Journal: Veterinary research

Article Title: Getah virus nonstructural protein 2 suppresses interferon-beta production by interrupting interferon regulatory factor 3 activation.

doi: 10.1186/s13567-025-01547-3

Figure Lengend Snippet: Figure 2 Inhibition of the activity of transcription factors on specific PRDs of the IFN‐β promoter by GETV Nsp2. PK-15 cells were transfected with Nsp2 (200 ng) for 12 h before being co-transfected with pRL-TK plasmid (50 ng) and AP-1&NF-κB-Luc (50 ng) or IRF3-Luc (50 ng) reporter plasmids. 24 h later, cells were infected or stimulated with or without SeV (A, B) or poly (I:C) (C, D). Luciferase activity was measured. Statistical significance of the presented data was determined as follows: * P < 0.05.

Article Snippet: Antibodies and reagents The following antibodies were used in this study: TBK1 Monoclonal antibody (Cat No. 67211-1-Ig), PhosphoTBK1 (Ser172) Recombinant antibody (Cat No. 82383- 1-RR), IRF3 Monoclonal antibody (Cat No. 66670-1-Ig), Phospho-IRF3 (Ser396) Polyclonal antibody (Cat No. 29528-1-AP), all purchased from Proteintech.

Techniques: Inhibition, Activity Assay, Transfection, Plasmid Preparation, Infection, Luciferase

Journal: Veterinary research

Article Title: Getah virus nonstructural protein 2 suppresses interferon-beta production by interrupting interferon regulatory factor 3 activation.

doi: 10.1186/s13567-025-01547-3

Figure Lengend Snippet: Figure 3 Getah virus Nsp2 suppresses IRF3 activation. A HEK293 T cells were co-transfected with IFN-β reporter plasmid, viral protein expressing plasmid, and stimulator plasmid RIG-I-CARD, MDA5, MAVS, IRF3 and IRF3/5D. An empty plasmid was used as a control. Cells were assayed for luciferase activity at 24 h. The data were analysed by normalising the Firefly luciferase activity to the Renilla luciferase activity and then normalising it to non-stimulated samples to obtain the fold induction. Empty vector control was set to 100%. B–F HEK293 T cells were transfected with an IFN-β reporter plasmid, along with a control plasmid or with an increasing amount of plasmids expressing Nsp2, together with plasmids expressing RIG-I-CARD, MDA5, MAVS, IRF3 and IRF3/5D. At 24 h post-transfection, cells were infected with SeV for 12 h, then the luciferase activity was measured. The statistical significance of the presented data was determined as follows: * P < 0.05, ** P < 0.01. G. PK-15 cells were transfected with the Nsp2-expressing plasmid. After 24 h, cells were treated with poly(I:C) for 12 h. The cells were fixed and subjected to laser scanning confocal microscopy. Scale bar, 10 μm.

Article Snippet: Antibodies and reagents The following antibodies were used in this study: TBK1 Monoclonal antibody (Cat No. 67211-1-Ig), PhosphoTBK1 (Ser172) Recombinant antibody (Cat No. 82383- 1-RR), IRF3 Monoclonal antibody (Cat No. 66670-1-Ig), Phospho-IRF3 (Ser396) Polyclonal antibody (Cat No. 29528-1-AP), all purchased from Proteintech.

Techniques: Virus, Activation Assay, Transfection, Plasmid Preparation, Expressing, Control, Luciferase, Activity Assay, Infection, Confocal Microscopy

Journal: Veterinary research

Article Title: Getah virus nonstructural protein 2 suppresses interferon-beta production by interrupting interferon regulatory factor 3 activation.

