Specificity and in vivo function of the Naip/Nlrc4 inflammasomes

描述（由申请人提供）：免疫学中的一个基本问题是先天免疫系统如何检测病原微生物。宿主的健康关键取决于先天免疫系统对病原体快速和选择性反应的能力，同时避免对自身或无害的体内微生物的不适当反应。我们的目标是描述如何在分子水平上实现这种快速，灵敏和准确的病原体检测。我们一直特别感兴趣的一个家庭的胞质病原体探测器称为炎性小体。炎性小体包括响应于有害和感染性刺激而在细胞溶质中组装的多蛋白复合物家族。炎性小体通过激活半胱天冬酶-1蛋白酶启动先天免疫应答。一般来说，人们对炎性小体如何识别病原体并组装以激活Caspase-1知之甚少。为了解决这些问题，我们的研究集中在称为Naip/Nlrc 4炎性小体的炎性小体亚家族。Naip蛋白（其中在C57 BL/6小鼠中有四个）和Nlrc 4是称为NLR（核苷酸结合结构域，富含亮氨酸的重复序列的蛋白）的更广泛的蛋白质超家族的成员。在过去的资助期间，我们发现Naip 5介导细菌鞭毛蛋白的特异性胞质检测，而Naip 2介导来自不同细菌III型分泌系统的内杆蛋白的特异性胞质检测。我们还开发了新的生物化学测定来剖析配体结合和炎性体寡聚化，并使用这些新的测定来显示Naips的关键生物化学功能是识别配体并诱导Nlrc 4的下游寡聚化。在这个更新的申请中，我们描述了三个目标，将解剖配体结合，寡聚化，和下游体内效应功能的Naip/Nlrc 4炎性小体。目的1试图确定特定的细菌配体是如何被Naip蛋白识别的。这将是重要的，因为它将代表任何哺乳动物NLR蛋白的配体结合的第一次分析。目的2旨在剖析炎性小体组装的生化和细胞机制，这是一个关键但知之甚少的过程。最后，目标3将研究炎性小体如何在体内启动效应子应答。特别是，我们试图建立一个新的联系炎症体激活和类花生酸脂质介质的生产在体内。已知类二十烷酸是炎症的关键介质，但它们的产生以前没有与炎性小体相关。我们在体内表明，炎性小体激活导致类花生酸的产生和严重的血管渗漏综合征，可以迅速致命。总之，我们对Naip/Nlrc 4炎性小体的研究将为配体识别、组装和这个关键的胞质免疫检测器家族的体内功能提供新的见解。

DESCRIPTION (provided by applicant): A fundamental question in immunology is how the innate immune system detects pathogenic microbes. Host health depends critically upon the ability of the innate immune system to respond rapidly and selectively to pathogens, while avoiding inappropriate responses to self or harmless commensal microbes. Our goal is to describe how this rapid, sensitive, and accurate detection of pathogens is achieved at a molecular level. We have been particularly interested in a family of cytosolic pathogen detectors called inflammasomes. Inflammasomes comprise a family of multiprotein complexes that assemble in the cytosol in response to noxious and infectious stimuli. Inflammasomes initiate innate immune responses by activating the Caspase-1 protease. In general, it is poorly understood how inflammasomes recognize pathogens and assemble to activate Caspase-1. To address these questions, our studies have focused on a sub-family of inflammasomes called the Naip/Nlrc4 inflammasomes. Naip proteins (of which there are four in C57BL/6 mice) and Nlrc4 are members of a broader superfamily of proteins called NLRs (nucleotide- binding domain, leucine rich repeat-containing proteins). In the past funding period, we found that Naip5 mediates specific cytosolic detection of bacterial flagellin, whereas Naip2 mediates specific cytosolic detection of the inner rod protein from diverse bacterial type III secretion systems. We have also developed novel biochemical assays to dissect ligand binding and inflammasome oligomerization, and have used these novel assays to show that a key biochemical function of the Naips is to recognize ligands and induce downstream oligomerization of Nlrc4. In this renewal application, we describe three aims that will dissect ligand binding, oligomerization, and the downstream in vivo effector functions of the Naip/Nlrc4 inflammasomes. Aim 1 seeks to determine how specific bacterial ligands are recognized by Naip proteins. This will be significant because it will represent the first analysis of ligand binding by any mammalian NLR protein. Aim 2 seeks to dissect the biochemical and cellular mechanisms of inflammasome assembly, a critical but poorly understood process. Lastly, Aim 3 will investigate how inflammasomes initiate effector responses in vivo. In particular, we seek to establish a novel link between inflammasome activation and eicosanoid lipid mediator production in vivo. Eicosanoids are known to be critical mediators of inflammation, but their production has not previously been linked to inflammasomes. We show in vivo that inflammasome activation results in eicosanoid production and a severe vascular leakage syndrome that can be rapidly fatal. Taken together, our studies of the Naip/Nlrc4 inflammasomes will provide novel insights into ligand recognition, assembly and in vivo function of this critical family of cytosolic immune detectors.