Controlling inflammation and cellular damage is the key to preventing and treating multiple organ failure and death associated with sepsis and septic shock. Despite improvements in our understanding of mechanisms underlying sepsis and its complications, there are currently no drugs approved to specifically treat severe sepsis. Our overarching goal is ultimately to develop new therapeutics for sepsis patients based on our identification of regulatory mechanisms of inflammation during sepsis that may serve as promising drug targets. In this proposal we will investigate novel roles for inducible nitric oxide synthase (iNOS) and nitric oxide (NO) during sepsis. NO and iNOS are integral components of host responses to pathogens, and have been shown to have protective effects in organs such as the liver during sepsis. We have had a long-standing interest in the role of iNOS/NO particularly in the liver, and how these effects can be protective during sepsis. In this grant application we propose to investigate the pathways that transmit signals from bacterial components, through pattern recognition and cytokine receptors, such as Toll-like receptors (TLR) and IL1- receptor, to regulate iNOS expression and lead to the cleavage of a soluble cytokine receptor, TNFR1, from the cell surface of hepatocytes. Our preliminary data has identified a key novel role for TLR/IL1R-mediated iNOS in activating the enzyme responsible for cleavage of TNFR1, TNF-converting enzyme (TACE or ADAM17). Activation of TACE and release of TNFR1 are important mechanisms in regulating the inflammatory effects of the cytokine TNF, high levels of which have been associated with increased organ damage and patient death during sepsis. Our data are novel in that they describe a specific effect of iNOS-upregulation and a novel, unexpected feedback of NO on iNOS to regulate receptor shedding, as well as an important novel role for iNOS as part of the activating complex of proteins at the cell surface in both human and mouse liver. Understanding the role of iNOS in the regulation of TNFR1 release may ultimately result in development of drugs designed to specifically affect these pathways to improve patient outcome during sepsis. Our specific aims are to determine the role of Myddosome-signaling via TLR/IL1R in iNOS regulation and TNFR1-shedding from hepatocytes and liver during sepsis, and also determine how iNOS activates TACE to allow a complex to form at the cell surface to cleave TNFR1 in the liver and hepatocytes. We will also investigate whether iNOS regulates other TACE-mediated functions during sepsis, and what the specific role for iNOS is within the TACE/TNFR1 complex at the cell surface. Our experiments will allow us to clearly identify the effects of the novel TLR-iNOS-mediated pathways important in regulating inflammation and organ damage during sepsis.