Synoviocyte innate immune responses: II. Pivotal role of IFN regulatory factor 3

Abstract

Innate immune responses contribute to synovial inflammation in rheumatoid arthritis. The present study was designed to investigate the contribution of IFN regulatory factor (IRF)3 and IRF7 to type I IFN-regulated gene expression in synoviocytes. Fibroblast-like synoviocytes were stimulated with polyinosinic-polycytidylic acid (poly [I-C]) after transfection with IRF3 or IRF7 small interfering RNA to knockdown transcription factor expression. Western blots, luciferase assay after transfection with reporter constructs, quantitative PCR, and AP-1 DNA binding ELISA were performed to evaluate the role of IRF3 and IRF7 in poly (I-C)-induced signaling and synoviocyte gene expression. IRF3 regulates IFN-stimulated response element (ISRE) promoter activity as well as IFN-beta, IRF5, IRF7, RANTES, IFN-inducible protein-10, MCP-1, and MIP1alpha gene expression in response to poly (I-C). IRF7 knockdown modestly decreased a subset of genes and ISRE activity, although the results were not statistically significant. Surprisingly, IRF3 knockdown almost completely blocked expression of additional genes in which the ISRE is not traditionally considered a dominant promoter site in fibroblast-like synoviocytes, including matrix metalloproteinase (MMP)3, MMP9, IL-6, and IL-8. Transcription factor activation studies demonstrated a role for IRF3 in regulation of c-Jun phosphorylation and AP-1 binding. IRF3 rather than IRF7 regulates poly (I-C)-induced type I IFN responses in human synoviocytes by increasing ISRE promoter activity. IRF3 also partially regulates expression of other cytokines and MMP through activation of c-Jun and the AP-1 promoter site. Targeting synoviocyte IRF3 represents a potential approach to suppress diverse mediators while limiting suppression of IRF7-mediated immune responses.

Figure 1

Western blot analysis of IRF3 phosphorylation and IRF7 induction. FLS were stimulated for 18h with poly (I-C), IFNβ, IL-1, LPS, or PGN. Lysates were then analyzed by Western blot using anti-P-IRF3 ser 396, anti-IRF7, and anti-GAPDH antibodies. Jurkat cell lysate was included as a positive control. Stimulation with poly (I-C) resulted in a significant increase in phosphorylation of IRF3 (2.67 ± 0.32, n=4). Induction of IRF7 was also increased by poly (I-C) stimulation (3.27 ± 0.27, n=4) and this TLR3 ligand was used for stimulation of the type I IFN response by cultured FLS. Top panel shows a representative Western blot and the bottom panel shows combined results for 3 separate FLS lines.

Figure 2

Time course of IRF activation in poly (I-C)-stimulated FLS. Cells were incubated with poly (I-C) for up to 24h and analyzed by Western blot analysis. Poly (I-C) increased phosphorylated IRF3 levels within 2–4h, which persists for at least 24h. IRF7 induction was detected between 4–6h and increased for at least 24h time. Total IRF3 and GAPDH levels were constant. Figure is representative of two independent experiments.

Figure 3

Effect of siRNA knockdown on IRF3 and IRF7 protein expression. Cultured FLS were transfected with Smartpool control siRNA (sc), IRF3, IRF7, or IRF3 + IRF7 (IRF3/7). Three days later, cells were stimulated with poly (I-C) for 18 h. Western blot analysis shows knockdown of IRF3 and P-IRF3 and decreased IRF7 accumulation by IRF3 siRNA. IRF7 siRNA only decreased IRF7, with no effect on IRF3. Figure is representative of three independent experiments.

Figure 4

Effect of IRF3 and IRF7 deficiency on ISRE promoter activity. IRF3, IRF7, IRF3+IRF7 (IRF3/7) or scrambled (sc) siRNA-treated FLS were co-transfected with an ISRE luciferase reporter construct. Synoviocytes were stimulated with poly (I-C) (20μg/ml) overnight and cell lysates were assayed for luciferase activity normalized to R. reniformis luciferase. FLS transfected with sc siRNA were used as control. ISRE promoter activity was decreased to baseline by IRF3 and IRF3/7 siRNA (p<0.03, n=3), but no additional effect was detected when comparing IRF3 and IRF3/IRF7 double knockdown FLS. A trend towards modest suppressive effect of IRF7 knockdown was observed but this was not statistically significant. These data confirm the key role of IRF3, with no additional decrease in promoter activity by IRF7 knockdown. The values are the mean ± SEM from 3 independent experiments.

Figure 5

Figure 5A. Inhibition of IFN-response gene expression by IRF3 knockdown. Q-PCR was performed to determine relative expression of IFN-regulated genes (ISRE is the dominant promoter element) after IRF3, IRF7, or IRF3+IRF7 (IRF3/7) siRNA knockdown. After transfection, cells were stimulated for 18h with poly (I-C) followed by quantitative analysis of IFNβ, IRF5, IRF7, RANTES, IP-10, MCP-1 and MIP1α mRNA. Percent inhibition for IRF siRNA compared with scrambled siRNA was calculated (see Material and Methods). IRF3 deficiency markedly decreased IFNβ, IRF5, IRF7, RANTES, IP-10, MCP-1 and MIP1α gene expression (p<0.04 for each gene, n=3 separate FLS lines). IRF7 inhibition decreased IRF7 expression levels but did not significantly decrease other IFN-regulated genes compared with IRF3. Figure 5B. Inhibition of pro-inflammatory and MMP gene expression by IRF3 knockdown. Q-PCR was performed to determine relative expression of cytokine and MMP genes after IRF3, IRF7, or IRF3+IRF7 (IRF3/7) siRNA knockdown. Surprisingly, IRF3 inhibition decreased gene expression of poly (I-C)-induced MMP3 and MMP9, as well as IL-6 and IL-8 (p<0.03 for each gene, n=3 separate FLS lines), while IRF7 siRNA had minimal effect.

Figure 6

IRF3 inhibition decreases activated c-Jun binding to AP-1. We investigated a role for IRF3 in AP-1 activation because this promoter is present in all four of the genes (IL-6, IL-8, MMP3, MMP9) inhibited by IRF3 knockdown. IRF3 deficiency decreased AP-1 binding by 52% compared with control (n=3, p <0.02). The figure shows the mean ± SEM from 3 independent experiments.