Characterization of inflammasome activation in anthrax lethal toxin-mediated cyto

描述（由申请人提供）：我们的目标是确定炎性体在炭疽致命毒素（LT）介导的疾病中的作用。炎性小体是先天免疫的重要组成部分，被许多与生物防御相关的病原体激活，包括炭疽杆菌。引人注目的是，LT介导的细胞杀伤受炎性小体激活控制。然而，目前还不清楚LT如何触发炎性小体，并导致随后的细胞病变和致病作用。小鼠B。炭疽模型对于研究炎性小体参与微生物细胞杀伤非常有用，因为炎性小体组分Nalp 1b与鼠巨噬细胞的LT敏感性有关。将小鼠分成含有对LT杀伤敏感或抵抗的巨噬细胞的品系。易感巨噬细胞表达显性Nalp 1b等位基因，这似乎赋予LT的敏感性，而耐药巨噬细胞表达隐性Nalp 1b等位基因，已链接到LT抗性。在过去的4年中，我们已经获得了许多有价值的工具和方法，以解决炎症体和Nalp 1b在炭疽外毒素LT介导的细胞杀伤中的作用。这些药物包括来自易感和非易感鼠株的标记Nalp 1b等位基因，以及缺乏炎症体不同成分的细胞系。因此，我们在解决炭疽和其他微生物疾病的长期问题方面处于非常有利的地位。基于我们的观察和其他人的发现，我们假设特定Nalp 1b等位基因的表达决定了造血细胞对LT杀伤的易感性，并且Nalp 1b激活caspase-1是这一过程所必需的。我们的目标是了解Nalp 1b和caspase-1激活在LT杀伤中的作用，特别是LT如何触发Nalp 1b刺激和细胞病变效应。 在这里，我们将测试是否转导Nalp 1b等位基因来源于易感小鼠巨噬细胞赋予敏感性LT耐药巨噬细胞。嵌合Nalp 1b构建体，通过交换来自易感和非易感小鼠巨噬细胞的Nalp 1b等位基因的相应片段产生，将用于鉴定LT反应性的Nalp 1b决定簇。为了了解Nalp 1b的激活，我们将分析LT处理和未处理的巨噬细胞中炎性小体复合物的蛋白质组成。Nalp 1b和炎性体结合蛋白将使用免疫沉淀和蛋白质组学进行鉴定。将测试Nalp 1b结合蛋白在炎性小体活化和细胞杀伤中的作用。这些研究将阐明炎性小体在LT杀伤中的作用，并有助于阐明涉及炎性小体的微生物致病性的一般机制。 我们计划研究炎性小体在炭疽发病机制中的作用。我们将确定如何刺激炎性小体，这是免疫反应的一部分，可能导致细胞死亡和炭疽病。

DESCRIPTION (provided by applicant): Our goal is to determine the role of the inflammasome in disease mediated by anthrax lethal toxin (LT). The inflammasome, a critical part of innate immunity, is activated by numerous pathogens relevant to biodefense, including Bacillus anthracis. Strikingly, LT-mediated cell killing is controlled by inflammasome activation. It is unclear, however, how LT triggers the inflammasome, and causes subsequent cytopathic and pathogenic effects. The murine B. anthracis model is very useful for studying inflammasome involvement in microbial cell killing, since the inflammasome component Nalp1b has been linked to LT sensitivity of murine macrophages. Mice are divided into strains that harbor macrophages that are either susceptible or resistant to LT killing. Susceptible macrophages express a dominant Nalp1b allele, which appears to confer LT sensitivity, while resistant macrophages express a recessive Nalp1b allele that has been linked to LT resistance. Over the last 4 years we have acquired many valuable tools and methods to address the role of the inflammasome and Nalp1b in cell killing mediated by the anthrax exotoxin LT. These agents include tagged Nalp1b alleles derived from susceptible and nonsusceptible murine strains, as well as cell lines lacking different components of the inflammasome. We are therefore in an excellent position to address longstanding questions in anthrax and other microbial diseases. Based on our observations and findings by others, we hypothesize that expression of specific Nalp1b alleles determines the susceptibility of hematopoietic cells to LT killing, and that activation of caspase-1 by Nalp1b is required for this process. Our goal is to understand the role of Nalp1b and caspase-1 activation in LT killing, and specifically how LT triggers Nalp1b stimulation and cytopathic effects. Here we will test whether transducing Nalp1b alleles derived from susceptible murine macrophages confer sensitivity to LT resistant macrophages. Chimeric Nalp1b constructs, generated by exchanging corresponding fragments of Nalp1b alleles derived from susceptible and nonsusceptible murine macrophages, will be used to identify Nalp1b determinants for LT responsiveness. To understand of Nalp1b activation, we will analyze the protein composition of the inflammasome complex in LT-treated and untreated macrophages. Nalp1b and inflammasome-binding proteins will be identified using immunoprecipitation and proteomics. The role of Nalp1b-binding proteins in inflammasome activation and cell killing will be tested. These studies will shed light on the role of the inflammasome in LT killing, and contribute to elucidating general mechanisms in microbial pathogenicity involving the inflammasome. We plan to study the role of the inflammasome in anthrax pathogenesis. We will determine how stimulation of the inflammasome, which is part of the immune response, could lead to cell death and morbidity in anthrax.