Methods: To examine the effect of infected hepatocytes on other cell types in the absence of cell-to-cell contact we used HCV Jc1-infected Huh-7 cells that stably express TLR3, which is known to RXDX-106 price recognize HCV dsRNA. We also generated an Huh-7 cell line refractory to HCV entry through shRNA knockdown of CD81 to simulate uninfected hepatocytes. These cells were cultured in conditioned media (CM) from TLR3-positive HCV-infected cells, as were PH5CH8 cells, stellate cells and cells harbouring a subgenomic replicon
with a luciferase reporter. To examine the effect of infected TLR3-positive Huh-7 cells in cell contact with other hepatocytes we developed a CD81-negative cell line with stable mCherry expression on the cell surface (mCherry fused to transferrin receptor membrane targeting signal),
allowing magnetic bead-based separation from HCV-infected cells after co-culture. Gene expression was evaluated by qRT-PCR and Affymetrix GeneChip Trametinib clinical trial analysis. Results: Reintroduction of TLR3 into Huh-7 cells and stimulation with either poly(I:C) or infection with HCV Jc1 significantly upregulated mRNA expression of a number cytokines and chemokines (RANTES, MIP1β, IP-10) 上海皓元 in comparison to control cells (ΔTIR), as demonstrated by qRT-PCR and microarray analysis. These results indicate that TLR3-positive Huh-7 cells successfully recognized dsRNA and activated the TLR3 pathway. CM from HCV-infected TLR3-positive Huh-7 cells was used to stimulate CD81-negative Huh-7 cells and PH5CH8 cells, however little transcriptional response was demonstrated by microarray analysis, suggesting no impact
on the gene expression in uninfected cells in the absence of cell-to-cell contact. However incubation of cells harbouring a luciferase reporter replicon in CM demonstrated a reduction in viral replication, suggesting an antiviral effect of soluble factors secreted from infected cells. The transcriptional profile of mCherry-positive cells co-cultured with and separated from HCV-infected TLR3-positive Huh-7 cells is being evaluated to determine the impact of cell-to-cell contact on uninfected cells. Incubation of stellate cells in CM has also been performed and results are under analysis. Conclusion: These studies will help define the bystander effect of HCV-infected hepatocytes on uninfected hepatocytes, infected hepatocytes and stellate cells with the ultimate aim to identify mediators responsible for the pathogenesis of HCV-related liver disease.