Fig 1: Co-culture of JFH-1-infected Huh7.5.1 cells and pDC-GEN2.2 cells leads to the upregulation of IFN genes and viral control.A) Huh7.5.1 cells were infected for 24 hours with JFH-1 and then resting pDC-GEN2.2 cells were added for 24 hours. mRNA from the CD45+ cells was isolated and examined for IFN gene expression. Gene fold increase is shown for each gene after normalization to the reference gene GAPDH and the uninfected condition. B) Huh7.5.1 cells were infected for 24 hours with JFH-1 and then pDC-GEN2.2 cells that had been mock transfected, transfected with X-region RNA or pU/UC RNA for 8 hours were added for 4 days (5 days total infection). RNA was isolated and examined for JFH-1 copy number. Normalized HCV copy number is shown where the infection control condition HCV copy number is set to 1 and other conditions are expressed as normalized HCV copy number compared to infection control. Normalized HCV Copy Number = (Absolute copy number for condition/absolute copy number for infection control) 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: 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 3: HA and CB inhibit Influenza-induced production of type I interferons in PBMC and mice.(a–c) mRNA levels of IFN-α (a), IFN-β (b) and IFN-λ2/3 (c) from PBMC pre-incubated with histamine, and CB and stimulated overnight with Influenza, were measured by RT-qPCR and normalized to RPL13A. Data shown as the means of three independent experiments ±s.e.m. (d–f) 29S8 mice were infected with X31 (800 TCID50) and IFN-α (d), IFN-β (e) and IFN-λ2/3 (f) levels in BAL fluid were measured by ELISA (as described in methods). Data shown as the means ±s.e.m. ***P<0.0001, **P<0.001, *P<0.01 as measured by two-way ANOVA with Bonferroni post-tests.
Fig 4: The inhibitory effect of amines on pDC requires the binding to CXCR4.(a) Jurkat cells were incubated with CXCL12 (100 nM), HA or CB (1 mM) at 4 °C for 30 min before staining with 12G5 antibody (anti-CXCR4) to assess compound fixation on CXCR4 by flow cytometry. (b) Confocal microscopy of purified pDC was performed to assess the internalization of CXCR4. Cells were incubated with 12G5 antibody at 4 °C before treatment with CB. The cells were stained with secondary antibody before or after being fixed and permeabilized, to observe extracellular or intracellular CXCR4 expression respectively. Cells were mounted with Fluoromount G with DAPI (blue). Scale bar, 7 μm. (c) Quantification of the intensity of fluorescence of 15 purified pDC after treatment or not. (d) Jurkat cells were incubated with CXCL12 (250 nM) or CB (10 μM) at 37 °C for 30 min before being stained with 1D9 antibody (anti-CXCR4) to assess internalization of CXCR4 by the compounds by flow cytometry. (e) Confocal microscopy of purified pDC after pre-incubation with FFN-511 (green). Cells were then stained with anti-CXCR4 (12G5) (red) and mounted with Fluoromount G with DAPI (blue). Scale bar, 12 μm. (f) Quantification of the colocalization between CXCR4 and FFN-511 in the cytoplasm of 20 purified pDC. Mander's Coefficient was obtained after analysis by JACoP pluging in ImageJ. M1 represents FFN-511 overlapping CXCR4 and M2 represents CXCR4 overlapping FFN-511. (g) Affinity of ligands for CXCR4 receptor were evaluated with the Tag-lite assay on HALOtag-CXCR4 receptors. Cells expressing HALO-tag CXCR4 were incubated with HALOtag- Lumi4-Tb (100 nM) for 1 h, then incubated in the presence of CXCL12-red tracer (5 nM) and increasing concentration of competitors. FRET signal were measured after overnight incubation at 4 °C (ref. 65). (h) mRNA levels of IFN-α/β from purified pDC pre-incubated with CB (10 μM) or CXCL12 (62.5 nM) for different times and stimulated overnight with HIV, were measured by RT-qPCR and normalized to RPL13A. For (c) and (f) the box represents the average±the s.d. The middle line represents the average. The whiskers represent the average±1.96 times the s.d. Data shown as the means of three independent experiments±s.e.m. P values (p) were determined using a two-tailed Student's t test. ***P<0.001, **P<0.01, *P<0.05.
Fig 5: 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/IFN?3 genotype (2 subjects IL28A/IFN?2, IL28B/IFN?3, IL29/IFN?1 and IFNß1; 1 subject IFNa2; RIG-I SNPs GG and GA); middle: CT IL28B/IFN?3 genotype (1 subject, RIG-I SNP AA); Bottom: TT IL28B/IFN?3 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.
Supplier Page from PBL Assay Science for VeriKine Human Interferon Alpha Multi-Subtype ELISA Kit