doi: 10.1186/s13567-025-01547-3

Figure Lengend Snippet: Figure 4 Getah virus Nsp2, combined with TBK1, decreases IRF3 phosphorylation. A PK-15 cells were co-transfected with TBK1-expressing plasmid and varying amounts of Nsp2-encoding plasmids. After 24 h, western blot was used to analyse the cell lysates for phosphorylated TBK1, total TBK1, phosphorylated IRF3, total IRF3, and β-actin. B.HEK293 T cells were co-transfected with plasmids expressing TBK1 and HA-tagged Nsp2. After 24 h, cell lysates and immunoprecipitates were immunoblotted with the indicated antibodies. C. Schematic representation of GETV Nsp2 domains according to its amino acid sequence. HEK-293 T cells were transfected with HA-tagged Nsp2 truncated fragments and Flag-TBK1 for 24 h. Cell lysates and immunoprecipitates were subjected to immunoblotting with the indicated antibodies. D Schematic representation of TBK1 domain positions. HEK-293 T cells were transfected with Flag-tagged TBK1 truncated fragments and HA-Nsp2, and co-immunoprecipitation and immunoblot analysis were performed after 24 h. E Docked complex of Nsp2-TBK1. Wheat shows the TBK1, and light blue represents the Nsp2; TBK1-binding residues are shown in green, and Nsp2-binding residues are displayed in cyan. Red represents the hydrogen bonds.

Article Snippet: Antibodies and reagents The following antibodies were used in this study: TBK1 Monoclonal antibody (Cat No. 67211-1-Ig), PhosphoTBK1 (Ser172) Recombinant antibody (Cat No. 82383- 1-RR), IRF3 Monoclonal antibody (Cat No. 66670-1-Ig), Phospho-IRF3 (Ser396) Polyclonal antibody (Cat No. 29528-1-AP), all purchased from Proteintech.

Techniques: Virus, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Western Blot, Sequencing, Immunoprecipitation, Binding Assay

Journal: Veterinary research

Article Title: Getah virus nonstructural protein 2 suppresses interferon-beta production by interrupting interferon regulatory factor 3 activation.

doi: 10.1186/s13567-025-01547-3

Figure Lengend Snippet: Figure 5 Getah virus Nsp2 interrupts the interaction of IRF3 with KPNA3 and KPNA4. A PK15 cells were transfected with or without Nsp2 for 24 h, then stimulated with or without poly(I:C) for 12 h. Cells were harvested, and the expression levels of KPNA3 and KPNA4 were analysed by western blotting. B, C HEK293 T cells were co-transfected with the KPNA3-Flag or KPNA4-Flag construct and ΔN-Nsp2 plasmid (50 ng, 200 ng,) or empty vector. The cells were harvested and lysed at 36 h post-transfection. Immunoprecipitation was performed with Flag antibody, and western blotting was performed with the indicated antibodies D, E The pale green shade shows the Nsp2, and the light pink shade represents the KPNA3; Nsp2-binding residues are shown in cyan, and KPNA3-binding residues are displayed in yellow. The yellowish colour is the hydrogen bond between Nsp2 and KPNA3 (D). The wheat shade represents Nsp2, and the blue and white shades represent KPNA4. The residues involved in Nsp2 binding are marked in green, and those participating in KPNA4 binding are displayed in cyan. A close-up view of the interaction site further emphasises hydrogen bonds, highlighted in yellow (E). F HEK293 T cells were co-transfected with an HA-tagged Nsp2-expressing plasmid and a Flag-tagged KPNA1-6 6 plasmid or an empty plasmid. At 24 h, Co-immunoprecipitations were performed with whole-cell lysates using the indicated antibodies.

Article Snippet: Antibodies and reagents The following antibodies were used in this study: TBK1 Monoclonal antibody (Cat No. 67211-1-Ig), PhosphoTBK1 (Ser172) Recombinant antibody (Cat No. 82383- 1-RR), IRF3 Monoclonal antibody (Cat No. 66670-1-Ig), Phospho-IRF3 (Ser396) Polyclonal antibody (Cat No. 29528-1-AP), all purchased from Proteintech.

Techniques: Virus, Transfection, Expressing, Western Blot, Construct, Plasmid Preparation, Immunoprecipitation, Binding Assay

Journal: Veterinary research

Article Title: Getah virus nonstructural protein 2 suppresses interferon-beta production by interrupting interferon regulatory factor 3 activation.

