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Bromodomain- and extra-terminal domain (BET) proteins are epigenetic reader proteins that regulate transcription of their target genes by binding to acetylated histone side chains. Small molecule inhibitors, such as I-BET151, have anti-inflammatory properties in fibroblast-like synoviocytes (FLS) and in animal models of arthritis. Here, we investigated whether BET inhibition can also affect the levels of histone modifications, a novel mechanism underlying BET protein inhibition. On the one hand, FLSs were treated with I-BET151 (1 µM) for 24 h in absence and presence of TNF. On the other hand, FLSs were washed with PBS after 48 h of I-BET151 treatment, and the effects were measured 5 days after I-BET151 treatment or after an additional 24 h stimulation with TNF (5 d + 24 h). Mass spectrometry analysis indicated that I-BET151 induced profound changes in histone modifications, with a global reduction in acetylation on different histone side chains 5 days after treatment. We confirmed changes on acetylated histone side chains in independent samples by Western blotting. I-BET151 treatment reduced mean TNF-induced levels of total acetylated histone 3 (acH3), H3K18ac, and H3K27ac. In line with these changes, the TNF-induced expression of BET protein target genes was suppressed 5 d after I-BET151 treatment. Our data indicate that BET inhibitors not only prevent the reading of acetylated histones but directly influence overall chromatin organization, in particular after stimulation with TNF.

Additional file 1: Supplementary Methods. Adaptation algorithm for spinal radiographic scores. Imputation of missing covariate data. Supplementary Table 1. Multivariable analysis for identification of factors associated with radiographic progression defined as an increase of ≥ 2 mSASSS units per 2 years in the cervical and in the lumbar spine. Supplementary Figure 1. Cumulative probability plot of 2-year progression in the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) by spinal segments, illustrating the change in mSASSS values in patients already treated with TNFi at start of the respective interval, stratified by the ASDAS cut-off level reached at baseline.

OBJECTIVE We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extra-terminal motif (BET) proteins translate epigenetic changes into transcription. Here we used a BET inhibitor to target inflammatory tissue priming and reduce flare severity in experimental arthritis. METHODS BALB/c mice were treated intraperitoneally or locally into the paw with I-BET151, which blocks interaction of BET proteins with acetylated histones. Effect of I-BET151 on acute arthritis and/or inflammatory tissue priming was assessed in a model of repeated injections of monosodium urate crystals or zymosan into the paw. I-BET151 was given either from before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. Transcriptomic (RNA-Seq), epigenomic (ATAC-Seq) and functional analysis (invasion, cytokine production, migration, senescence, metabolic flux) was performed on murine and human SFs treated with I-BET151 in vitro or in vivo. RESULTS Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic and pathogenic phenotype of SFs. CONCLUSION Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, which makes them promising therapeutic targets for preventing arthritis flares in previously affected joints.

Fibroblast specific protein 1 (FSP1/S100A4) is an intracellular calcium binding protein which has been linked to epithelial-mesenchymal transition, tissue fibrosis, pulmonary vascular disease, metastatic tumour development, increased tumour cell motility and invasiveness. This protein is reported to be uniquely expressed in newly formed and differentiated fibroblasts and has been used in various studies to demonstrate epithelial-mesenchymal transition (EMT). However, S100A4 immunoreactivity does not solely fit the distribution and shape of fibroblasts; therefore, we aimed to characterize S100A4 positive cells in different human tissue compartments. We found S100A4 expression in a wide range of cells, including cells of haematopoietic lineage, namely CD4 and CD8 positive T-lymphocytes, but not B-lymphocytes. All investigated monocytes, macrophages and specialised histiocytes were positive for S100A4. Only a limited number of fibroblasts and most smooth muscle actin positive myofibroblasts expressed S100A4. Even some epithelial cells of the kidney and bladder were positive for S100A4. Expression was also found in the vasculature. Here, cells of the subendothelial space, tunica adventitia and some smooth muscle cells of the tunica media were positive for S100A4. Expression was also detected in the stroma of the prostate, adipocytes and in skeletal muscle. In summary, S100A4 is expressed in various cell types of different lineage. It is therefore not possible to equate the expression of this protein to fibroblasts. Hence, studies that used this marker as evidence of transition from epithelia to fibroblasts must be revisited.