Journal: PLoS Pathogens

Article Title: Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells

doi: 10.1371/journal.ppat.1003316

Figure Lengend Snippet: 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 . 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.

Article Snippet: Recombinant Interferon proteins (IL-28A/IFNλ2, PeproTech, 300-02K: 1500 pg/mL; IL-29/IFNλ1 PeproTech, 300-02L: 500 pg/mL; IL-28B/IFNλ3, R&D Systems, 5259-IL-025: 10 pg/mL) were added to the infected Huh7.5.1 cultures 24 hours post-infection and the experiment was performed as described above.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2

doi: 10.1101/2021.02.17.431591

Figure Lengend Snippet: Nasal ALI cultures were pre- treated for 16 h with IFNβ (1000 IU/mL) or IFNλ1 (100 ng/mL) prior to infection (MOI 0.01). (A) Immunoblot of whole-cell lysates prepared from nasal ALI cultures at 48 hpi (representative of experiments in n=4 donors). (B) Plaque assay of apical washes showing significant reduction in infectious particle release at 48 hpi if pre-treated with IFNβ (1000 IU/mL) or IFNλ1 (100 ng/mL) (same experimental conditions as A; n=5 donors, mean ± SEM; **** P < 0.0001, ANOVA with Dunnett’s post- test correction compared to untreated control). (C-D) Immunoblot of whole-cell lysates prepared at 48 hpi. Nasal ALI cultures were either pre-treated with IFNβ (1000 IU/mL, C) or IFNλ1 (100 ng/mL, D) for 16h prior to infection with SARS-CoV-2 or IFN treatment was applied at 6 or 24 hpi. Results representative of experiments in n=3 donors. (E) Plaque assay on apical washes collected at 48 hpi from experiments in C-D (n=3 donors, mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ANOVA with Dunnett’s post-test correction compared to untreated control). Dotted line indicates lower limit of assay detection.

Article Snippet: Cytokines/inhibitors were used at the following concentrations: human recombinant IFNβ1 (1000 ng/mL; Avonex, NDC 59627-002-06, Biogen Inc, USA); IFNλ1 (100 ng/mL; 1598-IL-025, R&D Systems, USA); and Ruxolitinib (10 μM; S1378, Calbiochem, USA) alongside the appropriate dilution of DMSO vehicle.

Techniques: Infection, Western Blot, Plaque Assay, Control

Journal: Frontiers in Immunology

Article Title: IRF1 Maintains Optimal Constitutive Expression of Antiviral Genes and Regulates the Early Antiviral Response

doi: 10.3389/fimmu.2019.01019

Figure Lengend Snippet: IRF1 is required for early expression of types I and III IFNs and ISG expression. (A) Parent BEAS-2B and IRF1 KO cells were transfected with poly I:C, and expression of IFNβ, IFNλ1, or IFNλ2 transcripts were examined by RT-qPCR at 6 h or 24 h after poly I:C transfection. Data represent mean ± SEM from four independent experiments. (B) Parent BEAS-2B and IRF1 cells were transfected with a plasmid expressing firefly luciferase under the control of the IFNβ promoter or a plasmid constitutively expressing Renilla luciferase. Cells were then transfected with poly I:C and luciferase expression was examined at 6 h or 24 h afterwards. Firefly luciferase expression was normalized to Renilla luciferase expression and expressed as relative light units (RLU). Data shown are mean ± SD from three independent experiments. (C) IRF1 KO and parent cells were transfected with poly I:C, and cell lysates were immunoblotted for STAT1 phosphorylation (Y701). (D) Experimental protocol is same as A except that ISG expression was measured by RT-qPCR. Relative gene expression (2 −ΔΔ Cq ) is shown. Data represent mean ± SD from three independent experiments. * Statistically significant.

Article Snippet: Human IFNλ1 (1598-IL) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Control, Phospho-proteomics, Gene Expression

Journal: Frontiers in Immunology

Article Title: IRF1 Maintains Optimal Constitutive Expression of Antiviral Genes and Regulates the Early Antiviral Response

doi: 10.3389/fimmu.2019.01019

Figure Lengend Snippet: IRF1 is dispensable for IFN-mediated restriction of VSV in BEAS-2B cells. Parent and IRF1 KO cells were pre-treated with IFNβ (A,B) or IFNλ1 (C,D) for 6 h, then infected with VSV in the presence of IFN, and harvested at 20 hpi for flow cytometry. Data represent mean ± SD from three independent experiments. Percent cells infected is shown in (A,C) and normalized infection is shown in (B,D) . (E) Parent and IRF1 KO BEAS-2B cells were either treated with vehicle control or with B18R or Y136 proteins 24 h before infection. Cells were then infected with VSV at 0.01 MOI in the presence of B18R or Y136 and percent cells infected at 18 hpi was analyzed by flow cytometry. Data represent mean ± SD from three independent experiments. (F) Parent and IRF1 KO BEAS-2B cells were either treated with vehicle control or with JAK inhibitors 24 h before infection and then infected with VSV at 0.01 MOI in the presence of the drugs. Percent cells infected at 18 h was analyzed by flow cytometry. Data represent mean ± SD from three independent experiments. * Statistically significant.

Article Snippet: Human IFNλ1 (1598-IL) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: IRF1 Maintains Optimal Constitutive Expression of Antiviral Genes and Regulates the Early Antiviral Response

doi: 10.3389/fimmu.2019.01019

Figure Lengend Snippet: IRF1 regulates basal expression of a subset of ISG in BEAS-2B cells. (A–C) Parent and IRF1 KO cells were either treated with IFNβ (0.2 ng/ml) or IFNλ1 (5 ng/ml) for 24 h and gene expression was analyzed by RNA-seq. Heat map (A) and Venn diagram (B) for IRF1 dependent- and IFN inducible genes are shown. (C) Validation of IRF1 dependent, non-ISGs (top row), IRF1 dependent ISGs (middle row) and IRF1-independent ISGs (bottom row) expression by RT-qPCR using comparative C q method (2 −ΔΔ Cq ). Data represent mean ± SEM from three independent experiments. * Statistically significant.

Article Snippet: Human IFNλ1 (1598-IL) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Gene Expression, RNA Sequencing, Biomarker Discovery, Quantitative RT-PCR