TLR–3 stimulated non-parenchymal liver cells are potent suppressors of HCV replication

Aims and Aims: Hepatitis C virus (HCV) is an enveloped, positive-stranded RNA virus that belongs to the Flaviviridae and is the major cause of chronic hepatitis worldwide. While a substantial amount of data has been generated with respect to the role of the adaptive immune system (T cells, B cells), only little is known about the role of non- parenchymal liver cells (NPC) of the hepatic sinusoid (Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC)) in the antiviral defense against HCV. Therefore, the aim of this study was to further elucidate their role in the local innate immune response against HCV in the liver using a murine replicon system.

Materials and methods: MH1 and MH2 (kindly provided by C. Seeger) are mouse hepatoma cell lines (Hepa1–6) harbouring a subgenomic HCV replicon based on the HCV-Con1 replicon I377/NS3–3´ containing several adaptive mutations. To study the effect of type I and II interferons (IFN) in the absence of NPC, the cells were cultured in the presence of absence from various concentrations of IFN-α, IFN-β and IFN-γ. In addition, the cells were cultured with cell culture supernatants from freshly isolated murine KC and LSEC stimulated with various concentrations of TLR 1–9 agonists for 6h. Then, total RNA of the MH1 cells was isolated and the HCV replicon concentration was measured by quantitative real-time PCR.

Results: The replicon RNA is increasing over a time course of 72 hours with a fold change about four, independent from the passage number and confluence of the cells. Treatment of these cells with IFN-α, -β or -γ led to decreased HCV replicon replication in a dose dependent manner with half maximal inhibitory concentrations (IC50) that were comparable to data from human HCV replicon systems (5–10 U/ml). All three IFNs led to a maximal suppression of HCV replication of about 80%. Cultivation of MH1 cells with supernatants from TLR 3-stimulated KC or LSEC also led to a marked inhibition of HCV replication. While TLR 3-stimulated KC could suppress HCV replication by about 80% in our system, supernatants TLR 3-stimulated LSEC could suppress HCV-replication by about 60%, respectively. All other TLR agonists had no effect in this system despite the presence of all TLR receptors and KC and LSEC. The antiviral effect could be blocked by antibodies against IFN-α.

Conclusions: In conclusion, these results show that the HCV-Con1 Replicon I377/NS3–3´, when replicated in a mouse hepatic cell system, is sensitive to IFN-α, -β and -γ. Furthermore, TLR 3-stimulated NPC are potent suppressors of HCV-replication which is mediated through IFN-β. These novel findings are of particular relevance for the control of HCV replication by the innate immune system of the liver.