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症小体在炭疽致死毒素（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，可被许多与生物防御相关的病原体激活，包括炭疽芽孢杆菌。引人注目的是，lt介导的细胞杀伤是由炎性小体激活控制的。然而，目前尚不清楚LT如何触发炎性体，并导致随后的细胞病变和致病作用。小鼠炭疽杆菌模型对于研究炎症小体参与微生物细胞杀伤非常有用，因为炎症小体成分Nalp1b与小鼠巨噬细胞的LT敏感性有关。小鼠被分为含有巨噬细胞的菌株，这些巨噬细胞对LT杀伤敏感或耐药。易感巨噬细胞表达显性Nalp1b等位基因，这似乎赋予了LT敏感性，而耐药巨噬细胞表达隐性Nalp1b等位基因，这与LT耐药性有关。在过去的4年里，我们已经获得了许多有价值的工具和方法来解决炎症小体和Nalp1b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小鼠菌株的标记Nalp1b等位基因，以及缺乏炎症小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方面的长期问题方面处于有利地位。根据我们的观察和其他人的发现，我们假设特异性Nalp1b等位基因的表达决定了造血细胞对LT杀伤的易感性，并且Nalp1b激活caspase-1是这一过程所必需的。我们的目标是了解Nalp1b和caspase-1激活在LT杀伤中的作用，特别是LT如何触发Nalp1b刺激和细胞病变作用。

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.