TNF-induced inflammatory genes escape repression in fibroblast-like synoviocytes: transcriptomic and epigenomic analysis

Abstract

ObjectiveWe investigated genome-wide changes in gene expression and chromatin remodelling induced by tumour necrosis factor (TNF) in fibroblast-like synoviocytes (FLS) and macrophages to better understand the contribution of FLS to the pathogenesis of rheumatoid arthritis (RA).MethodsFLS were purified from patients with RA and CD14+ human monocyte-derived macrophages were obtained from healthy donors. RNA-sequencing, histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposable accessible chromatin by high throughput sequencing (ATAC-seq) were performed in control and TNF-stimulated cells.ResultsWe discovered 280 TNF-inducible arthritogenic genes which are transiently expressed and subsequently repressed in macrophages, but in RA, FLS are expressed with prolonged kinetics that parallel the unremitting kinetics of RA synovitis. 80 out of these 280 fibroblast-sustained genes (FSGs) that escape repression in FLS relative to macrophages were desensitised (tolerised) in macrophages. Epigenomic analysis revealed persistent H3K27 acetylation and increased chromatin accessibility in regulatory elements associated with FSGs in TNF-stimulated FLS. The accessible regulatory elements of FSGs were enriched in binding motifs for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interferon-regulatory factors (IRFs) and activating protein-1 (AP-1). Inhibition of bromodomain and extra-terminal motif (BET) proteins, which interact with histone acetylation, suppressed sustained induction of FSGs by TNF.ConclusionOur genome-wide analysis has identified the escape of genes from transcriptional repression in FLS as a novel mechanism potentially contributing to the chronic unremitting synovitis observed in RA. Our finding that TNF induces sustained chromatin activation in regulatory elements of the genes that escape repression in RA FLS suggests that altering or targeting chromatin states in FLS (eg, with inhibitors of BET proteins) is an attractive therapeutic strategy.

Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration

“…Next, we compared the scRNA-seq between healthy meniscus and degenerated meniscus (figure 4B–E). As a result, we detected significant changes in the proportions of degenerated meniscus cell clusters, including three new clusters: (1) monocyte-derived dendritic cells (MoDC, expressing CD14 and S100A9),2324 (2) hypertrophic chondrocytes (HTC, expressing CCL20 and EREG)25 26 and (3) degenerated meniscus progenitor cells (DegP), which are found in degenerated meniscus and express skeletal stem cell marker, such as GREM127 (figure 4C–G). Moreover, the proportion of EC and FCP expression was found to decrease in degenerated meniscus (figure 4E).…”

IRF9 Affects the TNF-Induced Phenotype of Rheumatoid-Arthritis Fibroblast-Like Synoviocytes via Regulation of the SIRT-1/NF-κB Signaling Pathway

“…Excessive FLS growth is considered to be a key characteristic of RA, which would promote hyperplasia of synovial tissue and lead to cancer-like and aggressive invasion into the bone and cartilage, and thus to joint malformations and dysfunction [Turner and Filer, 2015;Busta-mante et al, 2017]. Previous studies found that TNF, secreted by macrophages and other cells, could activate RA-FLS, which then proliferated rapidly and produce copious amounts of cytokines, proteases, adhesion molecules, and growth factors [Lee et al, 2013;Donlin et al, 2014;Sohn et al, 2015;Loupasakis et al, 2017;Loh et al, 2019]. Besides, TNF has been shown to induce sustained inflammatory responses in RA-FLS by regulating its receptors (TNFRI and TNFRII) and enhancing NF-κB activity [Zhang and Xiao, 2017].…”

Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory

“…Recent large-scale genome-wide studies have incontrovertibly established that FLS transformation is associated with epigeneticremodeling. However, which of these changes are reversible and which of them are permanent is not fully understood (5,14,16) A recent study identified that multiple proinflammatory genes in human RA FLS escaped transcriptional repression for at least up to period of 72h of TNF treatment. These escaped genes were suggested to contribute to the persistence of synovitis observed in RA patients.…”

“…We further compared these results with the genome-wide data from 9 human RA FLS samples treated with TNF for varying time periods. Some of these data were generated in our laboratory and others were accessed from the public repositories (14,16). This exercise provided a critical insight into the molecular understanding of the sustained epigenetic remodeling.…”

“…Previous studies comparing human RA-FLS to macrophages showed that TNF treatment could induce sustained gene expression in RA-FLS but not in macrophages (10,12,14,15). Recent studies suggest that treatment with TNF prolonged chromatin accessibility and histone modifications for up to 72h in the RA-FLS (10).…”