Regulation of LPS-responses by ZBP1

项目摘要 本研究的目的是探讨ZBP 1介导的对LPS的反应机制， 由ZBP 1提供的细菌感染。我们发表的和初步的数据描述了一种新的 Caspase 8对LPS的非典型激活，导致细胞凋亡。 巨噬细胞在进一步的研究中，我们鉴定了含有CASP 8促死亡复合物II的蛋白， 对LPS的反应，不依赖于TNFR 1，这是迄今为止广泛接受的，而是依赖于TRIF，从而使 TRIF是其激活途径的核心成分.在复合体II中反映对TRIF的要求 由于CASP 8的形成和活化，我们将这种新的热解诱导复合物称为“TRIFosome”。在 进一步的机制调查，我们发现，核酸传感器，ZBP 1，迄今已牵连 仅在病毒感染中，在TRIFosome复合物的形成中起重要作用，通过穿梭RIPK 1到 TRIF。复合物II形成对ZBP 1的依赖性提出了一个问题，即ZBP 1是否介导炎症反应。 对LPS的反应也是如此。为了支持这一假设，我们的初步数据表明，ZBP 1介导LPS-1。 诱导炎症反应，从而为进一步在体内研究ZBP 1的作用提供了理论基础。 可能在LPS和耶尔森氏菌感染的反应中起作用。为了解决ZBP 1介导的对LPS的反应是否 提供保护免受细菌感染，我们提出2个目标。在目标1中，我们将研究ZBP 1- 使用双敲除和基因沉默方法介导对LPS的体外应答。我们将使用ZBP 1- 特异性免疫沉淀以确定ZBP 1-相互作用体的组分。确定 ZBP 1对LPS诱导的免疫应答的贡献，其将包括阐明ZBP 1在LPS诱导的免疫应答中的特异性作用。 介导TRIF依赖性和TRIF不依赖性反应。它还将包括界定以下机构的作用： ZBP 1在对LPS的RIPK 1特异性应答中的作用。包括了解功能域的作用， ZBP 1在该途径中的作用，ZBP 1募集到内体TRIFosome复合物中的分析，以及 表征宿主对体内细菌感染的应答中的ZBP 1依赖性变化。在第二 目的，我们将利用我们的初步研究结果，并将进一步研究如何反应的动力学 由ZBP 1赋予的LSP赋予对LPS诱导的毒性I的体内敏感性，以及这种敏感性是否 在体内提供对革兰氏阴性感染的保护。这些研究将有助于确定ZBP 1的作用 在宿主对细菌感染的应答中，ZBP 1将定位于不同免疫应答的交叉点 途径。

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.