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

We 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). FLS 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. We 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. Our 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.

Loh C ，Park SH ，Lee A ，Yuan R ，Ivashkiv LB ，Kalliolias GD ... -  《-》

Age-associated B cells contribute to the pathogenesis of rheumatoid arthritis by inducing activation of fibroblast-like synoviocytes via TNF-α-mediated ERK1/2 and JAK-STAT1 pathways.

Age-associated B cells (ABCs) are a recently identified B cell subset, whose expansion has been increasingly linked to the pathogenesis of autoimmune disorders. This study aimed to investigate whether ABCs are involved in the pathogenesis and underlying mechanisms of rheumatoid arthritis (RA). ABCs were assessed in collagen-induced arthritis (CIA) mice and patients with RA using flow cytometry. Transcriptomic features of RA ABCs were explored using RNA-seq. Primary fibroblast-like synoviocytes (FLS) derived from the synovial tissue of patients with RA were cocultured with ABCs or ABCs-conditioned medium (ABCsCM). IL-6, MMP-1, MMP-3 and MMP-13 levels in the coculture supernatant were detected by ELISA. Signalling pathways related to ABCs-induced FLS activation were examined using western blotting. Increased ABCs levels in the blood, spleen and inflammatory joints of CIA mice were observed. Notably, ABCs were elevated in the blood, synovial fluid and synovial tissue of patients with RA and positively correlated with disease activity. RNA-seq revealed upregulated chemotaxis-related genes in RA ABCs compared with those in naive and memory B cells. Coculture of FLS with RA ABCs or ABCsCM led to an active phenotype of FLS, with increased production of IL-6, MMP-1, MMP-3 and MMP-13. Mechanistically, ABCsCM-derived TNF-α promoted the upregulation of interferon-stimulated genes in FLS, with elevated phosphorylation of ERK1/2 and STAT1. Furthermore, blockage of ERK1/2 and Janus Kinase (JAK)-STAT1 pathways inhibited the activation of FLS induced by ABCsCM. Our results suggest that ABCs contribute to the pathogenesis of RA by inducing the activation of FLS via TNF-α-mediated ERK1/2 and JAK-STAT1 pathways.

Qin Y ，Cai ML ，Jin HZ ，Huang W ，Zhu C ，Bozec A ，Huang J ，Chen Z ... -  《-》

Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay.

Grabiec AM ，Korchynskyi O ，Tak PP ，Reedquist KA ... -  《-》

Silencing aquaporin 1 inhibits autophagy to exert anti-rheumatoid arthritis effects in TNF-α-induced fibroblast-like synoviocytes and adjuvant-induced arthritis rats.

Fibroblast-like synoviocytes (FLS) are key players in rheumatoid arthritis (RA) by resisting apoptosis via increased autophagy. Elevated synovial aquaporin 1 (AQP1) affects RA FLS behaviors, but its relationship with FLS autophagy is unclear. We aim to clarify that silencing AQP1 inhibits autophagy to exert its anti-RA effects. We studied the effects and mechanisms of AQP1 silencing on autophagy in TNF-α-induced RA FLS and examined the crucial role of autophagy inhibition in its impacts on RA FLS pathogenic behaviors. We explored whether silencing synovial AQP1 relieved rat adjuvant-induced arthritis (AIA) by reducing synovial autophagy. TNF-α stimulation increased AQP1 expression and autophagy levels in RA FLS, with a positive correlation between them. AQP1 silencing inhibited autophagy in TNF-α-stimulated RA FLS, along with suppressing proliferation, promoting apoptosis, and mitigating inflammation. Notably, the inhibitory effects of AQP1 silencing on RA FLS pathogenic behaviors were cancelled by autophagy activation with rapamycin (Rapa) but enhanced by autophagy inhibition using 3-Methyladenine. Mechanistically, silencing AQP1 enhanced the binding of Bcl-2 to Beclin1 by decreasing Beclin1-K63 ubiquitination, thus inhibiting RA FLS autophagy. In vivo, silencing synovial AQP1 relieved the severity and development of rat AIA, alongside reducing Ki67 expression, promoting apoptosis, and decreasing autophagy within AIA rat synovium. Expectedly, the Rapa co-administration nullified the anti-AIA effects of silencing synovial AQP1. These findings reveal that silencing AQP1 inhibits RA FLS pathogenic behaviors and attenuates rat AIA through autophagy inhibition. This study may help clarify the pathogenic role of AQP1 in enhancing autophagy during RA development.

Zhang MY ，Wang MQ ，Huang Y ，Gu SL ，Zhou MY ，Xu ZS ，Li LL ，Lv M ，Cai L ，Li R ... -  《-》

Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes.

Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS). RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays. HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11. Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.

Angiolilli C ，Kabala PA ，Grabiec AM ，Van Baarsen IM ，Ferguson BS ，García S ，Malvar Fernandez B ，McKinsey TA ，Tak PP ，Fossati G ，Mascagni P ，Baeten DL ，Reedquist KA ... -  《-》