doi: 10.1186/s13567-025-01547-3

Figure Lengend Snippet: Figure 6 Overexpression of KPNA3 or KPNA4 recruits IRF3 activity, which is inhibited by GETV Nsp2. A A plasmid expressing ΔN-Nsp2 was co-transfected into PK-15 cells with vector plasmid (–) or a KPNA construct (+) and the IRF3-dependent reporter system. At 24 h post-transfection, cells were stimulated with poly(I·C) for 12 h before a dual-luciferase assay was used to determine IRF3 Activity. Statistical significance of the presented data was determined as follows: * P < 0.05. B PK-15 cells were transfected or co-transfected with a plasmid encoding GETV ΔN-Nsp2 and a plasmid expressing KPNA3 or KPNA4 for 24 h, then stimulated with poly(I·C) for another 12 h. Protein levels of IRF3 in the cytoplasmic extract and nuclear extract were analysed by western blotting. C Vero cells were co-transfected with a plasmid encoding GETV Nsp2 and a KPNA construct and the IRF3-dependent reporter system. At 24 h post-transfection, cells were stimulated with poly(I·C) for 12 h before a dual-luciferase assay was used to determine IRF3 activity. The statistical significance of the presented data was determined as follows: ** P < 0.01.

Article Snippet: Antibodies and reagents The following antibodies were used in this study: TBK1 Monoclonal antibody (Cat No. 67211-1-Ig), PhosphoTBK1 (Ser172) Recombinant antibody (Cat No. 82383- 1-RR), IRF3 Monoclonal antibody (Cat No. 66670-1-Ig), Phospho-IRF3 (Ser396) Polyclonal antibody (Cat No. 29528-1-AP), all purchased from Proteintech.

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Expressing, Transfection, Construct, Luciferase, Western Blot

Journal: Veterinary research

Article Title: Getah virus nonstructural protein 2 suppresses interferon-beta production by interrupting interferon regulatory factor 3 activation.

doi: 10.1186/s13567-025-01547-3

Figure Lengend Snippet: Figure 7 Schematic diagram of GETV Nsp2 inhibiting the RLR signalling pathway by regulating IRF3 activation. Nsp2 binds TBK1 to suppress IRF3 phosphorylation. Meanwhile, Nsp2 competitively inhibited the interaction of pIRF3 with KPNA3 and KPNA4, to inhibit IRF3 nuclear translocation.

Article Snippet: Antibodies and reagents The following antibodies were used in this study: TBK1 Monoclonal antibody (Cat No. 67211-1-Ig), PhosphoTBK1 (Ser172) Recombinant antibody (Cat No. 82383- 1-RR), IRF3 Monoclonal antibody (Cat No. 66670-1-Ig), Phospho-IRF3 (Ser396) Polyclonal antibody (Cat No. 29528-1-AP), all purchased from Proteintech.

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Experimental workflow diagram. Flow chart depicting multiomics analysis (scRNA-seq, ChIP-seq, and RNA-seq) and functional validation (in vitro siRNA knockdown, in vivo CIA model) to identify IRF7 as a therapeutic target in RA

Article Snippet: The sections were pretreated with citrate buffer (pH 6.0) for antigen retrieval and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD68 antibody (28058-1-AP, Proteintech; 1:200), rabbit anti-S100A12 antibody (16630-1-AP, Proteintech; 1:100), rabbit anti-IRF7 antibody (22392-1-AP, Proteintech; 1:100), and rabbit anti-PTGS2 antibody (27308-1-AP, Proteintech; 1:100).

Techniques: ChIP-sequencing, RNA Sequencing, Functional Assay, Biomarker Discovery, In Vitro, Knockdown, In Vivo

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: IRF7 is a specific transcriptional regulator of CD48 high S100A12 + macrophages. A Venn diagram showing overlapping transcription factors (TFs) identified by triplicate SCENIC analyses, with the CD48 high S100A12 + subcluster enriched for NFIL3, TGIF1, FOSL2, IRF7, and STAT1. B Heatmap of regulon activity scores (RASs) for TFs across macrophage subclusters. C Ranking of TFs in CD48 high S100A12 + macrophages by the regulon specificity score (RSS, calculated via Jensen‒Shannon divergence). D UMAP dimensionality reduction of TF activity profiles across subclusters. E – F UMAP plots highlighting spatial overlap between the CD48 high S100A12 + subcluster. ( E ) and cells with elevated IRF7 regulon activity ( F ). G Representative images of immunofluorescence staining for CD68 and IRF7 in knee synovial tissues from RA and OA patients. Scale bar: 100 μm

Article Snippet: The sections were pretreated with citrate buffer (pH 6.0) for antigen retrieval and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD68 antibody (28058-1-AP, Proteintech; 1:200), rabbit anti-S100A12 antibody (16630-1-AP, Proteintech; 1:100), rabbit anti-IRF7 antibody (22392-1-AP, Proteintech; 1:100), and rabbit anti-PTGS2 antibody (27308-1-AP, Proteintech; 1:100).

