Tumor necrosis factor α induces sustained signaling and a prolonged and unremitting inflammatory response in rheumatoid arthritis synovial fibroblasts

Abstract

Objective: The nonresolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the tumor necrosis factor α (TNFα)-driven inflammatory program in human FLS.

Methods: FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-κB signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the interleukin-6 (IL-6) promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays.

Results: In FLS, TNFα induced prolonged transcription of messenger RNA (mRNA) for IL-6 and progressive accumulation of IL-6 protein over 4 days. Similarly, induction of mRNA for CXCL8/IL-8, CCL5/RANTES, matrix metalloproteinase 1 (MMP-1), and MMP-3 after TNFα stimulation was sustained for several days. This contrasted with the macrophage response to TNFα, which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity, as indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL-6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors A20-binding inhibitor of NF-κB activation 3 (ABIN-3), IL-1 receptor-associated kinase M (IRAK-M), suppressor of cytokine signaling 3 (SOCS-3), and activating transcription factor 3 (ATF-3), which terminate inflammatory responses in macrophages.

Conclusion: TNFα signaling is not effectively terminated in FLS, which leads to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS in response to synovial TNFα contribute to the persistence of synovial inflammation in RA.

Figure 1. TNFα induces sustained production of IL-6, chemokines and MMPs in FLS

FLS were cultured in time course experiments in the presence or absence of TNFα (10ng/ml) that was added on the first day on culture and was not replenished. A, IL-6 protein in culture supernatants was measured by ELISA. B-C, IL-6 mRNA and IL6 primary transcripts (primers specific for the fourth intronic region of IL6 gene) and D, CXCL8/IL-8 mRNA, CCL5/RANTES mRNA, and MMP1 mRNA were measured by qPCR and normalized relative to GAPDH mRNA. Statistical analysis was performed using the two-way analysis of variance test (ANOVA) followed by the Bonferroni test. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001)

Figure 2. TNFα induces a robust but transient inflammatory response in human Mϕ

CD14+ cells from healthy donors’ blood were differentiated in vitro into Mϕ by stimulation with M-CSF (20ng/ml) for 48h. Mϕ were cultured in time course experiments in the presence or absence of TNFα (10ng/ml) that was added the first day on culture and not replenished. TNFα (A), IL-1b (B), CXCL8/IL-8 (C), and IL-6 mRNA (D) were measured by qPCR and normalized relative to GAPDH mRNA. Statistical analysis was performed using the two-way analysis of variance test (ANOVA) followed by the Bonferroni test. (*=p<0.05, ***=p<0.0001)

Figure 3. The sustained inflammatory response requires ongoing TNFα signaling

FLS were stimulated with TNFα on day 0. A, TNFα protein was measured with ELISA in culture supernatants (± TAPI-1). B, Etanercept or hIgG (Control) was added on days 0, 1, 2, or 3. Cells were harvested on day 4 and IL-6 mRNA was measured with qPCR. Statistical analysis was performed using the two-tailed, paired Student t test. C, IL-6 protein was measured with ELISA in culture supernatants at 48h and 72h with or without Infliximab (IFX) added at 48h. D, On day 3, etanercept, infliximab, anakinra, anti-gp130 or CP690550 were added and IL-6 mRNA were measured on day 4 with qPCR. Dashed line represents TNFα-stimulated control FLS that were set to 100%. Statistical analysis was performed using the one-way analysis of variance test (ANOVA) followed by the Bonferroni test or the two-tailed, paired Student t test. (*=p<0.05, ***=p<0.001, ****=p<0.0001, n.s.= not significant)

Figure 4. The prolonged induction of IL-6 by TNFα is dependent on sustained NF-κB signaling

FLS were cultured in the presence or absence of TNFα (10ng/ml). IκBα (A) and nuclear p65 (B) protein were measured with western blot and bands were quantified by densitometry (right panels). C, DMSO, Bay-11, IKK inhibitors II/XII, or MG132 were added on culture day 3 for 3h and IL6 primary transcripts (IL-6 PT) were measured with qPCR. Statistical analysis was performed using the two-tailed, paired Student t test (**=p<0.01) D, Expression levels of p55 or p75 protein on the cell surface were measured by FACS. Representative results of three independent experiments are shown.

Figure 5. Regulatory mechanisms that control TNFα-induced signaling and gene transcription in Mϕ are not effectively induced in FLS

FLS and Mϕ were cultured in the presence or absence of TNFα (10ng/ml) in time course experiments. ABIN3 (A), IRAK-M (B), SOCS3 (C) and ATF3 (D) mRNA were measured with qPCR (Mϕ: n=6, FLS: n=5). mRNA amounts were normalized relative to GAPDH mRNA, which was comparable in FLS and Mϕ. ND: Not Done. Statistical analysis was performed using the two-way analysis of variance test (ANOVA) followed by the Bonferroni test. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001)

Figure 6. TNFα, in FLS, induces sustained chromatin remodeling, histone acetylation, p65 and Pol II recruitment on the IL6 gene promoter

FLS and Mϕ were cultured in the presence or absence of TNFα (10ng/ml) in time course experiments. A, Nuclei from control and TNFα- or LPS-treated cells were digested with SspI (restriction enzyme accessibility assay for IL6 promoter). Purified genomic DNA was analyzed by Southern blot. Representative results from two independent experiments are shown. B-D, 72h after TNFα stimulation, FLS were harvested and ChIP assay was performed with anti-Histone 4 (H4), anti-acetylated H4 (H4Ac) (B), anti-p65 (C), or anti-Polymerase II (Pol II) (D) antibodies. The enrichment of immunoprecipitated proteins is shown as % of input and levels of H4Ac were normalized to total H4 levels at the promoters of IL6 and Hemoglobin B (HBB) (negative control). Representative results of three independent experiments are shown.