NLRP11 in non-canonical inflammasome activation in response to Shigella flexneri and cytosolic LPS.

福氏志贺氏菌是引起人类细菌性痢疾的胞内细菌病原体。确定 宿主细胞如何检测胞浆内细菌对促进我们对先天细菌的理解至关重要 免疫系统。福氏志贺氏菌感染结肠可引起免疫反应，从而触发新城疫的招募 中性粒细胞和巨噬细胞进入组织。当福氏志贺氏菌进入巨噬细胞时，它会激活 炎性小体，导致上睑下垂，这是一种促炎症细胞死亡。研究得最好的炎症组 NLRP3途径是典型的NLRP3炎症体，它由(1)节点样受体(NLR)NLRP3，(2) 接头蛋白ASC；(3)炎症性caspase-1。一种非正规性炎症性组织，包括 Caspase-4/5(Caspase-11，在小鼠中)已被描述；到目前为止，还没有鉴定出NLR蛋白。已激活 我们的初步数据显示，任一途径的炎性半胱氨酸激活Gasdermin D。 福氏杆菌感染NLRP11是有效的细胞死亡以及caspase-4和Gasdermin D激活所必需的 对于人类来源的巨噬细胞，我们的数据也表明胞浆内毒素(CLPs)足以触发NLRP11 这些细胞中的依赖性细胞死亡。此外，我的初步数据显示，NLRP11与内毒素结合， NLRP11和原caspase-4的相互作用不依赖于内毒素。基于我们的数据，我们提出了一个新的角色 NLRP11在检测非典型炎症体途径CLP中的作用。我的总体假设是 NLRP11(1)用于检测与caspase-4结合的CLP，(2)结合并激活caspase-4，以及(3)是关键的 非典型炎症组分。我建议通过以下目的来检验这一假设： 1.明确NLRP11在非典型性炎症小体激活中的分子功能 2.确定NLRP11与caspase-4相互作用的要求 3.确定内毒素、NLRP11和caspase-4是否形成三元复合体。 这一提议将产生对NLRP11如何在非典范炎症中发挥作用的机械性见解。它 极有可能在非典型炎症小体中发现新的功能相互作用，并加深我们的 对先天免疫系统的一般了解。拟议的研究将在Marcia博士的 戈德堡在马萨诸塞州综合医院(MGH)和哈佛医学院(HMS)的实验室，与 我的联合赞助人朱迪·利伯曼博士的指导。MGH和HMS都是严格的学术环境 有充足的资源支持我的研究和事业发展。我将有很多机会正式 介绍我的研究，参加科学会议，参加职业发展课程。建议数 研究将为我提供微生物学和先天免疫学方面的新专业知识，并使我处于理想状态 实现我的职业目标，成为一名独立研究员的职位。

Shigella flexneri are intracytosolic bacterial pathogens that cause bacillary dysentery in humans. Determining how intracytosolic bacteria are detected by the host cell is critical for advancing our understanding of the innate immune system. Infection of the colon by S. flexneri leads to an immune response that triggers recruitment of neutrophils and macrophages into the tissue. When S. flexneri enters macrophages, it activates inflammasomes, leading to pyroptosis, a type of pro-inflammatory cell death. The best studied inflammasome pathway is the canonical NLRP3 inflammasome, which consists of (1) the NOD-like receptor (NLR) NLRP3, (2) the adaptor protein ASC, and (3) the inflammatory caspase-1. A non-canonical inflammasome that consists of caspase-4/5 (caspase-11, in mice) has been described; as yet, no NLR protein has been identified. Activated inflammatory caspases from either pathway activate gasdermin D. Our preliminary data show that during S. flexneri infection NLRP11 is required for efficient cell death as well as caspase-4 and gasdermin D activation in human-derived macrophages, Our data also show that cytosolic LPS (cLPS) is sufficient to trigger NLRP11 dependent cell death in these cells. In addition, my preliminary data show that NLRP11 binds LPS and that NLRP11 and pro-caspase-4 interact independent of LPS. Based on our data, we propose a novel role for NLRP11 in the detection of cLPS in the non-canonical inflammasome pathway. My overall hypothesis is that NLRP11 (1) functions to detect cLPS with caspase-4, (2) binds and activates caspase-4, and (3) is a critical component of the non-canonical inflammasome. I propose to test this hypothesis with the following aims: 1. Define the molecular function of NLRP11 in non-canonical inflammasome activation 2. Define the requirements for the interaction of NLRP11 with caspase-4 3. Determine whether LPS, NLRP11, and caspase-4 form a ternary complex. This proposal will generate mechanistic insights into how NLRP11 acts in the non-canonical inflammasome. It is highly likely to uncover novel functional interactions in the non-canonical inflammasome and deepen our understanding of the innate immune system in general. The proposed studies will be conducted in Dr. Marcia Goldberg's laboratory at Massachusetts General Hospital (MGH) and Harvard Medical School (HMS), with the guidance of my co-sponsor, Dr. Judy Lieberman. Both MGH and HMS are rigorous academic environments with ample resources support my research and career development. I will have many opportunities to formally present my research, attend scientific meetings, and attend career development courses. The proposed studies will provide me with new expertise in microbiology and innate immunology and place me in an ideal position of reaching my career goals of being an independent researcher.