Hepatocyte injury and cell death underlie the development of hepatic failure and progression to the complications of cirrhosis and hepatocellular carcinoma in human liver disease. Despite recent advances, the mechanisms of hepatocyte injury and death remain poorly understood. Liver injury results largely from the effects of factors generated by the excessive sterile inflammatory response that accompanies hepatocyte injury and death. Over the previous funding period we demonstrated that conditions that predispose to liver injury are associated with decreased function of the degradative pathway of autophagy in both hepatocytes and macrophages. We and others have also shown that autophagy functions to prevent liver injury and decrease hepatic inflammation, but the mechanisms of autophagy?s protective effects remain unclear. The objective of this proposal is to delineate novel mechanisms by which impaired autophagy in hepatocytes and macrophages promotes liver injury and inflammation. Preliminary studies indicate that macrophages and hepatocytes with decreased autophagy have an increased proinflammatory response to toll-like receptor 2 (TLR2) stimulation that causes liver injury. In addition, hepatocytes with impaired autophagy become sensitized to cytokine toxicity from the synergistic effects of IL-1? and TNF, and once injured actively secrete proinflammatory damage-associated molecular patterns (DAMPs) in exosomes. Based on these and other preliminary findings, the central hypothesis of this proposal is that during liver injury the effects of decreased hepatic autophagy on cell signaling pathways both amplify the proinflammatory activation of macrophages and hepatocytes by increasing the TLR2-mediated sterile inflammatory response, and sensitize hepatocytes to IL-1?-dependent cell death which further sustains inflammation. We will test this hypothesis through investigations contained in three Specific Aims that will examine liver injury in cell culture and mouse models with a genetic decrease in autophagy. First, we will test the hypothesis that impaired autophagy promotes liver injury by hyperactivation of a TLR2-dependent innate immune response in both macrophages and hepatocytes through the mechanism of decreased docking protein 1 degradation which results in reduced extracellular signal-regulated kinase 1/2 signaling. Second, we will test the hypothesis that with decreased autophagy the energy demands of the proinflammatory cytokines IL-1? and TNF induce ATP depletion, lysosome permeabilization and cathepsin L-dependent hepatocyte necrosis. Third, we will test the hypothesis that with decreased autophagy injured hepatocytes induce a local and systemic innate immune response through the active secretion of exosomes containing DAMPs that are normally sequestered and degraded by autophagy. The ultimate goal of the investigations is to understand how impaired autophagy promotes liver disease through altered innate immunity and hepatocyte death signaling in order to develop new treatment modalities for human liver disease.
(PUBLIC HEALTH RELEVANCE) The injury and death of hepatocytes underlie the development of hepatic failure and progression of liver disease to cirrhosis and hepatocellular carcinoma. Despite recent advances, the process by which hepatocytes undergo injury and cell death is still poorly understood. The objective of this proposal is to increase our knowledge of the molecular mechanisms of hepatocyte death with the ultimate goal of developing new therapies to prevent and treat human liver disease.
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