Neutrophil Accumulation in Bacterial Pneumonia

描述(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部有效的先天免疫反应。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反应包括促进病原体清除的促炎反应和控制过度的全身炎症宿主反应的抗炎反应之间的微妙平衡。总体而言，人们对这些通路如何在调节宿主防御的同时将炎症组织损伤降至最低还知之甚少。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，我们将重点放在主要病原体肺炎克雷伯菌上，因为这种胞外革兰阴性杆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。虽然肺炎克雷伯菌通过Toll样受体(TLR)-4和TLR-9传递信号，但也有少数研究表明Nod样受体(NLRs)作为胞浆免疫感受器参与其中。一些被称为“炎症体”的NLR可以激活caspase-1以裂解IL-1�和IL-18。我们先前已经展示了含有4(NLRC4；IPAF)的NLR家族卡域作为人和小鼠巨噬细胞中肺炎克雷伯菌的新传感器，调节依赖于caspase-1的炎症细胞因子IL-1�和IL-18的成熟。IL-1R1基因敲除(KO)小鼠比NLRC4KO小鼠对肺炎克雷伯菌肺内感染的易感性更高，这表明其他更突出的NLR蛋白参与其中。我们的初步数据表明：(1)细菌性肺炎患者肺组织中含有6(NLRP6)炎症体的NLRP6家族结构域有较高的表达；(2)中性粒细胞在静止期和细菌感染时表达最高；(3)与其他NLR(NOD1、NOD2、NLRP3和NLRC4)相比，NLRP6在肺炎克雷伯菌肺炎时对肺内细菌的清除能力最强；(4)中性粒细胞在细菌性肺炎时在肺内产生IL-17A和IL-17F；以及(5)NLRP6击倒人肺泡巨噬细胞后，IL-1�和IL-18蛋白水平降低。我们的主要发现支持NLRP6在克雷伯氏菌感染过程中的一个关键但尚未被认识的功能。这项更新建议旨在解决核心假设，即NLRP6是通过诱导IL-17诱导革兰氏阴性细菌性肺炎期间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。这些研究的目的是：(1)确定NLRP6对肺炎克雷伯菌攻击后细菌清除和中性粒细胞功能的影响；(2)确定依赖NLRP6的IL-17产生在宿主对细菌性肺炎抵抗中的作用。(3)确定NLRP6干扰在细菌性肺炎中引起的肺泡巨噬细胞的功能变化。(4)探索在肺炎克雷伯菌肺炎过程中操纵NLRP6信号是否可以增强宿主抵抗。体内和体外系统的独特组合，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，将被用于解决这些目标。证明特定的炎症体在肺部炎症和宿主防御中发挥关键作用将导致重大范式的转变，并最终导致治疗细菌性肺炎ALI和ARDS的新的治疗和预防策略，因为以前对NLRs和其他细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.