Ribavirin Contributes To Hepatitis C Virus Suppression By Augmenting PDC Activation And Type 1 IFN Production

Ribavirin(RBV) has been used for the treatment of chronic hepatitis C for decades. Compared with IFN-monotherapy, the combination of IFN and RBV doubled the SVR. With the advent of direct-acting antiviral drugs, the treatment for HCV has been IFN-free. And some clinical experiments show that RBV still plays a role in IFNfree therapy. Its mechanism of action, however, is not clear. The most recent analysis of gene expression in patients on DAA sofosbuvir plus RBV revealed that restoration of endogenous IFN?2.However, it is unclear which cell population is mainly responsible for restoration of endogenous IFN-I and whether RBV participates in this process. In infected hepatocytes, HCV efficiently blocks IFN-I production by NS3-4A mediated cleavage of MAVS and TRIF.Therefore, uninfected bystander hepatocytes or virus-stimulated non-parenchymal cells are proposed to be the source of IFN-I in infected liver. Among those cells, p DCs are the most abundant source of IFN-I. In vitro studies have shown that p DCs do not respond directly to HCV but cell-to-cell contact of infected hepatocytes with p DCs results in TLR7-mediated production of IFN?. During the antiviral therapy p DCs are exposed to both HCV-infected cells and RBV, therefore we hypothesize RBV can potentially influence the activation of p DCs and modulate the host response to HCV infection.Part Ⅰ Study the effect of RBV on the activation of p DCs.Using the p DC-Gen2.2 cell line, stimulate the cells with TLR7/9 ligands separately in absence or presence of RBV, test IFN-a production and the expression of IFN-a、IFN- β at indicated time points to examine the effect of RBV on p DCs; Test the production of IFN-a on a single cell by FACS; Stimulate PBMC and p DC-depleted PBMCwith Cp G A,to compare the production of IFN-a.To study the mechanism of the effect of RBV on p DCs, test the IRF-7、IRF-9 、STAT1 and ISGs at m RNA level by RT-PCR and IRF-7 protein by IF. Pretreat the cells with IFNAR-Ab, then stimulate with ligands in absence or presence of RBV to study the effect of IFNAR-Ab.The results are: All ligands(R848/Cp G A/Cp G B) activated p DC-Gen2.2 to produce IFN?. For each tested ligand, the co-stimulation with RBV resulted in twofold higher level of IFN? than stimulation with ligand alone. When applied alone, RBV was not able to activate p DC-Gen2.2. RBV enhancement of IFN? production was dose and time dependent. Interestingly, Cp GA and RBV treatment increased the level of IFN-a 、 IFNβ as measured by m RNA expression, but had no effect on expression of type II(IFN?) and III(IL29-λ1 and IL28β-λ3) IFNs. On a single cell basis, RBV can enhance Cp G A-induced IFN-a production in p DC-Gen2.2.RBV enhancement of IFN? production was reproduced in total PBMC stimulated with Cp GA. The depletion of p DCs from PBMC reduced the responsiveness to Cp GA.RBV increased IFNα production to the same extent in the presence and absence of IFNAR-Ab. RBV can enhance IRF-7 、IRF-9、STAT1 and some ISGs m RNA expression in p DC-Gen2.2 when applied with Cp GA.Part II Ribavirin contributes to hepatitis C virus suppression by augmenting p DC activationThe H77 S.3/Gluc2 A infection system allowed for robust propagation of HCV RNA in the culture of human hepatoma(Huh7.5) cells. The extent of viral replication in that reporter system was quantified by changes in secreted Gaussia luciferase(GLuc) activity. Evaluate HCV sensitivity to IFNα using this system; Test the status of p DCGen2.2 when cocultivated with H77 S.3/Gluc2 A cells; Cocultivate p DCs-Gen22 with H77 S.3/Gluc2 A infected cells in the presence and absence of Cp GA and RBV, examine the effect of Cp G A/RBV stimulated p DCs-Gen2.2on HCV replication.The results are: Addition of IFNα to the culture medium inhibited the viral replication in a dose-dependent fashion. The low dose of IFNα(10u/ml) was efficient to inhibit the HCV replication by 50% by day3. The doses of 100 and 1000u/ml inhibited the HCV replication to the same extent. RBV alone didn’t reducethe HCV replication.In co-culture system, activation of p DC-Gen2.2 with Cp GA significantly reduced the viral replication. Remarkably, the simultaneous activation of p DC-Gen2.2 with Cp GA and RBV potentiated the inhibition by 2 –fold compared to Cp G alone. Consistently, co-stimulation with Cp GA and RBV augmented the production of IFNα.In conclusion, using the p DC-Gen2.2 cell line we found RBV alone was not able to activate p DC-Gen2.2, but it could enhance TLR-7 and TLR-9-mediated IFN-I responses. Using the HCV infection and replication system H77 S.3/Gluc2 A, we showed that RBV did not directly inhibit HCV replication, but it enhanced the ability of activated p DCs to inhibit HCV replication, correlated with elevated induction of IFNα. Our findings provide novel evidence that RBV contributes to HCV inhibition by augmenting p DCs activation and IFN-I production.