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: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Differentially expressed genes (DEGs, P-FDR < 0.05) were identified by RNA-seq analysis in IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1 KO HepG2 cells after 72 hrs of induction by dox, comparing to controls (dox-conditions). Cutoff threshold (fold change > +/-1.5) is indicated by dotted lines. (A) Analysis of all DEGs (n=3251) detected for IFN-λ4-GFP or IFN-λ4-GFP-IFNLR1 KO cells. In blue - DEGs (n=2,735) specific to IFN-λ4-GFP and considered IFNLR1-dependent. In black - DEGs (n=145) shared between both groups and considered IFNLR1-independent. In orange - DEGs (n=371) specific to IFN-λ4-GFP-IFNLR1 KO . (B) DEGs of IFN-λ4-GFP analyzed in IFN-λ3-GFP transcriptome. In black - DEGs (n=1,506) shared in IFN-λ4-GFP and IFN-λ3-GFP and in blue - IFN-λ4-signature DEGs (n=1,229) detected in IFN-λ4-GFP but not in IFN-λ3-GFP producing cells. Additional details are provided in Fig. S3 and Table S6. (C , D) Cell cycle analysis of cells synchronized by 24 hrs of serum starvation, treated with or without dox (0.5 µg/ml) for 72 hrs and analyzed by flow cytometry after PI staining. The plot shows a representative picture and the percentage of cells in each phase of the cell cycle. All data are shown as mean± SEM from triplicate experiments. *, P < 0.05. (E , F) Bromodeoxyuridine (BRDU, %) incorporation indicating cell proliferation in HepG2 cells expressing IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1KO. Cells were cocultured with HepG2 cells labeled with Far Red proliferation dye, dox-induced for 72 hrs and treated with BRDU for 3 hrs before analysis. Gates show HepG2 cells exposed to IFN-λs (IFN-λ treated cells) and HepG2 expressing IFN-λs. P-values compare corresponding dox+ vs. dox-HepG2 cells, ** p<0.01, Student’s T-test. Graphs represent one of three independent experiments, each in biological triplicates.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: RNA Sequencing, Cell Cycle Assay, Flow Cytometry, Staining, BrdU Incorporation Assay, Expressing, Labeling

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: (A) Unsupervised clustering of activities of 54 regulons that were significantly and directionally enriched with HepG2-DEGs in the set of 885 of TCGA-LIHC regulons. The heatmap shows differences in activity scores (dES) for the IFN-λ4-enriched regulons organized by GSEA-2T results for the IFN-λ4-GFP, IFN-λ3-GFP and IFN-λ4-GFP-IFNLR1 KO DEG signatures. Cluster I: regulons with dES > 0 in IFN-λ4-GFP; cluster II: regulons with dES < 0 in IFN-λ4-GFP. Table S7 provides the regulon activity scores presented in Figure 2A. (B-G) GSEA-2T plots for IRF1 and IRF2, respectively, in DEG signatures for (B-C) IFN-λ3-GFP, ( D-E ) IFN-λ4-GFP, and ( F-G ) IFN-λ4-GFP-IFNLR1 KO . (H) Inhibition of proliferation in IFN-λ4-GFP HepG2 cells, for one of three independent experiments. Cells were treated with IRF1 siRNA for 24 hrs, labeled with Far Red proliferation dye and dox-induced at indicated concentrations for 72 hrs. Proliferation was assessed by flow cytometry with a graph representing the geometric mean expression of Far Red proliferation dye with higher values indicating reduced cell proliferation. P-values compare dox-treated control siRNA with dox-treated IRF1 siRNA. ** p<0.01, Student’s T-test. Below: Western blots showing IRF1 protein levels following siRNA knockdown.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Activity Assay, Inhibition, Labeling, Flow Cytometry, Expressing, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Representative confocal images of HepG2 cells transduced with a mammalian baculovirus delivery system (BacMam) of GFP-tagged proteins targeting specific organelles - lysosomes, Golgi, early and late endosomes. After transduction for 6 hrs, cells were transiently transfected with Halo-tagged constructs for IFN-λ4 or control for indicated times, stained with cell-permeant Halo-tag ligand TMR (red), and imaged. (A) Confocal images showing IFN-λ4 accumulation in lysosomes but not in early endosomes. (B) Late endosomal trafficking of IFN-λ4, with the inset showing larger magnification. (C) Unfolded protein response (UPR) is represented by lysosomal enlargement after protein accumulation. (D) Live images of IFN-λ4-expressing HepG2 cells undergoing apoptosis, characterized by membrane blebbing and cell death. Images were scanned every minute for 12 hrs. Scale bars – 10 um. (E) Apoptosis detection with ApoTox-Glo assays in corresponding untreated and dox-induced cells for indicated time points. RLU, relative luminescence units. (F) Graph showing counts from colony formation assay for HepG2 cells expressing IFN-λ4 or IFNLR1 KO grown in 6-well plates with or without dox for 13 days. Cell colonies were stained with crystal violet and manually counted. The graph represents the number of colonies as a percentage of initial plated counts. (G,H) mRNA (G) and protein levels ( H ) of DDIT3 after siRNA knockdown tested by qRT-PCR and Western blot assays, respectively. (I-J) Apoptosis (I) and cell viability (J) assays were performed after siRNA knockdown of DDIT3 in dox-induced IFN-λ4-GFP cells. * p<0.05, ** p< 0.01, *** p<0.001.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Transduction, Transfection, Construct, Control, Staining, Expressing, Membrane, Colony Assay, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Clinical and Experimental Immunology

