Fig 1: Replicative control of HCV in JFH-1/Huh7.5.1 system with conditioned media (CM) from pU/UC-transfected pDC-GEN2.2 cells.Huh7.5.1 cells were infected for 24 hours prior to the addition of CM (A–D) or rIFNs (E) then 4 days later (5 days post-infection), cells were lysed and examined for HCV Copy number by qRT-PCR (see methods). A) Normalized JFH-1 copy number (see methods and below for calculation) after treatment with CM from Mock (negative; white bars)-, X-region (gray bars)- or pU/UC (dark gray bars)-stimulated pDC-GEN2.2 cells after 8 hours of RNA stimulation. B) Dose-dependent response of viral replication control. CM from the HCV PAMP-stimulated pDC-GEN2.2 cells was added to JFH-1 infected cells at the following dilutions: 1∶1 (Dark gray bars), 1∶10 (gray bars) or 1∶100 (white bars). C) Type I IFN dependence was determined using the Vaccinia protein B18R which blocks Type I IFN responses. D) Blocking IL-28B/IL-29 (IFNλ3/IFNλ1) with a blocking, cross-reactive antibody demonstrates dependence on Type III IFNs for a portion of the viral control. E) Recombinant Type III Interferons in the absence of CM at the same concentrations as found in the CM (IL-28A/IFNλ2: 1500 pg/mL; IL-28B/IFNλ3: 10 pg/mL; IL-29/IFNλ1: 500 pg/mL) were added to JFH-1 infected Huh7.5.1 cells. Normalized HCV Copy Number is shown where the infection control condition HCV copy number is set to 1 (except panel A where the Mock condition is set to 1) and other conditions are expressed as normalized HCV copy number compared to infection control (or compared to Mock in panel A). Normalized HCV Copy Number = (Absolute copy number for condition/absolute copy number for infection control). Combined data from 3 (A, C and E), 5 (B, D) independent experiments p values represent the Mann-Whitney result of the comparison. * p<0.05 ** p<0.01 *** p<0.001 # p≤0.0001. Bars represent the mean and error bars are +/− SEM.
Fig 2: pDC-GEN2.2 cells sense HCV PAMP and produce Type I and Type III IFNs.A) Cartoon of the 3′ end of the HCV genome indicating the location of the poly U/UC (pU/UC, HCV PAMP) and the X-region in the 3′ UTR. Adapted from reference [20]. B) Kinetics of interferon gene upregulation in GEN2.2 cells following transfection with the pU/UC RNA. Fold increases for each gene at each condition are shown after normalization to reference gene GAPDH and are compared to transfection with the X-region RNA (dashed line). Levels of IFN expression at 2 (white bars), 4 (gray bars), 8 (hashed bars) or 24 (black bars) hours are shown for each gene. C) Kinetics of PRR genes and ISGs show upregulation by pU/UC stimulation. Kinetics are shown as indicated in A for the IFN genes. D–G) Secretion of Type I and III IFNs by pDC-GEN2.2 cells following PAMP-stimulation. Increased production of D) IFNα, E) IFNβ, F) IL-28A/IFNλ2 and G) IL-29/IFNλ1 as detected by ELISA from pDC-GEN2.2 cells that have been stimulated with HCV PAMP compared to the X-region RNA. H) Western Blot of pDC-GEN2.2 cell lysates for IL-28B/IFNλ3 after 24 hours of HCV PAMP stimulation. The antibody was specific for IL-28B/IFNλ3 as it recognized low levels of recombinant IL-28B/IFNλ3 (rIL-28B; 10 ng) but failed to recognize recombinant IL-28A/IFNλ2 (rIL-28A; 5 µg). I) pU/UC-stimulation increases PRR signaling proteins in accordance to gene expression data. Western blots of listed PRR proteins after 8 or 24 hours stimulation with HCV PAMP RNA. B–C) Combined data from 5 independent experiments. D–I) Combined data from 3 independent experiments except for the gene expression graphs which are 5 independent experiments. H–I) Representative blots from 3 independent experiments. Densitometry shows relative density of each band after normalization to the reference protein. For gene expression graphs, p values are the Wilcoxon signed rank result for each gene and time point compared to the X-region stimulation from the same gene and time point. For ELISA and Densitometry graphs, p values are the Mann-Whitney result for the pU/UC condition compared to the X-region condition. * p<0.05 ** p<0.01 *** p<0.001 # p≤0.0001 Bars represent the mean and error bars are +/− SEM.
Fig 3: Ex vivo pDCs upregulate Type I and III Interferon genes in response to the HCV PAMP.A) Gene expression changes in ex vivo pDCs after HCV PAMP RNA stimulation. Top: subjects with the CC IL28B/IFNλ3 genotype (2 subjects IL28A/IFNλ2, IL28B/IFNλ3, IL29/IFNλ1 and IFNβ1; 1 subject IFNα2; RIG-I SNPs GG and GA); middle: CT IL28B/IFNλ3 genotype (1 subject, RIG-I SNP AA); Bottom: TT IL28B/IFNλ3 genotype (1 subject RIG-I SNP GG). Top graph is combined data from 2 independent experiments while middle and bottom graphs are data from a single independent experiment each. B) Same gene expression data as in A graphed together. Compared to the non-CC genotypes, the CC subjects had significantly more IFN mRNA. p values are the Wilcoxon signed rank result for (A) each gene compared to the X-region stimulation (dashed line) from the same gene, and (B) each gene CC genotype compared to each gene non-CC genotype. * p<0.05 ** p<0.01 *** p<0.001 # p≤0.0001. Bars represent the mean and error bars are +/− SEM.
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