Figure 1.
Antiviral responses elicited by LIC-pIC in chimeric mice with humanized livers.
(A, B) Serum HCV RNA levels, relative to the baseline, in HCV genotype 1b-infected (A) or 1a-infected (B) chimeric mice following the indicated treatments, as determined by qRT-PCR (n = 3–5 per group). (C) Liver HCV RNA levels on Day 4 of LIC-pIC treatment (0.1 mg/kg/day), as determined by qRT-PCR. (D) Immunostained liver tissues of HCV genotype 1b-infected chimeric mice treated with saline (Upper) and LIC-pIC (0.1 mg/kg/day) (Lower) at Day 4. (E) Serum HBV DNA levels relative to the baseline following the indicated treatments, as determined by qPCR (n = 3–4 per group). (F) HBV DNA levels of chimeric mouse livers following LIC-pIC treatment, as determined by qPCR (n = 1 per each time point, the results indicates the mean of two measurements) (G) HCV RNA levels relative to the baseline following the indicated treatments, as determined by qRT-PCR (n = 3–4 per group). In all cases, bars indicate SD. **P<0.01, treatment vs. saline control by Dunnett’s multiple comparison test.
Figure 2.
Expression of genes encoding IFN-α, IFN-β, and IFN-γ following induction by LIC-pIC in chimeric mice with humanized livers.
(A) Experimental schedule in chimeric mice infected with HCV. (B) Serum concentrations (in chimeric mice) of human and mouse IFN-β and IFN-α, as determined by ELISA (n = 3–5 for no treatment or LIC-pIC group; n = 6–8 for PegIFN-α group). Bars indicate the mean concentration of human (red) and mouse (green) IFNs averaged across the entire group. The results indicates the representative of two measurements (C) The liver mRNA levels of genes encoding IFN-β, IFN-α2 and IFN-γ (human genes: IFNB1, IFNA2 and IFNG, respectively; mouse genes: Ifnb1, Ifna2 and Ifng, respectively), as determined by qRT-PCR (n = 3–4 per group). The bars indicate the mean mRNA levels of human (red) and mouse (green) IFN genes averaged across the entire group. The results indicates the representative of two measurements (D) Effectiveness of recombinant human or mouse IFN-β against HCV in FLR 3-1 HCV-replicon cells. The bars indicate SD (n = 4 per group). **P<0.01, treatments vs. the vehicle by Dunnett’s multiple comparison test.
Figure 3.
IFN expression following induction of innate immunity by LIC-pIC.
(A) Experimental schedule in the chimeric mice infected with HCV. (B) Microarray analysis of IFN family gene expression. Log2 ratio values of gene expression were calculated with respect to control (untreated and HCV-infected) chimeric mice. (C) Liver mRNA levels of human IFN genes (n = 3–5 per group), as determined by qRT-PCR. (D) Serum concentration of human IFN-λ1, IFN-λ2, IFN-β, and IFN-α (n = 3 per group), as determined by ELISA. (E) Serum HCV RNA levels, relative to the baseline, in HCV-infected chimeric mice treated with human IFN-λ, LIC-pIC, or control saline (n = 3–5 per group), as determined by qRT-PCR. (F) The HCV RNA levels in the serum and liver of HCV genotype 1a-infected chimeric mice collected on Day 4 of LIC-pIC treatment, as determined by qRT-PCR. The mice were additionally treated with either a control antibody or a neutralizing antibody against IFN-λ (n = 3 per group). Bars indicate SD. **P<0.01, treatments vs. saline controls on the respective day, by Dunnett’s multiple comparison test.
Figure 4.
IFN expression in cell lines following the induction of innate immunity by various stimuli.
(A, B) The indicated cell lines were stimulated by exposure to LIC-pIC (1 µg/ml). (A) The mRNA levels of IFN-β- and IFN-λ1-encoding genes (IFNB1 and IFNL1 (IL29), respectively) before and after (12 h) stimulation, as determined by qRT-PCR. (B) The concentration of IFN-λ1 (IL-29) in the culture medium 24 h after stimulation, as determined by ELISA. (C, D) The indicated cell lines were stimulated by viral infection with NDV or VSV. (C) The mRNA levels of IFN-β- and IFN-λ1-encoding genes (IFNB1 and IFNL1 (IL29), respectively) in HepG2 cells and MRC-5 cells in the absence or presence (12 h) of NDV or VSV, as determined by qRT-PCR. (D) The concentration of IFN-λ1 protein in the culture medium at 24 h post-infection, as determined by ELISA. (E, F) Expression in HepG2 cells of genes encoding MAVS, TICAM-1, and IFN-λ1 (MAVS, TICAM1, and IFNL1 (IL29) respectively), as determined by qRT-PCR, after 12 h stimulation with LIC-pIC (1 µg/ml). Cells were pre-treated for 48 h with siRNAs against MAVS, TICAM-1, or a non-target control. Bars indicate SD (n = 3 per group). (G) Schematic outline of the proposed mechanism of the antiviral response induced by LIC-pIC.