Septic shock occurs in more than 750,000 patients each year in the US, nearly 215,000 of who die. The current renewal is a continuing evolution of studies focused upon identifying molecular mechanisms that negatively regulate Toll-like receptor (TLR) function and inflammation in sepsis. During the past funding cycle several major findings have provided a new direction and focus. The new direction is, in part, derived from our findings that mice deficient in the heterotrimeric quanine nucleotide inhibitory protein (G1i2) exhibit an enhanced pro- inflammatory phenotype to endotoxemia and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Activation of G1i protein by specific ligands is beneficial in endotoxemia and sepsis and suppresses TLR activation. During the course of evaluating factors that regulate G1i protein function, we discovered a novel role of the G protein-coupled receptor regulators, 2 arrestin 1 and 2. We demonstrated that 2 arrestins function as negative regulators of sepsis induced inflammation and severity. 2 arrestins also function as negative regulators of TLR induced NF:B signaling and inflammatory cell activation. Collectively, these observations prompt the overarching hypothesis that the heterotrimeric G1i2 protein and 2-arrestins 1 and 2 are negative regulators of the signaling pathways that contribute to sepsis-induced inflammation and severity.
Two specific aims will address this hypothesis. The first specific aim will determine the role of G1i2 protein and 2 arrestins 1 and 2 in regulation of the inflammatory process in murine polymicrobial sepsis by loss of function and gain of function studies. Specifically these studies will examine CLP induced sepsis severity and tissue signaling changes in mice with genetic deficiencies of G1i2, 2 arrestin and 2 arrestin. As a gain of function approach we will examine the susceptibility of genomic knock in of regulator G protein signaling (RGS) insensitive mutated G1i2G184S mice to CLP-induced sepsis. The importance of gain or loss of function of G1i2 and loss of function of 2-arrestin 1 or 2 in myelopoietic lineage cells in chimeric mice subjected to CLP-induced sepsis will be determined. In the second specific aim the in vitro cellular responses of PMNs and splenic MX in response to loss of function G1i2(-/-), 2-arrestin 1(-/-), 2-arrestin 2(-/-) and gain of function G1i2G184S mice to TLR ligand activation will be investigated. The latter studies will also examine changes in cell signaling kinetics and compartmentalization of signaling. To determine the molecular mechanisms whereby G1i2 and 2-arrestin 1 and 2 regulate cytoplasmic and nuclear signaling, we will employ 2-arrestin 1 and 2 double knockout (DKO) and 2-arrestin 1 or 2 reconstituted DKO mouse embryonic fibroblasts and CD14/TLR4/MD2 permanently transfected HEK293 cells. Understanding the molecular mechanisms of G1i2 and 2 arrestin 1 and 2 regulation and how they function conjointly or independently in reducing sepsis severity will lead to novel and more rational therapeutic approaches to treat septic shock.
this project will test the hypothesis that the heterotrimeric G1i2 protein and 2-arrestins 1 and 2 are negative regulators of the signaling pathways that contribute to sepsis induced inflammation and severity. This hypothesis is based upon in vivo studies that G1i2(-/-) and 2-arrestin 2(-/-) mice demonstrate enhanced susceptibility to polymicrobial sepsis, and in vitro studies demonstrating a pro-inflammatory phenotype of cells from G1i2(-/-) and 2-arrestin 2(-/-) mice. The current proposal will employ a series of in vivo and in vitro gain and loss of G1i2 function and loss of 2-arrestin 1 or 2 functions to identify the molecular mechanisms by which G1i2 and 2-arrestin 1 or 2 negatively regulate TLR activation and sepsis induced inflammation.
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