- 1. Gene Expression In Hepatitis C Virus Related Liver Disease: Identification Of Novel Antiviral And Pathogenesis Related Genes
Infection with HCV results in a chronic infection that over a period of 2-3 decades can progress to liver failure and in some cases liver cancer (HCC). How HCV evades the immune response and how the liver responds to infection are not well understood. Viral infection of cells results in a host antiviral response and the induction of anti-viral genes that attempt to clear viral infection. We have used DNA microarray technology to investigate the expression of thousands of genes in the HCV infected liver. In spite of the chronic nature of HCV infection we have found that the liver responds to infection with an antiviral response suggesting that HCV has evolved mechanisms to evade host defence mechanisms. How HCV does this and the cellular antiviral genes upregulated in the HCV infected liver is a research focus of our laboratory. We have identified a number of genes expressed as a result of infection with HCV. One termed Viperin was cloned by our laboratory and found to have antiviral activity against HCV in vitro. We are currently investigating how Viperin exerts its antiviral effect that may have implications for novel antiviral therapeutics. A number of other novel upregulated genes are being evaluated for their anti HCV activity.
One of the features of hepatitis C is a chronic immune infiltrate into the liver, however the signals that recruit these cells are not well understood. We have identified I-TAC as significantly expressed in the HCV infected liver. I-TAC is a molecule that is responsible for immune cells recruitment and we believe that it plays an important role in HCV-related liver disease, I-TAC belongs to a family of chemokines known as the CXCR3 chemokines of which IP-10 mad Mig are also members. Interestingly IP-10 and Mig are also upregulated in the HCV infected liver. We are currently developing an adenovirus induced hepatitis mouse model that will allow us to determine the relative roles of the CXCR3 chemokines in liver disease through the use of specific CXCR3 anatagonists.
Analysis of differentially expressed genes in the HCV infected liver will increase our knowledge of how we respond to HCV infection and will potentially help in the development of novel therapeutic strategies, antiviral therapies and biomarkers of disease progression.
- 2. Osteopontin as a Biomarker of HCV Related Liver Disease and its Role in Hepatocarcinogenesis
Our microarray studies have also identified osteopontin (OPN) as significantly expressed in the chronic hepatits C and HCC. OPN is a multifunctional protein involved in cell migration, survival, chemotaxis and fibrogenesis, and can be detected in both serum and urine, indicating its potential as a biomarker of disease. As a result we used real time RT-PCR and ELISA to investigate OPN expression in liver tissue, patient sera and cultured cells. Interestingly we found that OPN mRNA levels and protein (ELISA) increased with advanced liver disease. In the HCV infected liver and in Huh-7 cells, OPN was expressed as three splice variants (OPN: A, B, C). Stable expression of each OPN variant in Huh-7 cells resulted in secretion of OPN into the culture medium, however some cells retained OPN resulting in a distinct sub-cellular localization for each variant. Stable expression of all variants resulted in accelerated cellular growth rates in vitro and similar results have been obtained in vivo using injection of cells subcutaneously in nude mice. This work has identified that OPN is significantly expressed in advanced HCV-related liver disease and HCC as three distinct splice variants. Each of the variants are secreted from cells in culture, however they differ in sub-cellular localization suggesting that each may play a different role in hepatocyte biology. Expression of OPN variants significantly increased the growth rate of Huh-7 cells in vitro and in vivo suggesting a role for OPN in dysregulation of hepatocyte growth and a possible role in oncogenesis. Furthermore, elevated OPN detected in sera of patients with advanced liver disease may be a useful biomarker of disease progression in chronic hepatitis C.
- 3. Ethanol Potentates HCV Replication and Blocks the Action of Interferon
It is well established that liver disease associated with HCV infection is exacerbated by alcohol consumption. The mechanisms for this are unknown, however, it has been shown that alcohol consumption can increase HCV RNA levels both in the serum and liver. We have initiated a study to determine the effect of ethanol metabolism on HCV replication and liver cell biology. Hepatocytes in culture loose the ability to metabolise ethanol through the loss of the ethanol metabolising enzyme cytochrome P450 2E1 (CYP2E1). We have introduced the CYP2E1 gene back into hepatocytes in culture harbouring HCV and treated these cells with physiological levels of ethanol (10-100mM). Interestingly, HCV RNA levels increase approximately 3-4 fold after 72 hrs of ethanol stimulation. The molecular basis of this increase is unknown, however, if we treat cells with the anti-oxidant NAC, HCV RNA levels are restored to baseline. We have also shown that cells harbouring HCV replication and CYP2E1 have increased levels of oxidative stress when metabolising ethanol compared to control cells. Together these observations suggest that HCV replication may be enhanced by low-level oxidative stress and could explain the increased viral loads in HCV infected individuals who drink alcohol and have more severe liver disease. We have also shown in vitro that the antiviral effects of Interferon, the only treatment for chronic hepatitis C, is blocked in the presence of ethanol. We have recently shown that alcohol acts to block certain cellular interferon signalling pathways resulting in a block of interferon action. This may explain why those individuals with HCV and who consume alcohol have a worse prognosis and accelerated liver disease.
- Funding Sources
- The University of Adelaide
- The Royal Adelaide Hospital Florey Research Fund