diy human ifn lambda 2 3  (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: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: (A) Duolink PLA was performed to examine interactions of FLAG-Iso1 and FLAG-Iso2 with IL10RB after IFNL3 stimulation (15min). Yellow puncta represent complex formation; nuclei were counterstained with DAPI (blue) and imaged by scanning confocal microscopy. Scale bar, 20µm. (B) Quantitation of mean puncta per cell showing comparable ability of FLAG-Iso1 and -Iso2 to form ligand-induced complexes with IL10RB. Three images per condition were analyzed, mean ± SEM shown. n=43 cells per image. ( C ) Representative brightfield and fluorescent images of dox-induced FLAG-Iso1 and –Iso2 cells pre-incubated with anti-FLAG antibody at 4°C, then shifted to 37°C in the presence or absence of IFNL3 showing redistribution of FLAG-specific fluorescent signal (green) over time. Images selected at each time point represent cells with comparable peripheral FLAG-specific fluorescent signal that corresponded with the mean observed for the total population. (D) Quantitation of FLAG-specific signal at the cell surface, normalized to the respective time point zero. n= 5000 live events, mean ± SEM shown. *p≤ 0.05, comparing FLAG-Iso1 to –Iso2 at each timepoint.

Article Snippet: WT- and IFNLR1 -KO-iHeps harboring FLAG-Iso1, FLAG-Iso2, and EV constructs were +/-dox-induced (100ng/ml, 24h) then mock, IFNL3 (100ng/ml), or IFNA2 (100ng/ml, PBL Assay Science) stimulated at 37°C for 15min, then collected and lysed on ice in RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors (Pierce).

Techniques: Confocal Microscopy, Quantitation Assay, Incubation

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: Western blot analysis of whole cell lysates from ( A ) WT iHeps and ( B ) IFNLR1 -KO iHeps +/-dox-induced for 24h then treated +/-IFNL3 or IFNA2 for 15min. GAPDH served as an indicator of equivalent protein loading per lane. The ratio of phosphorylated to total protein for ( C ) dox-uninduced WT iHeps and ( D ) dox-induced IFNLR1 -KO iHeps +/-IFNL3 is shown as a percentage, based on integrated band intensity determined in ImageJ.

Article Snippet: WT- and IFNLR1 -KO-iHeps harboring FLAG-Iso1, FLAG-Iso2, and EV constructs were +/-dox-induced (100ng/ml, 24h) then mock, IFNL3 (100ng/ml), or IFNA2 (100ng/ml, PBL Assay Science) stimulated at 37°C for 15min, then collected and lysed on ice in RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors (Pierce).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: Shown are MX1 and CXCL10 expression in iHeps pre-treated with JAK1-upadacitinib; IC 50 47nM) or TYK2-(deucravacitinib; IC 50 0.2nM) inhibitors prior to IFNL3 stimulation (100ng/ml, 24h) relative to mock-treated cells. ( A ) Comparison of MX1 expression in WT-Iso1 (+/-dox) and KO-Iso1 (+dox) iHep lines and ( B ) WT-Iso2 (+/-dox) and KO-Iso2 (+dox) iHep lines. ( C ) Comparison of MX1 expression in WT-Iso1 and WT-Iso2 iHep lines (-dox) and ( D ) KO-Iso1 and KO-Iso2 iHep lines (+dox). ( E ) Comparison of CXCL10 expression in WT-Iso1 (+/-dox) and KO-Iso1 (+dox) iHeps. Biological replicates were assayed in technical duplicate and mean ± SEM is shown relative to GAPDH . Results are representative of two independent experiments. Percent gene expression was calculated relative to respective mock treated samples. *, +, and _ indicate p≤ 0.05 by Student’s t-test.

Article Snippet: WT- and IFNLR1 -KO-iHeps harboring FLAG-Iso1, FLAG-Iso2, and EV constructs were +/-dox-induced (100ng/ml, 24h) then mock, IFNL3 (100ng/ml), or IFNA2 (100ng/ml, PBL Assay Science) stimulated at 37°C for 15min, then collected and lysed on ice in RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors (Pierce).

Techniques: Expressing, Comparison, Gene Expression

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: Top pathways up-regulated by IFNL3 treatment in dox-uninduced WT-iHeps expressing FLAG-Iso1 ( A ) or FLAG-Iso2 ( B ) relative to similarly treated WT-EV iHeps. Pathways with at least 10 genes identified in the dataset represented within the indicated pathway are shown. Pathways identified in both datasets are indicated in red, with the number of genes from the dataset represented within each individual pathway indicated.

