The Role of Serine Protease Inhibitors in Regulating Hepatotoxicity During Viral Infection

Major hepatitis viruses, such as HBV and HCV, are not directly cytopathic; instead liver injury is due to a vigorous immune response. Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells kill virally infected and malignant cells by two major pathways, the perforin/granzyme pathway and the Fas and/or TNF death receptor pathways. In contrast to other target cells, virally infected hepatocytes are resistant to killing by perforin and granzyme-dependent cytotoxic effector pathways. This results in a more prominent role for Fas and TNFR-mediated killing of infected hepatocytes and clearance of hepatic viral infections. Human proteinase inhibitor 9 (PI-9/ serpinB9) and the murine orthologue, serine proteinase inhibitor 6 (SPI-6/ serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, antigen-presenting cells, and cytotoxic T cells protects these cells against the actions of granzyme B and when expressed in tumor cells, confers resistance to killing by CTL and NK cells. Thus, one potential explanation for hepatocyte resistance to the perforin/granzyme pathway would be expression of PI-9/ SPI-6 within human or murine liver cells. The present studies were designed to assess whether factors present during hepatic viral infections as well as the associated antiviral immune responses, might induce the expression of SPI-6 in murine liver and thus, confer resistance of virally infected hepatocytes to perforin and granzyme B dependent hepatotoxicity. To this extent, we examined SPI-6 expression after IFN-alpha treatment and during in vivo adenoviral infection of the liver. To detect changes in SPI-6 gene expression, mRNA and cytosolic protein was isolated from liver and real time PCR and western blotting were performed. The results indicated that SPI-6 is the only PI-9/serpinB9 homologue that is significantly up-regulated in liver post IFN-alpha stimulation or during the course of viral infection. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL, as reflected by increased lymphocyte surface receptor mRNA levels within the liver. Additional experiments using virally infected, genetically altered mice unable to process or express granzyme B indicated that SPI-6 is a cytoprotective gene that is selectively up-regulated by hepatocytes in response to activated granzyme B. Knockdown of SPI-6 gene expression in vivo by hydrodynamic injection of siRNA specifically targeting SPI-6 resulted in a significant increase in serum alanine amino transferase (ALT) levels, a measure of hepatocellular injury, during early time points after viral infection. In addition, inhibition of SPI-6 resulted in accelerated clearance of AdCMV-LacZ encoded transgene products from the liver. However, expression during high dose AdCMV-LacZ infection resulted in early onset of lethal, acute liver failure. Given these results, we conclude that up-regulation of SPI-6 gene expression in hepatocytes protects against perforin/ granzyme B mediated killing during hepatic viral infection and thereby slows the rate of immune elimination of virally infected hepatocytes.