Journal: Hepatology (Baltimore, Md.)

Article Title: IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

doi: 10.1002/hep.25897

Figure Lengend Snippet: Fig. 2. Type I IFNs are minimally, but type III IFNs are robustly induced during acute HCV infection. (A) Serum HCV RNA titers (filled circles) and ALT levels (gray-colored area) were previously reported20,21 and are shown for reference purposes. (B-D) Intrahepatic type I (B) and III IFN (C) mRNA levels were determined by qPCR, normalized to endogenous references, and presented as fold-increase over preinfection levels. Type III (D), but not type I, IFNs (not shown) were detectable by ELISA in plasma.

Article Snippet: In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 lg/mL each of anti-IFN-a [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-b [R&D Systems, Minneapolis, MN]) or type III IFNs (10 lg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Hepatology (Baltimore, Md.)

Article Title: IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

doi: 10.1002/hep.25897

Figure Lengend Snippet: Fig. 3. ISG response of PHH to dsRNA is associated with weak type I, but strong type III IFN induction. PHHs were trans- fected with polyI:C to mimic intracellular viral RNA. ISG (A) and IFN mRNA (C) and protein levels (B and D) were determined by qPCR in transfected cells and by ELISA in culture supernatants. mRNA levels were normalized to endogenous references and presented as fold-increase over pretrans- fection levels. Mean and standard error of the mean are shown (A and C).

Article Snippet: In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 lg/mL each of anti-IFN-a [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-b [R&D Systems, Minneapolis, MN]) or type III IFNs (10 lg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

doi: 10.1002/hep.25897

Figure Lengend Snippet: Fig. 4. ISG response of PHH to HCV infection is associated with weak type I, but strong type III IFN induction. PHHs were infected with HCV at an MOI of 0.4-2.7. ISG mRNA (A) and protein (B) levels as well as IFN mRNA (C) and pro- tein (D) levels are presented as in Fig. 3. (E and F) HCV RNA levels in PHHs were compared to ISG (E) and IFN levels (F). Mean and standard error of the mean are shown (A, C, and E).

Article Snippet: In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 lg/mL each of anti-IFN-a [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-b [R&D Systems, Minneapolis, MN]) or type III IFNs (10 lg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).

Techniques: Infection

Journal: Hepatology (Baltimore, Md.)

Article Title: IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

doi: 10.1002/hep.25897

Figure Lengend Snippet: Fig. 5. Type III IFNs produced by HCV-infected PHH inhibit HCV repli- cation. (A) Recombinant type I (IFN-aA2 and IFN-b; 500 pg/mL) and type III (IL-28A, IL-28B, and IL-29; 100 ng/mL) IFNs decreased lucifer- ase activity of Huh7-HCV-Luc cells, and this effect was blocked by Ab- mediated neutralization of the respective cytokines. An isotype control Ab did not change RLU values (not shown). (B) Recombinant type I and III IFNs mediated a dose-dependent antiviral effect when added for 30 hours to Huh7-HCV-Luc cells. (C) Supernatant of HCV-infected PHH, harvested 72 hours postinfection and containing an average of 526 pg/ mL of type III IFN proteins, reduced HCV replication of Huh7-HCV-Luc cells by 50%. NAbs against IL28A, IL-28B, and IL-29 (type III IFN) reversed antiviral activity to a significantly greater extent than NAbs against IFN-a and IFN-b (type I IFN; P ¼ 0.0244). Statistical analysis: mean plus standard error of the mean, paired Wilcoxon’s signed-rank test. RLU, relative light units; Sup, supernatant; Nabs, neutralizing Abs.

Article Snippet: In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 lg/mL each of anti-IFN-a [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-b [R&D Systems, Minneapolis, MN]) or type III IFNs (10 lg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).

Techniques: Produced, Infection, Recombinant, Activity Assay, Neutralization, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

doi: 10.1002/hep.25897

Figure Lengend Snippet: Fig. 6. IL-29, but not IL-28, is produced in a type I IFN-independ- ent manner in HCV-infected hepatocytes. Treatment of PHH before and during HCV infection with NAb against IFN-a and IFN-b reduced the level of secreted IL-28, but not IL-29, in most PHH cultures. Statistical analysis: paired Wilcoxon’s signed-rank test. Nab, neutralizing Ab.

Article Snippet: In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 lg/mL each of anti-IFN-a [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-b [R&D Systems, Minneapolis, MN]) or type III IFNs (10 lg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).

Techniques: Produced, Infection

Journal: Hepatology (Baltimore, Md.)

Article Title: IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.

doi: 10.1002/hep.25897

Figure Lengend Snippet: Fig. 7. Production of IL-29 by PHH is independent of direct contact with pDCs. (A) Experimental strategy: PHHs were infected with HCV for 30 hours, followed by medium change and a 24-hour coculture with or without pDCs or transwells. (B) IFN-a secretion depended on direct contact between pDCs and HCV-infected PHHs. CXCL10 and CXCL11, but not IL-29, secretion was enhanced by PHH/pDC contact. PHH/ pDC coculture in the absence of HCV infection did not induce cyto- kines (not shown). Mean and standard error of the mean are shown for CXCL10 and CXCL11.