Article Title: Expression of type III interferons (IFNλs) and their receptor in Sjögren's syndrome

doi: 10.1111/cei.12865

Figure Lengend Snippet: Expression of type III interferons (IFNs) (IFN‐λ1/interleukin (IL)−29, IFN‐λ2/IL‐28A and IFN‐λ3/IL‐28B) and their signalling receptor (IFN‐λR1/IL‐28Rα) (a) in minor salivary gland (MSG) tissues that belong to the three Sjögren's syndrome (SS) subgroups, as these classified by the grade of the inflammatory lesion (SS‐I: mild, n = 16; SS‐II: intermediate, n = 14; and SS‐III: severe, n = 16 MSG lesions. Sicca controls CT, n = 17), (b) in non‐malignant parotid glands of patients undergoing parotidectomy due to mixed tumours. Original magnification ×200.

Article Snippet: Rabbit polyclonal antibodies to human IFN‐λ1/IL‐29 (Santa Cruz, Dallas, Texas, USA), IFN‐λ2/IL‐28A (Antibodies‐online, Atlanta, GA, USA), IFN‐λ/IL‐28B (Bioss, Woburn, MA, USA), IFN‐λR1/IL‐28Ra (Novus Biologicals, Littleton, CO, USA) and CD3 (Dako, Glostrup, Denmark) were used.

Techniques: Expressing

Journal: Clinical and Experimental Immunology

Article Title: Expression of type III interferons (IFNλs) and their receptor in Sjögren's syndrome

doi: 10.1111/cei.12865

Figure Lengend Snippet: Representative double immunofluorescent staining for the common type III interferon (IFN) signalling receptor [IFN‐λR1/interleukin (IL)−28Rα] and markers of inflammatory cells [plasmatocytoid dendritic cells (pDCs): CD303 and CD123, macrophages: CD68, T lymphocytes: CD3 and B lymphocytes: CD20] in a minor salivary gland (MSG) tissue from a Sjögren's syndrome (SS) patient. Co‐localization is indicated with yellow arrows. Original magnification ×1000.

Article Snippet: Rabbit polyclonal antibodies to human IFN‐λ1/IL‐29 (Santa Cruz, Dallas, Texas, USA), IFN‐λ2/IL‐28A (Antibodies‐online, Atlanta, GA, USA), IFN‐λ/IL‐28B (Bioss, Woburn, MA, USA), IFN‐λR1/IL‐28Ra (Novus Biologicals, Littleton, CO, USA) and CD3 (Dako, Glostrup, Denmark) were used.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Type I and III interferons shape the retinal cytokine network and barrier function in an in vitro model of ocular toxoplasmosis

doi: 10.3389/fimmu.2023.1148037

Figure Lengend Snippet: The four cell lines express IFNLR1 receptor subunit. (A) Confocal microscopy image of RPE, astrocytes, microglia and Müller cells. Cells were plated on cover slides for 24h. The cells were then fixed, permeabilized and the IFNLR1 subunit and cell nuclei labeled with AlexaFLuor488 and Hoechst 33342, respectively. (B) Flow cytometry assay for IFNLR1 receptor subunit. Cells were harvested by trypsination and placed 24h under agitation to restore membranous receptor expression. Finally, they were labeled and analyzed as described in Material and Methods.

Article Snippet: Then, samples were labeled successively with 5µg/mL mouse anti-ZO-1 (Invitrogen, 33-9100) and goat anti-mouse IgG Alexa Fluor 555 (Invitrogen, A32727), or with 5µg/mL mouse anti-IFNLR1 Alexa Fluor 488 (RDsystems, FAB5260G).

Techniques: Confocal Microscopy, Labeling, Flow Cytometry, Expressing