Article Snippet: WT- and IFNLR1 -KO-iHeps harboring FLAG-Iso1, FLAG-Iso2, and EV constructs were +/-dox-induced (100ng/ml, 24h) then mock, IFNL3 (100ng/ml), or IFNA2 (100ng/ml, PBL Assay Science) stimulated at 37°C for 15min, then collected and lysed on ice in RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors (Pierce).

Techniques: Expressing

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: Heat map of RNA-seq data showing column-normalized expression for the 35 IFNL3-induced DEGs identified in the “Interferon alpha beta signaling” pathway in WT-iHeps expressing FLAG-Iso1 relative to EV (-dox, + IFNL3) shown in ( A ) WT and ( B ) KO iHep samples with dox and IFNL3 treatment as indicated. Rows depict individual biological duplicate samples clustered by cell line and treatment; red indicates higher expression and blue represents lower expression ( C-D ) Relative expression of select genes ( MX1 , IFITM1 , IFITM3 ) evaluated by qRT-PCR relative to GAPDH in WT ( C ) and KO ( D ) iHeps showed correlation with RNA-seq data. Data are representative of two biological replicates assayed in technical duplicates relative to GAPDH . Mean ± SEM, *p≤0.05 by Student’s t-test.

Article Snippet: WT- and IFNLR1 -KO-iHeps harboring FLAG-Iso1, FLAG-Iso2, and EV constructs were +/-dox-induced (100ng/ml, 24h) then mock, IFNL3 (100ng/ml), or IFNA2 (100ng/ml, PBL Assay Science) stimulated at 37°C for 15min, then collected and lysed on ice in RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors (Pierce).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: ( A ) Model depicting the mechanisms of distinct signaling outcomes imparted by ternary complexes composed of IFNL3, IL10RB and either IFNLR1 variant 1 or 2. Greater arrow width indicates higher association and/or phosphorylation (JAK1, STAT1, STAT2), greater internalization of receptor complexes, or higher induction of gene expression. IFNLR1 variant 1 containing heterodimers are depicted to have greater stability of JAK1 and/or pJAK1 binding and to be more prone to internalization than IFNLR1 variant 2 containing heterodimers. This correlates with variant 1 mediating more efficient phosphorylation of STAT1 and STAT2 and supporting higher expression of antiviral ISGs and de novo expression of proinflammatory ISGs compared to variant 2. ( B ) Model depicting assemblage of multimeric clusters containing multiple heterodimers of IFNL3-bound IFNLR1 variants in complex with IL10RB. In this model, proximity of JAK1 molecules bound to the cytoplasmic domains of IFNLR1 variants 1 and 2 could participate in TYK2-independent transphosphorylation. The relative abundance of IFNLR1 variants within multimeric complexes would influence the nature of receptor internalization, STAT phosphorylation, and downstream gene expression. The TYK2 dependence of signaling differs for antiviral vs. proinflammatory ISG expression and has a complex relationship with the relative abundance of IFNLR1 variants. Soluble variant 3, not studied in these experiments, is included in the model for consideration. Created in BioRender. Novotny, L. (2025) https://BioRender.com/24aqcgc .

Article Snippet: WT- and IFNLR1 -KO-iHeps harboring FLAG-Iso1, FLAG-Iso2, and EV constructs were +/-dox-induced (100ng/ml, 24h) then mock, IFNL3 (100ng/ml), or IFNA2 (100ng/ml, PBL Assay Science) stimulated at 37°C for 15min, then collected and lysed on ice in RIPA buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors (Pierce).

Techniques: Variant Assay, Phospho-proteomics, Gene Expression, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Modeling viral and bacterial infections in human lung organotypic systems reveals strain specific host responses

doi: 10.1101/2025.03.31.644992

Figure Lengend Snippet: (A) IFNβ and (B) IFNλ3 proteins detected in top channel washes recovered at 4 days post-infection. (n=5 independent experiments with 5 different organoid lines. Each dot color represents an organoid line. Median +/- interquartile range are plotted and Kruskal-Wallis tests are used for addressing statistically significant differences).

Article Snippet: For the IFNλ3 assay, the 21730-1 IgG1, kappa, mouse monoclonal antibody (PBL Assay Science) was used as a capture antibody after coating on paramagnetic beads (0.3 mg.mL -1 ), the MAB52591R IgG2a, mouse monoclonal antibody (R&D Systems, Minneapolis, MN, USA) was biotinylated (biotin/antibody ratio = 60/1) and used as the detector antibody at a concentration of 0.3 μg.mL -1 , and the 5259-IL-025/CF recombinant human IFNλ3 protein (R&D Systems) was used as calibrator.

Techniques: Infection