Article Snippet: In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 lg/mL each of anti-IFN-a [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-b [R&D Systems, Minneapolis, MN]) or type III IFNs (10 lg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: Surface plasmon resonance-derived binding constants and ISFG3 assay EC 50 values —, not determined.

Article Snippet: Reagents used in the assays included anti-IFNα (MMHA-2, PBL Assay Science), anti-IFNAR1 (ab10739, Abcam), and anti-IFNAR2 (MMHAR-2, PBL Assay Science) neutralizing antibodies, soluble receptors IFNAR1-FC (245-AB-050, R&D Systems) and IFNAR2-FC (4015-AB-050, R&D Systems), and viral type-I antagonist B18R (34-8185-85, eBioscience).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: SPR analysis of IFNϵ, IFNκ, IFNα2, and IFNω binding to IFNAR1-FC, IFNAR2-FC, and the IFNAR1/IFNAR2-FC heterodimer. SPR sensorgrams for each IFN-IFNAR interaction are shown in black. The calculated sensorgrams, derived from fitting the data to a 1:1 binding model, are shown in black (IFNα2), red (IFNκ), blue (IFNϵ), and green (IFNω). Kinetic and equilibrium constants derived from the data are shown in Table 2.

Article Snippet: Reagents used in the assays included anti-IFNα (MMHA-2, PBL Assay Science), anti-IFNAR1 (ab10739, Abcam), and anti-IFNAR2 (MMHAR-2, PBL Assay Science) neutralizing antibodies, soluble receptors IFNAR1-FC (245-AB-050, R&D Systems) and IFNAR2-FC (4015-AB-050, R&D Systems), and viral type-I antagonist B18R (34-8185-85, eBioscience).

Techniques: Binding Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: Molecular model of unique IFNϵ/κ and IFNα1 residues that modulate IFNAR2 and B18R binding. A and B, schematic models and structures of the (A) IFNα2/IFNAR1/IFNAR2 complex (PDB 3SE3) and the (B) IFNα2/B18R complex (IFNα2 from PDB 3S9D, and B18R derived from the C12R structure, PDB 3OQ3). C, ribbon diagram of the IFNα2 backbone with key residues that regulate IFNAR2 and B18R binding affinity, as discussed in the text, shown in yellow, orange, and magenta. D, superposition of IFNs in IFN/IFNAR2 and IFN/B18R structures with B18R D3 domain in red and the IFNAR2 D1 domain cyan. Three negatively charged residues conserved in IFNAR2 (green) and B18R (yellow) are positioned by IFNα arginines: Arg-35, Arg-56, and Arg=172 (magenta). E, amino acids within the AB loop region of IFNs that distinguish strong and weak IFNAR2 and B18R binders. Amino acid substitutions made to produce IFNα1 and IFNκ mutants are shown in bold.

Article Snippet: Reagents used in the assays included anti-IFNα (MMHA-2, PBL Assay Science), anti-IFNAR1 (ab10739, Abcam), and anti-IFNAR2 (MMHAR-2, PBL Assay Science) neutralizing antibodies, soluble receptors IFNAR1-FC (245-AB-050, R&D Systems) and IFNAR2-FC (4015-AB-050, R&D Systems), and viral type-I antagonist B18R (34-8185-85, eBioscience).

Techniques: Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

doi: 10.1101/2020.05.09.085456

Figure Lengend Snippet: PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p<0.05; **p<0.01; ***p<0.001).

Article Snippet: Cells were washed and incubated with antibodies to human IFN-α biotin (clone MMHA2; PBL), TNF-α-Pacific Blue (clone MAb11; Biolegend) or TNF-α FITC (clone cA2; Miltenyi Biotech), and Zenon-labeled (Invitrogen)-anti-IRF-7-AF488 (clone, G-8, Santa Cruz) or AF488 Zenon-labeled (Invitrogen) mouse IgG2a isotype control (clone UPC-10; Sigma-Aldrich,), for 30 minutes at RT.

Techniques: Incubation, Flow Cytometry, Inhibition, Control

Journal: bioRxiv

Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

doi: 10.1101/2020.05.09.085456

Figure Lengend Snippet: PBMC were incubated with SAHA at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed with intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 (MOI of 1) and IAV-induced IFN-α and TNF-α production in pDC by SAHA (500 nM) or DMSO vehicle control (1:1000) (A) . Pooled data of dose curve experiments showing a dosedependent effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α (B) and TNF-α (C) by SAHA (0-1000 μM) when compared to the respective vehicle control. (N = 3; 3 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p<0.05; **p<0.01; ***p<0.001).

Article Snippet: Cells were washed and incubated with antibodies to human IFN-α biotin (clone MMHA2; PBL), TNF-α-Pacific Blue (clone MAb11; Biolegend) or TNF-α FITC (clone cA2; Miltenyi Biotech), and Zenon-labeled (Invitrogen)-anti-IRF-7-AF488 (clone, G-8, Santa Cruz) or AF488 Zenon-labeled (Invitrogen) mouse IgG2a isotype control (clone UPC-10; Sigma-Aldrich,), for 30 minutes at RT.

Techniques: Incubation, Flow Cytometry, Inhibition, Control