IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes

Fig. 1. TNF-induced IRF1 expression in RA-FLSs. a Representative immunostaining for IRF1 (brown staining) in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue samples (upper panel). Staining with an isotype-matched control antibody (CTRL) is presented in the lower panel. b Synovial tissue samples from 12 RA and 8 OA patients evaluated for IRF1 expression using a semiquantitative score (0 = no staining, 3 = high staining). Lining: Mann–Whitney U test, **p = 0.0016; Sublining: Student’s t-test, ****p < 0.0001. c Quantitative RT-PCR analysis of the IRF1 mRNA levels in hind paws obtained from wild-type (WT) and hTNFtg mice. Mann-Whitney U test, ***p = 0.0008. d Immunohistochemical detection of IRF1 (brown staining) in hind paw tissue from WT and hTNFtg mice. e Western blot analysis of TNF-stimulated (10 ng/ml) RA-FLSs. Blots representative of at least five independent experiments with FLSs from different donors are shown. f RA-FLSs cultured in micromass organ cultures for 7 days in the presence or absence of TNF (10 ng/ml). Micromasses were fixed, sectioned, and stained with hematoxylin and a specific antibody against IRF1 (brown staining). Representative images from three…

Fig. 2. The TNF-driven interferon response in FLSs depends on IRF1. a Workflow outlining the RNA sequencing (RNA-seq) experiment. RA-FLSs from three different RA patients were transfected with nontargeting (siCTRL) or IRF1-targeting siRNA pools and stimulated with TNF (10 ng/ml) for 3 h. RNA was isolated and processed for transcriptomic profiling. b Representative immunoblots of IRF1 expression in RA-FLSs after siRNA transfection and stimulation with TNF (10 ng/ml) for 3 h. c Scatter-plot of the RNA-seq data (panel A) showing the impact of IRF1 knockdown on TNF-regulated genes. Dots in the lower right quadrant (red box) represent genes with expression upregulated by TNF, but impaired by the siRNA-mediated knockdown of IRF1 expression. Dashed blue lines indicate a twofold change in gene expression. d Network analysis of GO term (biological process, BP) enrichment among significantly regulated TNF-IRF1-dependent genes (as depicted in panel C, red box). The resulting network was calculated and visualized using EnrichmentMap. Groups of similar GO terms were manually circled. Line thickness is proportional to the similarity coefficient between the connected nodes. Node color is proportional…

Fig. 3. IRF1 is critical for the TNF-induced expression of CXCR3-binding chemokines and TNFSF13B. a–c FLSs were transfected with nontargeting (siCTRL) or IRF1-targeting siRNA pools. a Transfected FLSs from seven donors with RA were treated with TNF (10 ng/ml) for 6 h. Gene expression was determined by qPCR. Expression in the treated cells is presented relative to that in the unstimulated cells. Values are shown as the mean ± SEM. **p < 0.01, Wilcoxon matched-pairs test. b RA-FLSs were stimulated with TNF for 24 h. Representative western blots of at least four experiments with different RA-FLS cell lines are shown. c. IRF1 expression knockdown FLSs (n = 4) were treated with TNF (10 ng/ml) for 24 h. Supernatants were analyzed for CXCL10 expression by ELISA. Values are shown as the mean ± SEM. d qPCR was used to analyze gene expression in human RA-FLSs treated with TNF in the absence or presence of cycloheximide (CHX, 20 μg/ml). Each experiment was performed in technical triplicates (error bars, SEM of triplicates). Expression in the treated cells is presented relative to that in the unstimulated cells

Fig. 4. The TNF-induced interferon response in FLSs depends on IRF1. a Western blot analysis of IRF1 expression and STAT1 phosphorylation in TNF-stimulated (10 ng/ml) FLSs. Blots representative of three independent experiments with different RA-FLS cell lines are shown. b Western blots showing the IRF1 expression and STAT1 phosphorylation in TNF-treated (10 ng/ml; 3 h) RA-FLSs that were transfected with either nontargeting or IRF1-targeting siRNA pools. Blots representative of three independent experiments with FLSs from different RA donors are shown. c–e FLSs were transfected with nontargeting (siCTRL) or IFNβ-targeting siRNA pools and then stimulated with TNF (10 ng/ml). c Transfected FLSs stimulated with TNF for 24 h. Western blots representative of at least four experiments performed with different RA-FLS cell lines are shown. d Transfected FLSs from seven donors with RA treated with TNF (10 ng/ml) for 6 h. Gene expression was determined by qPCR. Expression in the treated cells is presented relative to that in the unstimulated cells. Values are shown as the mean ± SEM. *p < 0.05, **p < 0.01; Wilcoxon matched-pairs test. e Transfected RA-FLSs from seven different donors suffering from…

Fig. 5. JAKinibs inhibit the expression of CXCR3-binding chemokines and TNFSF13B. a RA-FLSs (n = 7) were pretreated with DMSO, baricitinib (250 nM) or tofacitinib (250 nM) for 1 h and then stimulated with TNF (10 ng/ml) for 6 h. Gene expression was determined by qPCR. Expression in the treated cells is presented relative to that in the unstimulated cells. Values are shown as the mean ± SEM. *p < 0.05, **p < 0.01; Wilcoxon matched-pairs test. b RA-FLSs were pretreated with DMSO, baricitinib (250 nM) or tofacitinib (250 nM) for 1 h and then stimulated with TNF (10 ng/ml) for 24 h. Western blots representative of at least four experiments with different RA-FLS cell lines are shown. c RA-FLSs were cultured in micromass organ cultures for 8 days. After serum starvation overnight, the FLSs were treated with DMSO (Unstimulated), TNF (10 ng/ml) + DMSO, TNF (10 ng/ml) + baricitinib (250 nM) or TNF (10 ng/ml) + tofacitinib (250 nM) for 24 h. Micromasses were fixed, sectioned and stained with hematoxylin and a specific antibody against TNFSF13B (brown staining). Images representative of four independent experiments performed with FLSs from four different RA patients are shown. d The schematic…