Regulation of LPS-responses by ZBP1

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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.