The bystander effect in hepatitis C virus infection: cellular interactions between infected cells and uninfected cells

Abstract

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide that often results in progressive liver disease in the form of fibrosis, cirrhosis and in some cases, hepatocellular carcinoma. The mechanisms responsible for progression to advanced liver disease are poorly understood, but this primarily occurs as a result of chronic hepatic inflammation. Despite universal involvement of the liver in this inflammatory and fibrogenic process, only a small percentage of hepatocytes are infected. We therefore hypothesised that the pathological effect of the virus is extended beyond the infected hepatocyte to uninfected ‘bystander’ cells by cellular interactions between these cells. To study this hypothesis, we developed in vitro cell culture model systems to observe the interactions between HCV-infected and uninfected Huh-7 cells and stellate cells. HCV permissive Huh-7 cells are relatively unresponsive to virus infection with regard to the innate immune response. This is due to a lack of expression of the pattern recognition receptor Toll-like receptor 3 (TLR3), which is known to play an important role in the innate immune response to HCV infection. To restore the response of infected Huh-7 cells to HCV we generated a Huh-7 cell line stably expressing functional TLR3. We subsequently demonstrated by microarray analysis upregulation of TLR3 response genes such as chemokines and classical interferon response genes (ISGs) in response to HCV infection of these cells. To prevent HCV infection of Huh-7 ‘bystander’ cells we also generated a line refractory to HCV infection by shRNA knockdown of the essential HCV entry receptor CD81. This cell line was also tagged with GFP to allow for FACS sorting of uninfected cells in co-culture. We subsequently employed these cell lines in conditioned media and co-culture model systems to examine the cell interactions mediated by soluble factors and cell-to-cell contact at the level of the transcriptome using Affymetrix microarray analysis. Although the effect of HCV-infected hepatocytes on uninfected ‘bystander’ hepatocytes was not dramatic, preliminary data suggested that suppressor of cytokine signalling 3 (SOCS3), a known inhibitor of endogenous interferon signalling pathways, is upregulated in uninfected Huh-7 cells co-cultured with HCV-infected TLR3-positive Huh-7 cells. Furthermore we also demonstrated that HCV-infected cells exert an antiviral effect on other infected cells, possibly via exosome-mediated signalling, and can increase expression of pro-fibrogenic markers in hepatic stellate cells. We also showed that TLR3-positive uninfected Huh-7 cells enhance chemokine expression in HCV-infected hepatocytes. In summary, we have generated stable cell lines that can be employed in an in vitro cell culture model system to study the interactions between HCV-infected hepatocytes and other resident liver cells such as uninfected hepatocytes and hepatic stellate cells. We have demonstrated bidirectional cross-talk between cell types, and the observed exerted effects are likely to contribute to the pathogenesis of chronic liver disease in HCV infection by recruiting uninfected cells into the proinflammatory and pro-fibrogenic response to HCV infection. The knowledge gained from this work contributes to our understanding of the mechanisms underlying progression of liver disease in HCV infection.