Techniques: Activity Assay, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: IRF7 directly regulates downstream inflammatory genes in M1 macrophages. A ChIP-seq peak heatmaps showing increased IRF7 binding to promoter/enhancer regions in LPS-stimulated M1 macrophages. B Venn diagram of 108 overlapping genes from the IRF7 ChIP-seq data and the SCENIC-predicted target genes. C Reactome pathway enrichment of IRF7-regulated genes, highlighting the involvement of NF-κB, TNF, and Toll-like receptor signalling (key genes: IL-1β, FOS, NF-κB1, PTGS2, and CXCL10). D Bulk RNA-seq heatmap showing the upregulation of IRF7 and target genes in M1-polarized macrophages ( GSE130011 , GSE154346 ). E UMAP plots of NFKB1, PTGS2, IL1B, and CXCL10 expression in the CD48 high S100A12 + subcluster. F RT‒qPCR analysis of IRF7 and M1 marker genes in siRNA-treated macrophages (performed in triplicate, with 3 distinct patient sources used for each repetition). G – H Western blot validation of IRF7 and downstream protein expression following IRF7 knockdown in M1-polarized macrophages (performed in triplicate, with 3 distinct patient sources used for each repetition). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way ANOVA with the Bonferroni post hoc correction)

Article Snippet: The sections were pretreated with citrate buffer (pH 6.0) for antigen retrieval and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD68 antibody (28058-1-AP, Proteintech; 1:200), rabbit anti-S100A12 antibody (16630-1-AP, Proteintech; 1:100), rabbit anti-IRF7 antibody (22392-1-AP, Proteintech; 1:100), and rabbit anti-PTGS2 antibody (27308-1-AP, Proteintech; 1:100).

Techniques: ChIP-sequencing, Binding Assay, RNA Sequencing, Expressing, Marker, Western Blot, Biomarker Discovery, Knockdown

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Local IRF7 knockdown alters the immune cell composition in CIA mice. A Schematic of intra-articular IRF7 siRNA treatment in collagen-induced arthritis (CIA) model mice. B – C Flow cytometry analysis of the CD86 and CD206 mean fluorescence intensities (MFIs) in F4/80 + macrophages from ankle joints ( n = 6). CD86: NC: 1241 ± 265.4, si-IRF7: 2469 ± 390.3, positive: 3489 ± 570.9, si-mock: 3689 ± 370.1. CD206: NC: 2225 ± 225.9, si-IRF7: 3395 ± 369.4, positive: 978.2 ± 147.9, si-mock: 2022 ± 170.6. D – E Frequencies of Foxp3 + Tregs among CD3 + CD4 + T cells ( n = 6). NC: 1.66% ± 0.15%, si-IRF7: 3.27% ± 0.28%, positive: 0.47% ± 0.17%, si-mock: 1.10% ± 0.22%. F Immunofluorescence staining for S100A12 + inflammatory macrophages in the ankle synovium of different groups. Scale bar: 100 μm. The data are presented as the means ± SDs. Statistical significance: *** P < 0.001, **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction)

Article Snippet: The sections were pretreated with citrate buffer (pH 6.0) for antigen retrieval and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD68 antibody (28058-1-AP, Proteintech; 1:200), rabbit anti-S100A12 antibody (16630-1-AP, Proteintech; 1:100), rabbit anti-IRF7 antibody (22392-1-AP, Proteintech; 1:100), and rabbit anti-PTGS2 antibody (27308-1-AP, Proteintech; 1:100).

