Toll-Like Receptors: Target of Hepatitis C Virus: A Dissertation

Abstract

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed…