Regulation of LPS-responses by ZBP1

项目总结 本研究的目的是研究ZBP1介导的内毒素应答和保护机制 由ZBP1提供的细菌感染。我们发表的和初步的数据描述了一部小说的特点 脂多糖对Caspase8的非规范激活，导致嗜热性细胞死亡 巨噬细胞。在进一步的研究中，我们发现含有CASP8亲死复合体II，它组装在 对内毒素的反应，并不像目前普遍接受的那样依赖于TNFR1，而是依赖于TRIF，从而使 Trif是该激活途径的中心组成部分。以反映复合体II对TRIF的要求 在CASP8的形成和激活过程中，我们将这种新的诱导焦虑症的复合体命名为“TRIFosome”。在……里面 进一步的机制调查，我们发现到目前为止被牵连的核酸传感器ZBP1 仅在病毒感染中，通过将RIPK1穿梭到 特里夫。复合体II的形成对ZBP1的依赖提出了一个问题：ZBP1是否介导炎症 对脂多糖的反应也是如此。为了支持这一假说，我们的初步数据显示，ZBP1介导了内毒素- 诱导炎症反应，从而为进一步体内研究ZBP1的作用提供了理论基础 可能在对内毒素和耶尔森氏菌感染的反应中发挥作用。为了解决ZBP1是否介导对内毒素的反应 提供对细菌感染的保护，我们提出了两个目标。在目标1中，我们将调查ZBP1- 采用双基因敲除和基因沉默的方法介导对内毒素的体外应答。我们将使用ZBP1- 特异性免疫沉淀，以确定ZBP1-相互作用组分。确定 ZBP1在脂多糖诱导的免疫应答中的作用，将包括阐明ZBP1在 调节TRIF依赖和非TRIF依赖的反应。它还将包括对 ZBP1在RIPK1对内毒素的特异性反应中。包括了解以下功能域的作用 ZBP1在这一途径中的作用，分析ZBP1在内体TRIFosome复合体中的募集，以及 体内细菌感染时宿主反应中依赖ZBP1的变化的特征。在第二个 目标，我们将利用我们的初步发现，并将进一步研究反应动力学如何 ZBP1编码的LSP在体内对脂多糖诱导的毒性反应具有敏感性，以及这种敏感性 防止体内的革兰氏阴性菌感染。这些研究将有助于确定ZBP1的作用 在宿主对细菌感染的反应中，会将ZBP1定位在不同免疫反应的交叉点 小路。

PROJECT SUMMARY The goal of this proposal is investigation of the mechanism of ZBP1-mediated response to LPS and protection from bacterial infection that is provided by ZBP1. Our published and preliminary data characterize a novel noncanonical activation fo Caspase 8 in response to LPS, which results in the pyroptotic cell death in macrophages. In further inquiry, we identified that containing CASP8 pro-death complex II that assembles in response to LPS, is not dependent on TNFR1 as was widely accepted so far, but rather on TRIF thus making TRIF a central component of theis activation pathway. To reflect the requirement for TRIF in complex II formation and activation of CASP8, we termed this new pyroptosis-inducing complex the “TRIFosome”. In further mechanistic inquiry, we found that the nucleic acid sensor, ZBP1, which thus far has been implicated only in viral infections, plays a significant role in the formation of the TRIFosome complex by shuttling RIPK1 to TRIF. The reliance of Complex II formation on ZBP1 raised a question whether ZBP1 mediates inflammatory responses to LPS as well. In support of this hypothesis, our preliminary data show that ZBP1 mediates LPS- induced inflammatory responses, thus providing rationale for further in vivo investigations of the role that ZBP1 might play in responses to LPS and Yersinia infection. To address whether ZBP1-mediated response to LPS provides protection from the bacterial infection, we propose 2 aims. In Aim 1, we will investigate ZBP1- mediated in vitro response to LPS using double knockout and gene-silencing approach. We will use ZBP1- specific immunoprecipitation in order to determine the components of ZBP1-interactome. determine the contribution of ZBP1 to LPS-induced immune response, which will include elucidation specific role of ZBP1 in mediating TRIF-dependent and TRIF-independent responses. It will also include delineation of the role of ZBP1 in the RIPK1-specific response to LPS. including understanding the roles of the functional domains of ZBP1 in this pathway, analysis of the recruitment of ZBP1 into the endosomal TRIFosome complex, and characterization of the ZBP1-dependent changes in host response to bacterial infection in vivo. In the second Aim, we will capitalize on our preliminary findings and will further investigate how the kinetics of response to LSP that is conferred by ZBP1 confers sensitivity to LPS-induced tosicity I vivo, and whether this sensitivity provides protection from gram-negative infection in vivo. These studies will help establishing the role of ZBP1 in host response to bacterial infection and will position ZBP1 at the crosstalk of different immune response pathways.