il28ra subunit  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: Analysis of IFN protein from HIEs

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: IFN response to inactivated HRV. HIEs (j3) were inoculated with replication-competent HRV (MOI of 10 FFU) or with an equivalent amount (in micrograms) of inactivated HRV particles. (A) Expression of type III IFN (IFNL1), type I IFN (IFNB1), and the ISG IFI44L was assessed at 6 hpi by RT-qPCR. Each data bar represents the geometric mean ± 95% CI of the geometric mean (n = 4 technical replicates). Statistical analyses were performed by Mann–Whitney U test. (B) HIEs were infected as described in A and, after 1 h, HIEs were washed and resuspended in medium containing type III IFN receptor-blocking antibody (25 μg/mL anti-IL28RA). The media was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SD of three or four technical replicates.

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: Effect of receptor-blocking antibodies on rotavirus infectivity and induction of OAS2 transcripts. (A) Stock aliquots of Ito-HRV were incubated with no antibody (control), the anti-type III IFN receptor-blocking antibody, or the anti-type I IFN system antibody mixture for 2 h. To assess the titer of HRV under these conditions, MA104 cells were infected with control Ito-HRV aliquots or antibody-treated Ito-HRV aliquots for 1 h, washed twice, and incubated overnight in DMEM for 18 h. Viral titer was subsequently obtained by FFA. (B) HIEs (j11) suspended in differentiation medium containing 0.5 mg/mL pancreatin were treated with or without blocking antibodies to the type III IFN receptor (anti-IL28RA) or the type I IFN system (anti-type 1 IFN mixture). After 1 h, 100 U/mL of IFN-λ1 or IFN-β1 was added to the HIEs. After 26 h, total RNA was extracted, and the transcriptional response was compared with HIE cultures that were neither antibody-treated nor IFN-treated. Transcript levels of the ISG OAS2 were first normalized to GAPDH levels, and the fold increase was calculated by using the 2−ΔΔCt method. Displayed above the bars is the percent reduction in OAS2 levels between the two groups being compared. Data in A and B are presented as geometric mean ± 95% CI of the geometric mean of three technical replicates. (C) HIEs (j3) were treated with or without blocking antibodies to the type III IFN receptor (anti-IL28RA) or the type I IFN system (anti-type 1 IFN mixture). After 2 h, HIEs were treated with 100 U/mL of IFN-λ1 or IFN-β1 for 24 h. After 24 h, HIEs were washed to remove the antibodies and IFN and inoculated with Ito-HRV (MOI of 0.1). The control samples consisted of HIEs which did not receive antibodies nor IFN before Ito-HRV infection. The increase in infectious virus was calculated by subtracting the titer at 1.5 hpi in the control infection from the titer at 24 hpi in each of the five test groups. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SEM of four technical replicates. Statistical analyses were performed by Mann–Whitney U test.

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques: Blocking Assay, Infection, Incubation, Control, Virus, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: Transcriptional profiling of PHH infected with HCVcc at an MOI of 0.2. (A) Relative expression of genes involved in the PAMP recognition pathway showing data from three replicates at 24 h or 48 h post-infection normalized to naive PHH at the same time points. HUGO nomenclature for the genes is to the left. Statistical analysis was performed using one-way ANOVA and P values reported in (B) Expression levels of IFNAR1 and IL28RA transcripts in naive (closed circle) and HCVcc-infected (open circle) PHH. The data points represent the RMA values from each individual analyte and horizontal bars indicate the mean levels from three biological replicates. The data shown is representative of two independent experiments.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Expressing

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: iHLCs were infected with HCVcc (MOI of 0.2), GT-1b HCVser or mock infected, and subsequently maintained in culture with medium replacement every 2 days. At 24 h post-infection, the NS3 PI ASV (0.5 μM) or vehicle control dimethyl sulfoxide (DMSO) was added to cell cultures during media replenishment. (A) Persistent HCVcc replication as measured by the detection of the virally-encoded core antigen and expression of type I (IFNAR1, IFNAR2) and type III IFN (IL28RA, IL10RB) co-receptor subunits in cells were monitored by Western immunoblotting at the indicated time points, with β-actin used as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVcc infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of three independent Western immunoblot analyses. (B) Co-localization of IFNAR1 and HCV-core positive iHLCs were assessed by fluorescence microscopy. On Day 6 post-infection, naive and HCVcc-infected iHLCs were fixed, permeabilized and stained with antibodies to IFNAR1 (red) and HCV core antigen (green) as indicated. Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (C) iHLC cultures infected in parallel with GT-1b HCVser or HCVcc were harvested at the indicated time points and IFNAR1 copy numbers estimated by quantitative RT-PCR following normalization to cellular GAPDH levels in each sample. Results are expressed as mean ± standard deviations (n = 4). Statistical analysis was performed by Bonferroni’s multiple comparison tests: **, P < 0.05; ***, P < 0.001. (D) Cell lysates were harvested at the indicated time points and protein levels of IFNAR1 and IL28RA were examined by Western immunoblotting. Susceptibility of HCVser to the NS3 PI ASV was determined using an antibody directed against NS3. Arrows indicate the presence of the processed and unprocessed forms of the HCV-encoded NS3/4A protease in infected iHLCs. Detection of β-actin served as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVser infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of two independent Western immunoblot analyses.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Control, Expressing, Western Blot, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Comparison

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Naive and HCVcc infected iHLC cultures maintained in presence or absence of the IL28RA nAb were treated for 15 minutes with 10 ng/mL or 100 ng/mL of alfa or Lambda. Cell lysates were then prepared, and equal amounts of proteins subjected to Western immunoblotting to examine the levels of STAT1 phosphorylation using an antibody directed against phospho STAT1 (pSTAT1; Tyr701). Detection of total STAT1 served as loading control to ensure that equivalent amounts of protein were analyzed among samples. (B) Phosphorylation of STAT1 in iHLCs was evaluated upon stimulation using the Luminex bead-based assay. MFI values were reported as mean values of three independent cultures. Error bars show the standard deviations. Two-way ANOVA statistical analysis was performed using Bonferroni post test: ***, P < 0.001.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Western Blot, Phospho-proteomics, Control, Luminex, Bead-based Assay

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Expression of Mx1 protein (red) and HCV-core antigen (green) was monitored in HCVcc-infected iHLC cultures maintained in presence or absence of the IL28RA nAb (10 μg/mL). Dual immunostaining was performed as described in the legend for . Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (B) Induction of Mx1 expression (red) was assessed in Naive iHLCs 24 h following stimulation with 10 ng/mL of alfa (a) or Lambda (b). iHLC cultures infected with HCVcc were maintained in the presence (left panels) or absence (right panels) of the IL28RA nAb (10 μg/mL). On Day 6 post-infection, cells were treated as indicated with 10 ng/mL of alfa or Lambda for 24 h, and then dual immunostaining was performed using antibodies directed against Mx1 (red) and the HCV-core antigen (green). Arrows indicate examples of different HCV-Mx1 co-localization patterns in overlaid optical field. Scale bar, 60 μm.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Expressing, Infection, Immunostaining

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: Transcriptional profiling of PHH infected with HCVcc at an MOI of 0.2. (A) Relative expression of genes involved in the PAMP recognition pathway showing data from three replicates at 24 h or 48 h post-infection normalized to naive PHH at the same time points. HUGO nomenclature for the genes is to the left. Statistical analysis was performed using one-way ANOVA and P values reported in (B) Expression levels of IFNAR1 and IL28RA transcripts in naive (closed circle) and HCVcc-infected (open circle) PHH. The data points represent the RMA values from each individual analyte and horizontal bars indicate the mean levels from three biological replicates. The data shown is representative of two independent experiments.

Article Snippet: The monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (clonal hybridoma E10889) was synthesized at ZymoGenetics, Inc.

