Tumor Necrosis Factor α Induces Sustained Signaling and a Prolonged and Unremitting Inflammatory Response in Rheumatoid Arthritis Synovial Fibroblasts

Abstract

Objective The non resolving 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 TNFα-driven inflammatory program in human FLS. Methods FLS obtained from synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by qPCR and ELISA. NF-κB signaling was evaluated using Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the IL6 promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays. Results In FLS, TNFα induced prolonged transcription of IL6 and progressive accumulation of IL-6 protein over four days. Similarly, induction of CXCL8/IL-8, CCL5/RANTES, MMP1 and MMP3 mRNA 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 pro-inflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors ABIN3, IRAK-M, SOCS3 and ATF3 that terminate inflammatory responses in macrophages. Conclusions TNFα signaling is not effectively terminated in FLS, leading 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.

SND-117, a sinomenine bivalent alleviates type II collagen-induced arthritis in mice

“…In a parallel study on translocation of NF-κB p-p65 from the cytoplasm into the nucleus, the data clearly exhibited that TNF-α induced a rapid nuclear translocation of p-p65, and nuclear p65 peaked after 30 min. This result was consistent with other reported results [47,48]. SND-117 clearly down-regulated the translocation of nuclear p65 in fibroblast like synovial cells.…”

Activated human B cells induce inflammatory fibroblasts with cartilage-destructive properties and become functionally suppressed in return

“…But since SF stayed activated as long as activated B cells were present, it cannot be excluded that under prolonged inflammatory conditions, SF stimulation might indeed lead to a stable phenotype as attributed to RA-SF before 64 65. Specifically, TNFα appears to be involved in inflammatory ‘imprinting’ as it promotes prolonged proinflammatory cytokine production by SF after initial exposure,66 well fitting our own observations. In the long run, this process has been shown to lead to characteristic chromatin changes in fibroblasts 67…”

Genetic polymorphism directs IL-6 expression in fibroblasts but not selected other cell types

“…Macrophages, in contrast, had less open chromatin with methylation patterns consistent with a poised, ready-to-respond state. This pattern supports prior work showing tight control of IL-6 production in macrophages but sustained production in synovial fibroblasts (34). Interestingly, although CpG DNA methylation is associated with low gene expression, our analysis showed poor correlation between IL-6 expression and promoter CpG methylation (Fig.…”