As shown

in Fig. 5E, both vitamin D3 and calcitriol treatment increased MxA protein expression in HCV-infected cells in a dose-dependent manner. The results shown here indicate that vitamin D or calcitriol treatment induces antiviral IFN-mediated signaling pathways in HCV-infected cells. To simulate in vitro the in vivo combination therapy of IFN-α and vitamin D3,22 we treated HCV-infected cells with a combination of both agents. In a preliminary experiment we evaluated the effective concentration of IFN-α that achieves 50% inhibition of virus production (EC50) by the FFU reduction assay and found it to be 0.1 ng/mL (data not shown). For combination treatment, Huh7.5 cells were treated with various concentrations of vitamin D3 or calcitriol in combination with a sub-EC50 concentration of IFN-α (0.025 ng/mL), which exert only minimal anti-HCV inhibitory effects. After 3 hours the cells signaling pathway were infected with the virus and treated as described above. Virus titer was determined by FFU assay 72 hours postinfection. As shown in Fig. 6A,B, calcitriol and vitamin D3 as single agents inhibited virus production in a dose-dependent manner as described above, whereas only minimal (10%-20%) inhibition of viral production was observed by treatment with 0.025 ng/mL

IFN-α alone. Treatment with low concentrations of vitamin D3 (0.1 μM) or calcitriol (0.1 nM), which inhibited viral production by ≤10%, combined with a low concentration of Z-IETD-FMK IFN-α (0.025 ng/mL), resulted in a synergistic effect attaining 70%-80% inhibition. At moderate concentrations of vitamin D (0.5-1 μM) or calcitriol (1-10 nM),

the addition of IFN increased the inhibition of virus production additively. To date, the association between circulating vitamin D levels and morbidity related to infectious disorders has been mainly based on epidemiological studies. These studies provide evidence that vitamin D deficiency may confer increased risk of viral infections such as influenza, respiratory tract infections, and HIV and suggested that vitamin D possesses antiviral activity. However, 上海皓元 this notion is mainly based on the known ability of vitamin D to up-regulate antimicrobial peptides.18, 35 The recent findings that low vitamin D serum levels are related to low responsiveness to IFN-based therapy in chronic hepatitis C21 and that supplementation of vitamin D significantly improved interferon therapy outcome in these patients22, 36 led us to surmise that vitamin D may have a direct antiviral effect. We here demonstrate for the first time that vitamin D has a direct inhibitory effect on viral production. This inhibition may be partially attributed to augmentation of the innate immune response, as treatment of HCV-infected cells with vitamin D or calcitriol up-regulated the expression of IFN-β, the immediate cellular response to viral infection.