The broad objective of this project is to elucidate the immune mechanisms responsible for killing of virally infected hepatocytes. Studies conducted as part of this project have found that hepatocytes are resistant to perforin and granzyme dependent mechanisms of cytotoxic T lymphocyte (CTL) killing and that clearance of virally infected hepatocytes from the liver is mediated predominately by alternative CTL effector mechanisms that utilize Fas/FasL, TNF/TNFR1 and/or other death receptor mediated pathways. These results lead us to hypothesize that hepatocyte resistance to the cytotoxic effects of the perforin and granzyme mediated CTL effector pathway is important in limiting the degree of liver injury during intrahepatic immune responses. To address this hypothesis and to explore the mechanisms responsible for the unique repertoire of CTL effector mechanisms employed during anti-viral immunity in the liver;we propose to explore mechanisms responsible for resistance of virally infected hepatocytes to the granule exocytosis pathway of CTL effector function. Preliminary findings indicate that cytokines produced during antiviral immune responses induce expression of the granzyme B specific inhibitors proteinase inhibitor 9 (PI-9) and serine proteinase inhibitor 6 (SPI-6) in human and mouse hepatocytes respectively. In proposed studies, we will determine the repertoire of serine proteinase inhibitors (serpins) expressed in normal and virally infected murine hepatocytes and determine the role of cytokines in regulating such serpin expression in vitro and in vivo. We will then explore the role that serpins and/or cathepsin B play in mediating hepatocyte resistance to granzymes and perforin, respectively. Finally, we will determine whether silencing of serpin and/or cathepsin B genes in vivo renders virally infected hepatocytes susceptible to perforin and granzyme dependent cytotoxicity and thereby accelerates immune clearance of virally infected hepatocytes and exacerbates viral hepatitis.
