL 1和IL 18的半胱天冬酶-1依赖性成熟。IL-1 R1敲除（KO）小鼠比NLRC 4 KO小鼠对肺内K. pneumoniae感染，表明其他更突出的NLR蛋白的参与。我们的初步数据表明：（1）人细菌性肺炎肺组织中NLR家族pyrin domain containing 6（NLRP 6）炎性小体表达增加;（2）中性粒细胞在静息期和细菌感染时NLRP 6表达增加;（3）与其他NLR相比（NOD 1、NOD 2、NLRP 3和NLRC 4），NLRP 6在克雷伯氏菌肺炎期间对于肺中的细菌清除是最突出的;（4）中性粒细胞在细菌性肺炎期间在肺中产生IL-17 A和IL-17 F;（5）NLRP 6敲低的人肺泡巨噬细胞在K. pneumoniae LPS攻击。我们的关键发现支持了NLRP 6在克雷伯氏菌感染期间的关键但未被认识的功能。该更新提案旨在解决核心假设，即NLRP 6是革兰氏阴性细菌性肺炎期间通过诱导IL-17进行宿主防御的关键介体;因此，NLRP 6是一种潜在的治疗靶点，可以增强宿主对急性呼吸道感染的防御。目的：（1）研究NLRP 6对K.肺炎的挑战。(2)确定NLRP 6依赖性IL-17的产生对宿主抵抗细菌性肺炎的影响。(3)确定细菌性肺炎中NLRP 6破坏引起的肺泡巨噬细胞功能改变以及（4）探索NLRP 6信号转导的操纵是否可以在肺炎克雷伯菌期间增强宿主抗性。将采用体内和体外系统的独特组合，包括条件性KO小鼠、慢病毒转导、过继转移和细胞因子恢复策略来解决这些目标。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范式转变，并最终导致治疗细菌性肺炎中的ALI和ARDS的新的治疗和预防策略，因为先前对NLR和其他胞质传感器的作用的研究仅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.