Techniques: Knockdown, Flow Cytometry, Fluorescence, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Local IRF7 inhibition attenuates joint inflammation and bone erosion in CIA mice. A Representative ankle joint images on day 42 postimmunization. B H&E staining and histological staining. C - E IHC staining for CD68, S100A12, and IRF7 in the ankle synovium. Scale bar: 100 μm. F Quantification of paw thickness at the ankle joint ( n = 6 per group). 42 Days after the first immunization: NC: 8.33 ± 0.02, si-IRF7: 10.39 ± 0.54, positive: 12.08 ± 0.80, si-mock: 12.65 ± 0.57, Statistical significance: **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction). G H&E staining and histological scoring of synovial hyperplasia and inflammation ( n = 6). NC: 0.00 (0.00–0.00), si-IRF7: 1.50 (1.00–2.25), positive: 2.50 (1.75–3.00), and si-mock: 3.00 (2.75–3.00). Data are shown as medians with 25% − 75% percentiles. Statistical significance: * P < 0.05 (Kruskal‒Wallis test, followed by post hoc Dunn’s test with Bonferroni correction for multiple comparisons). H - J Semiquantitative analysis analysis of CD68, S100A12, and IRF7 expression via IHC staining via ImageJ ( n = 6). Statistical significance: **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction)

Article Snippet: The sections were pretreated with citrate buffer (pH 6.0) for antigen retrieval and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CD68 antibody (28058-1-AP, Proteintech; 1:200), rabbit anti-S100A12 antibody (16630-1-AP, Proteintech; 1:100), rabbit anti-IRF7 antibody (22392-1-AP, Proteintech; 1:100), and rabbit anti-PTGS2 antibody (27308-1-AP, Proteintech; 1:100).

Techniques: Inhibition, Staining, Immunohistochemistry, Expressing

Journal: Immunology

Article Title: HIV infection suppresses TLR3 activation-mediated antiviral immunity in microglia and macrophages.

doi: 10.1111/imm.13181

Figure Lengend Snippet: Figure 3. Effect of human immunodeficiency virus (HIV) infection on PolyI:C-induced IFN regulating factors (IRFs) and signal transducer and activator of transcription protein (STAT)1/STAT3. HIV-infected microglial cells (HC695), uninfected microglial cells (C20) (a) and HIV-infected (day 4 post-infection) primary human macrophages (b) were treated with PolyI:C for 2 hr. Cellular proteins were then collected for the expres- sion of total and phosphorylated proteins of IRF3, IRF7, STAT1 and STAT3 by Western blot. Representative Western blots shown above are rep- resentative of three independent experiments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal control to normalize the target proteins expression. The relative intensity of each band is calculated based on the control band (untreated), which is defined as 1.

Article Snippet: We demonstrated that HMW PolyI:C is a potent TLR3 agonist to activate intracellular innate immunity.11 The primary antibodies against IRF3, IRF7, phospho-IRF7 (Ser477), signal transducer and activator of transcription protein (STAT)1, phospho-STAT1 (Tyr701), STAT3, phospho-STAT3 (Tyr705), ISG56, GBP5, Viperin, ISG15 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Virus, Infection, Western Blot, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 4. RelB deficiency triggers elevated basal expressions of T1-IFNs involving IRF7-mediated positive autoregulation. (A) GSEA comparing WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 5. T1-IFNs and IRF7 cooperatively restrict viral multiplication in Relb−/−cells. Plaque assay reveal- ing the titer of progeny virus particles produced by WT and Relb−/−cells, including primary MEFs (A) or BMDMs (C, left panel) or immortalized MEFs (C, right panel), upon infection for 12 h with the indicated MOI of CHPV. (B) RT-qPCR showing the relative abundance of viral genomic RNA and mRNAs encoding viral nucleo- capsid protein and phosphoprotein in WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Plaque Assay, Virus, Produced, Infection, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 6. Vitamin D modulates the T1-IFNIRF7 pathway through RelB to exert antiviral effects. Vitamin D naive or vitamin Dconditioned WT or Relb−/−primary MEFs were examined for the relative abundance of IRF7 mRNA (A) and protein (B). Similarly, vitamin Dinduced expressions of IFN-b mRNA were scored in Relb−/−MEFs (C) and immortalized IRF7DCRISPR MEFs (E). Virus propaga- tion was scored in vitamin D naive and vitamin Dconditioned Relb−/−MEFs (D) or immortalized IRF7DCRISPR MEFs (F) subsequent to cell infection with 0.1 or 2 MOI of CHPV. The RT-qPCR data represent mean ± SEM of three indepen- dent experiments. The immunoblots represent three experi- mental replicates. The plaque assay data represent mean ± SEM of four biological replicates. *p# 0.05, **p# 0.01.

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Virus, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay