anti human interferon lambda receptor 1 neutralizing antibody  (PBL Assay)

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Wild-type (WT) and Plscr1 -/- mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection. ( A ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( B ) Differential cell counts in BAL. ( C ) Representative lung sections stained with Hematoxylin and Eosin (H&E). Scale bars represent 3 mm (main) and 200 μm (inlays). ( D ) Whole lungs were analyzed for Ifna , Ifnb , Ifng, and Ifnl RNA by qRT-PCR. ( E ) Tnf-α and Ifn-λ concentrations in BAL by ELISA. Data are expressed as mean ± SEM of n=3–14 mice/group. All data were pooled from three independent experiments and described biological replicates. * p <0.05, ** p <0.01. dpi, days post-infection.

Article Snippet: In control Calu-3 groups, cells were first incubated with 1 μg/mL of anti-human interferon lambda receptor 1 neutralizing antibody (PBL Assay Science, cat #21885–1, clone #MMHLR-1) for 1 hr, and then treated with IFN-λ.

Techniques: Virus, Infection, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Wild-type (WT) and Plscr1-/- mice were intranasally given 2.5 μg/g of body weight of poly(I:C) (HMW) constitutively for 6 days and sacrificed on day 7. ( A ) Scheme of experiment. ( B ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( C ) Differential cell counts in BAL. ( D, F, G ) Whole lungs were analyzed for Ifna, Ifnb, Ifng, Ifnl ( D ); Plscr1 ( F ); and Ifnlr1 ( G ) RNA by qRT-PCR. ( E ) Representative lung sections stained with Hematoxylin and Eosin (H&E). Scale bars represent 3 mm (main) and 200 μm (inlays). Data are expressed as mean ± SEM of n=5–12 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, *p<0.05, ***p<0.001.

Article Snippet: In control Calu-3 groups, cells were first incubated with 1 μg/mL of anti-human interferon lambda receptor 1 neutralizing antibody (PBL Assay Science, cat #21885–1, clone #MMHLR-1) for 1 hr, and then treated with IFN-λ.

Techniques: Quantitative RT-PCR, Staining

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Plscr1 floxStop and Plscr1 floxStop ;Foxj1-Cre + mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection and sacrificed at 3 dpi. ( A ) Schematic representation of the experimental design of ciliated epithelial cell conditional Plscr1 KI mice. ( B ) Validation of Plscr1 overexpression in lungs of Plscr1 floxStop ;Foxj1-Cre + mice by qRT-PCR. ( C ) Representative immunofluorescent staining for Plscr1, Ifn-λr1, and Foxj1 in lungs. Scale bars represent 50 μm (main) and 10 μm (inlays).( D ) Mean relative weight of mice. ( E ) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. ( F ) Infectious viral titer in the lungs was assessed by plaque assays. ( G ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( H ) Neutrophil percentages in BAL. ( I ) Whole lungs were analyzed for Ifnlr1 RNA by qRT-PCR and Ifn-λr1 protein by western blot. ( J ) Whole lungs were analyzed for Ifna , Ifnb , Ifng , and Ifnl RNA by qRT-PCR. ( K ) Model depicting proposed mechanism of PLSCR1-regulated IFN-λ signaling. Data are expressed as mean ± SEM of n=3–10 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, * p <0.05, ** p <0.01, *** p <0.001. dpi, days post-infection. Figure 8—source data 1. PDF file containing original membrane for , indicating the relevant bands and treatments. The membrane was cut just under 50 kDa marker after transfer. The top part was incubated with α-Ifn-λr1 antibody and the bottom part was incubated with α-β-actin antibody. They were then incubated with corresponding secondary antibodies separately. The exposure time was adjusted to visualize Ifn-λr1 (top) or β-actin (middle). A colorimetric photo was taken to visualize the molecular weight markers (bottom). Lanes 2 and 3 were from an unrelated experiment. Figure 8—source data 2. Original membrane corresponding to .

Article Snippet: In control Calu-3 groups, cells were first incubated with 1 μg/mL of anti-human interferon lambda receptor 1 neutralizing antibody (PBL Assay Science, cat #21885–1, clone #MMHLR-1) for 1 hr, and then treated with IFN-λ.

Techniques: Virus, Infection, Biomarker Discovery, Over Expression, Quantitative RT-PCR, Staining, Western Blot, Membrane, Marker, Incubation, Molecular Weight

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Plscr1 floxStop and Plscr1 floxStop ;Lyz2-Cre + mice were exposed to sublethal (300 pfu) influenza A virus (IAV) (WSN) infection. ( A ) Validation of Plscr1 overexpression in lungs of Plscr1 floxStop ;Lyz2-Cre + mice by qRT-PCR. ( B ) Mean relative weight of mice. ( C ) Total Bronchoalveolar lavage (BAL) leukocyte numbers. ( D ) Differential cell counts in BAL. ( E ) Viral RNA load in the lungs was assessed by quantifying M gene by qRT-PCR. ( F ) Representative lung sections stained with Hematoxylin and Eosin (H&E). Scale bars represent 3 mm (main) and 200 μm (inlays). ( G–I ) Whole lungs were analyzed for Ifna , Ifnb , Ifng , Ifnl ( G ); Ifnlr1 ( H ); and Plscr1 ( I ) RNA by qRT-PCR. Data are expressed as mean ± SEM of n=15–16 mice/group for weight loss. For the rest analysis, n=3–7 mice/group. All data were pooled from three independent experiments and described biological replicates. ns, not significant, ** p <0.01. dpi, days post-infection.

Article Snippet: In control Calu-3 groups, cells were first incubated with 1 μg/mL of anti-human interferon lambda receptor 1 neutralizing antibody (PBL Assay Science, cat #21885–1, clone #MMHLR-1) for 1 hr, and then treated with IFN-λ.

Techniques: Virus, Infection, Biomarker Discovery, Over Expression, Quantitative RT-PCR, Staining

Journal: eLife

Article Title: Phospholipid scramblase 1 (PLSCR1) regulates interferon-lambda receptor 1 (IFN-λR1) and IFN-λ signaling in influenza A virus (IAV) infection

doi: 10.7554/eLife.104359

Figure Lengend Snippet: Influenza infection is usually detected by intracellular pattern recognition receptors (PRRs) such as TLR 3 and 7, RIG-I, and MDA5. These PRRs activate the expression of IFNL in early infection stage through IRF-3 and NK-κB-controlled transcriptions. IFN-λ secreted by the infected cells interacts with IL-10R2 and IFN-λR1 on neighboring cell surfaces, which results in activation of expression of various IFN-stimulated genes, including PLSCR1 . In ciliated airway epithelial cells, PLSCR1 can further enhance the transcription of IFNLR1 by directly binding to its promoter region as a transcriptional factor, or interact with IFN-λR1 on the cell membrane.

Article Snippet: In control Calu-3 groups, cells were first incubated with 1 μg/mL of anti-human interferon lambda receptor 1 neutralizing antibody (PBL Assay Science, cat #21885–1, clone #MMHLR-1) for 1 hr, and then treated with IFN-λ.

Techniques: Infection, Expressing, Activation Assay, Binding Assay, Membrane

Journal: Microbiology Spectrum

Article Title: Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells

doi: 10.1128/spectrum.01361-23

Figure Lengend Snippet: MeV is susceptible to exogenous IFN treatment. ( A ) HAE were infected at the basolateral surface with MeV expressing GFP upstream of N-protein (1 MOI) or ( B ) apical surface with RSV expressing GFP upstream of N-protein (1 MOI). Recombinant IFN-β (100 ng/mL), IFN-λ (2 µg/mL), or PBS vehicle (mock) were added to the apical surface of cultures at 18 hpi. Treatment lasted 24 h. Transwells were imaged 2–5 dpi. ( A ) Infectious center size or ( B ) fluorescence intensity was quantified using ImageJ software. Images were blinded in ImageJ prior to measurements using a blind-analysis tool. Measurements were unblinded just prior to GraphPad Prism input. n = 3 donors, indicated by color. Representative low power images for each treatment are shown. Scale bar = 500 µm. Significance was determined by one-way ANOVA with Tukey’s multiple comparison corrections.

Article Snippet: To induce IFN-blocking, MeV-infected HAE cultures were treated apically with 2 μg/mL recombinant B18R protein (StemCell Technologies cat# 78075) or 50 ng/mL monoclonal anti-human IFN-λ1 neutralizing antibody (clone MMHLR1, PBL Assay Science, Piscataway, NJ, cat# 21885-1).