Techniques: Infection, Expressing

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: iHLCs were infected with HCVcc (MOI of 0.2), GT-1b HCVser or mock infected, and subsequently maintained in culture with medium replacement every 2 days. At 24 h post-infection, the NS3 PI ASV (0.5 μM) or vehicle control dimethyl sulfoxide (DMSO) was added to cell cultures during media replenishment. (A) Persistent HCVcc replication as measured by the detection of the virally-encoded core antigen and expression of type I (IFNAR1, IFNAR2) and type III IFN (IL28RA, IL10RB) co-receptor subunits in cells were monitored by Western immunoblotting at the indicated time points, with β-actin used as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVcc infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of three independent Western immunoblot analyses. (B) Co-localization of IFNAR1 and HCV-core positive iHLCs were assessed by fluorescence microscopy. On Day 6 post-infection, naive and HCVcc-infected iHLCs were fixed, permeabilized and stained with antibodies to IFNAR1 (red) and HCV core antigen (green) as indicated. Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (C) iHLC cultures infected in parallel with GT-1b HCVser or HCVcc were harvested at the indicated time points and IFNAR1 copy numbers estimated by quantitative RT-PCR following normalization to cellular GAPDH levels in each sample. Results are expressed as mean ± standard deviations (n = 4). Statistical analysis was performed by Bonferroni’s multiple comparison tests: **, P < 0.05; ***, P < 0.001. (D) Cell lysates were harvested at the indicated time points and protein levels of IFNAR1 and IL28RA were examined by Western immunoblotting. Susceptibility of HCVser to the NS3 PI ASV was determined using an antibody directed against NS3. Arrows indicate the presence of the processed and unprocessed forms of the HCV-encoded NS3/4A protease in infected iHLCs. Detection of β-actin served as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVser infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of two independent Western immunoblot analyses.

Article Snippet: The monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (clonal hybridoma E10889) was synthesized at ZymoGenetics, Inc.

Techniques: Infection, Control, Expressing, Western Blot, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Comparison

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Naive and HCVcc infected iHLC cultures maintained in presence or absence of the IL28RA nAb were treated for 15 minutes with 10 ng/mL or 100 ng/mL of alfa or Lambda. Cell lysates were then prepared, and equal amounts of proteins subjected to Western immunoblotting to examine the levels of STAT1 phosphorylation using an antibody directed against phospho STAT1 (pSTAT1; Tyr701). Detection of total STAT1 served as loading control to ensure that equivalent amounts of protein were analyzed among samples. (B) Phosphorylation of STAT1 in iHLCs was evaluated upon stimulation using the Luminex bead-based assay. MFI values were reported as mean values of three independent cultures. Error bars show the standard deviations. Two-way ANOVA statistical analysis was performed using Bonferroni post test: ***, P < 0.001.

Article Snippet: The monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (clonal hybridoma E10889) was synthesized at ZymoGenetics, Inc.

Techniques: Infection, Western Blot, Phospho-proteomics, Control, Luminex, Bead-based Assay

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Expression of Mx1 protein (red) and HCV-core antigen (green) was monitored in HCVcc-infected iHLC cultures maintained in presence or absence of the IL28RA nAb (10 μg/mL). Dual immunostaining was performed as described in the legend for . Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (B) Induction of Mx1 expression (red) was assessed in Naive iHLCs 24 h following stimulation with 10 ng/mL of alfa (a) or Lambda (b). iHLC cultures infected with HCVcc were maintained in the presence (left panels) or absence (right panels) of the IL28RA nAb (10 μg/mL). On Day 6 post-infection, cells were treated as indicated with 10 ng/mL of alfa or Lambda for 24 h, and then dual immunostaining was performed using antibodies directed against Mx1 (red) and the HCV-core antigen (green). Arrows indicate examples of different HCV-Mx1 co-localization patterns in overlaid optical field. Scale bar, 60 μm.

Article Snippet: The monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (clonal hybridoma E10889) was synthesized at ZymoGenetics, Inc.

Techniques: Expressing, Infection, Immunostaining