Bromodomain Protein Inhibitors Reorganize the Chromatin of Synovial Fibroblasts.

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.

Reset of Inflammatory Priming of Joint Tissue and Reduction of the Severity of Arthritis Flares by Bromodomain Inhibition.

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 extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model. METHODS: BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of 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, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

Targeting the tissue-complosome for curbing inflammatory disease.

Hyperactivated local tissue is a cardinal feature of immune-mediated inflammatory diseases of various organs such as the joints, the gut, the skin, or the lungs. Tissue-resident structural and stromal cells, which get primed during repeated or long-lasting bouts of inflammation form the basis of this sensitization of the tissue. During priming, cells change their metabolism to make them fit for the heightened energy demands that occur during persistent inflammation. Epigenetic changes and, curiously, an activation of intracellularly expressed parts of the complement system drive this metabolic invigoration and enable tissue-resident cells and infiltrating immune cells to employ an arsenal of inflammatory functions, including activation of inflammasomes. Here we provide a current overview on complement activation and inflammatory transformation in tissue-occupying cells, focusing on fibroblasts during arthritis, and illustrate ways how therapeutics directed at complement C3 could potentially target the complosome to unprime cells in the tissue and induce long-lasting abatement of inflammation.

Stromal cell regulation of inflammatory responses.

In the last fifteen years it has become apparent that tissue-resident mesenchymal cells such as fibroblasts, which are the structural elements of all organs, play a cardinal role in the pathology of immune-mediated inflammatory diseases. We now know that all fibroblasts originate from universal pan-organ cellular ancestors and that they are diversified into more specific subsets according to the functional needs of their home tissue-and its activation state. In arthritis, a plethora of activated joint-resident and migrating fibroblast types have been recently described that are central for pathogenesis and persistence of inflammatory joint-disease. Here we provide a current overview on the multiple inflammatory and immune-related functions of fibroblasts and how they could be curbed to induce long-lasting abatement of disease.

Dietary Derived Propionate Regulates Pathogenic Fibroblast Function and Ameliorates Experimental Arthritis and Inflammatory Tissue Priming.

Short-chain fatty acids are gut-bacteria-derived metabolites that execute important regulatory functions on adaptive immune responses, yet their influence on inflammation driven by innate immunity remains understudied. Here, we show that propionate treatment in drinking water or upon local application into the joint reduced experimental arthritis and lowered inflammatory tissue priming mediated by synovial fibroblasts. On a cellular level, incubation of synovial fibroblasts with propionate or a physiological mixture of short-chain fatty acids interfered with production of inflammatory mediators and migration and induced immune-regulatory fibroblast senescence. Our study suggests that propionate mediates its alleviating effect on arthritis by direct abrogation of local arthritogenic fibroblast function.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.