Techniques: Infection, Expressing, Recombinant, Fluorescence, Software, Comparison

Journal: Microbiology Spectrum

Article Title: Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells

doi: 10.1128/spectrum.01361-23

Figure Lengend Snippet: Blocking IFN in HAE does not increase IC size. ( A ) HAE were infected at the basolateral surface with MeV expressing GFP upstream of N-protein (1 MOI) or ( B ) apical surface with RSV expressing GFP upstream of N-protein (1 MOI). Recombinant B18R (2 µg/mL), anti-λ1 blocking antibody (50 ng/mL), or PBS vehicle (mock) were added to the apical surface of cultures immediately post infection. Treatment lasted 24 h. Transwells were imaged 2–5 dpi. Infectious center size ( A ) or fluorescence intensity ( B ) was quantified using ImageJ software. Images were blinded in ImageJ prior to measurements using a blind-analysis tool. Measurements were unblinded just prior to GraphPad Prism input. n = 3 donors, indicated by color. Representative low power images for each treatment are shown. Scale bar = 500 µm. Significance was determined by one-way ANOVA with Tukey’s multiple comparison corrections.

Article Snippet: To induce IFN-blocking, MeV-infected HAE cultures were treated apically with 2 μg/mL recombinant B18R protein (StemCell Technologies cat# 78075) or 50 ng/mL monoclonal anti-human IFN-λ1 neutralizing antibody (clone MMHLR1, PBL Assay Science, Piscataway, NJ, cat# 21885-1).

Techniques: Blocking Assay, Infection, Expressing, Recombinant, Fluorescence, Software, Comparison

Journal: Microbiology Spectrum

Article Title: Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells

doi: 10.1128/spectrum.01361-23

Figure Lengend Snippet: Neither WT MeV nor V KO MeV induces basolateral release of IFN in HAE. HAE were either mock infected or infected with RSV, WT MeV, or V KO MeV (1 MOI) for 4 h. Basolateral media were collected 12 hpi and everyday up to 7 dpi. Basolateral medium was replenished for each culture with fresh media after collection. Samples were analyzed for the presence of ( A ) IFN-α2, ( B ) IFN-β, ( C ) IFN-λ1, and ( D ) IFN-λ2/3. In addition, antiviral cytokines ( E ) CXCL10 and ( F ) IL-6 were analyzed using a human antiviral response panel. Significance was determined by one-way ANOVA with Tukey’s multiple comparison corrections.

Article Snippet: To induce IFN-blocking, MeV-infected HAE cultures were treated apically with 2 μg/mL recombinant B18R protein (StemCell Technologies cat# 78075) or 50 ng/mL monoclonal anti-human IFN-λ1 neutralizing antibody (clone MMHLR1, PBL Assay Science, Piscataway, NJ, cat# 21885-1).

Techniques: Infection, Comparison

Journal: Microbiology Spectrum

Article Title: Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells

doi: 10.1128/spectrum.01361-23

Figure Lengend Snippet: IFN RNA levels are not elevated during MeV infection of HAE. ( A ) Single-cell RNA sequencing was performed on mock or MeV-infected cultures 3 and 7 dpi. Each MeV-infected culture was FACS sorted into GFP+ or GFP− populations. Intensity shows measure of raw gene transcript counts normalized for the number of cells and total transcript reads in each group (MNA). Every condition included 10 pooled, matched donor HAE cultures, and sequencing was performed with a 10× Genomics scRNA-seq. ( B ) A heat map is shown depicting expression levels for selected innate immune genes measured by RNAseq of MeV-infected HAE. Transcript abundances are expressed as transcripts per million (TPM). MNA and TPM are both measures of raw transcript reads; however, direct comparison of the color intensity between the two heat maps is not necessarily meaningful. HAE were infected with MeV and collected for sequencing at 0, 0.5, 1, 2, 3, and 5 dpi. n = 4 donors. Heatmaps were constructed using RStudio. ( C–H ) qRT-PCR results of indicated genes from MeV-infected HAE 0.25–5 dpi. n = 3 donors.

Article Snippet: To induce IFN-blocking, MeV-infected HAE cultures were treated apically with 2 μg/mL recombinant B18R protein (StemCell Technologies cat# 78075) or 50 ng/mL monoclonal anti-human IFN-λ1 neutralizing antibody (clone MMHLR1, PBL Assay Science, Piscataway, NJ, cat# 21885-1).

Techniques: Infection, Single Cell, RNA Sequencing, Sequencing, Expressing, RNA sequencing, Comparison, Construct, Quantitative RT-PCR