Improving treatment predictors of HCV therapy and the impact of steatosis on the hepatocyte transcriptome and anti-HCV action of interferon

Hepatitis C Virus (HCV) is a significant global health issue that leads to the development of chronic liver inflammation, and subsequent establishment of cirrhosis and hepatocellular carcinoma (HCC). Previously the standard therapy of chronic hepatitis C (CHC) was pegylated interferon α (IFN-α) and ribavirin, which had poor treatment success rates and was associated with significant side effects. Risk factors that have been shown to be associated with treatment failure include excess alcohol consumption, advanced age, diabetes and obesity. Although these negative predictors of treatment outcome have been well established in clinical practice, little is known regarding the molecular mechanism(s) of treatment failure. Obesity is another major health issue which is associated with numerous deleterious health issues, one of which being steatosis, or non-alcoholic fatty liver disease (NAFLD). NAFLD can progress to necroinflammation of the liver or non-alcoholic steatohepatitis (NASH), leading to fibrosis and development of cirrhosis. Given how common obesity is, clinicians are commonly faced with managing patients with CHC and concurrent steatosis. Understanding the molecular mechanism(s) of interferonbased treatment failure in patients with CHC with concurrent steatosis, may allow adjuvant therapy to be targeted to those with negative predictors of treatment outcome, thus resulting in an improved virological response. In this thesis, an in vitro model of steatosis has been adopted to investigate the effect of lipid loading on gene expression, in particular, interferon-stimulated genes (ISGs). Two different free fatty acids, oleic acid and palmitic acid, were used to induce steatosis in the Huh-7 hepatoma cell line. In this thesis, it was shown that steatosis was associated with a marked alteration of gene expression, some of which interestingly were classical ISGs. This was likely due to TLR2 mediated pathways, leading to subsequent downstream NF-κβ activation and gene expression. Through induction of steatosis by oleic and palmitic acid, it was also shown that Huh- 7 cells can accentuate the effect of interferon stimulation, leading to an increased ISG expression, which is believed to be secondary to the increase in STAT1 phosphorylation. Finally the effect of steatosis-induced ISG expression on HCV replication, as well as the responsiveness to IFN-α treatment, was investigated. Surprisingly, it was found that steatosis alone led to a modest reduction of HCV replication, with reduced interferon sensitivity, leading to a reduction in HCV knockdown when IFN-α was used. It was shown that the combination of OA:PA, HCV replication and IFN-α stimulation resulted in a significant increase in CXCL8 protein production, a cytokine known to have anti-IFN modulating activity. Moreover, exogenous addition of CXCL8 to cultured cells abrogated the anti-HCV actions of IFN-α. This highlighted a potential mechanism for IFN failure in the HCV infected liver with concurrent steatosis. In summary, the in vitro model of steatosis has revealed a much better understanding of the effects that free fatty acids have on gene expression in hepatocytes, as well as their relationship with HCV infection and IFN-α therapy. As reported in this thesis, new and unexpected data has been obtained, which may lead to a different way of thinking about host-virus interactions. In the future this will hopefully be translated to better treatment options for HCV infection.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015