4C), IFN-β protein levels remained below 4 pg/mL, and IFN-α protein was undetectable (Fig. 4D). In contrast, IL-28 and IL-29 peaked to significantly higher mRNA levels within 24-48 hours and protein levels reached up to 250 pg/mL 72 hours after HCV infection (Fig. 4C-D). Variations in ISG and IFN responses among donors did not correlate with HCV RNA levels in PHH cultures (Fig. 4E,F and Supporting Fig. 1) nor with IL28B SNPs (Supporting Fig. 2). Overall, our results demonstrate that transfection of PHH with double-stranded RNA (dsRNA) and infection with HCV reproduced the ISG and IFN expression

profile that we observed in the liver during acute HCV infection. To study the functional relevance of the strong type III IFN response during HCV infection, we examined its effect on HCV replication using Huh7-Lunet cells with a subgenomic HCV PLX3397 luciferase replicon as a readout.23 Recombinant type I (500 pg/mL) and type III IFNs (100 ng/mL) inhibited HCV replication by more than 90%, and this inhibition was almost completely abrogated by cytokine-specific neutralizing Abs (Fig. 5A). IL-29, IL-28A, and IL-28B showed 50% inhibition of HCV replication at 200-500 pg/mL (Fig. 5B), which is the maximum amount of type III IFNs that PHH produced 72 hours postinfection

(Figs. 4D and 6). Because 50% antiviral activity can also be mediated by very low concentrations of type I IFNs below the ELISA detection limit, we used an indirect method to further evaluate the relative effect of type I and III IFNs on HCV replication. Supernatants from PHH cultures harvested 72 hours post-HCV infection reduced click here HCV replication by 50% (Fig. 5C). Whereas neutralization of the supernatant with Abs against either type I (IFN-α and IFN-β) 4-Aminobutyrate aminotransferase or III IFNs (IL-28A, IL-28B, and IL-29) significantly increased HCV replication, neutralization of type III IFNs had a greater effect than neutralization of type I IFNs (P = 0.0244). This result suggests that type III IFNs contribute significantly to antiviral activity during

HCV infection. Experiments with hepatoma cell lines and TLR-stimulated macrophages suggest that type III IFNs are ISGs themselves and can be induced in a type I IFN-mediated manner.8, 24 To evaluate whether this holds true for HCV-infected PHH, we infected PHH with HCV in the presence or absence of neutralizing Abs against IFN-α and IFN-β. Whereas IL-28 secretion decreased in the presence of neutralizing Abs against type I IFNs (P = 0.0078), IL-29 production was not affected in the majority of cultures (P = not significant; Fig. 6). Thus, IL-29 can be induced independently from type I IFN signaling in HCV-infected hepatocytes. pDCs were recently shown to contribute to the induction of IFN-α and ISGs when cocultured with HCV-replicating hepatoma cells.7 We therefore asked whether this result extended to pDCs cocultured with HCV-infected PHH and to the production of type III IFNs. Less than 0.