Specificity and in vivo function of the Naip/Nlrc4 inflammasomes

描述（由申请人提供）：免疫学的一个基本问题是先天免疫系统如何检测致病微生物。宿主的健康主要取决于先天免疫系统对病原体做出快速和选择性反应的能力，同时避免对自身或无害的共生微生物做出不适当的反应。我们的目标是描述如何在分子水平上实现这种快速、敏感和准确的病原体检测。我们对一类细胞质病原体检测器特别感兴趣，它们被称为炎性小体。炎性小体包括一个多蛋白复合物家族，它们在细胞溶胶中聚集以响应有害和感染性刺激。炎性小体通过激活Caspase-1蛋白酶启动先天免疫反应。一般来说，人们对炎症小体如何识别病原体并聚集起来激活Caspase-1知之甚少。为了解决这些问题，我们的研究集中在炎症小体的一个亚家族上，称为Naip/Nlrc4炎症小体。Naip蛋白（在C57BL/6小鼠中有4个）和Nlrc4是一个更广泛的超家族蛋白NLRs（核苷酸结合域，富含亮氨酸的重复序列蛋白）的成员。在过去的资助期内，我们发现Naip5介导细菌鞭毛蛋白的特异性胞质检测，而Naip2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化分析方法来分析配体结合和炎症小体的低聚化，并利用这些新方法表明Naips的一个关键生化功能是识别配体并诱导Nlrc4的下游低聚化。在这个更新应用中，我们描述了三个目标，将剖析配体结合、寡聚化和Naip/Nlrc4炎症小体的下游体内效应功能。目的1旨在确定特异性细菌配体是如何被Naip蛋白识别的。这将具有重要意义，因为它将首次分析哺乳动物NLR蛋白的配体结合。目的2旨在剖析炎症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，Aim 3将研究炎症小体如何在体内启动效应反应。特别是，我们寻求在体内炎症小体激活和类二十烷类脂质介质产生之间建立一种新的联系。众所周知，类二十烷酸是炎症的关键介质，但它们的产生以前并没有与炎症小体联系起来。我们在体内表明，炎症小体激活导致类二十烷产生和严重的血管渗漏综合征，这可能是迅速致命的。综上所述，我们对Naip/Nlrc4炎症小体的研究将为这一关键细胞质免疫探测器家族的配体识别、组装和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.