Inflammatory activation of rheumatoid arthritis fibroblast-like synovopcytes. The role of epigenetic regulatory proteins and endoplasmic reticulum stress

Abstract

Rheumatoid arthritis (RA) is the most common autoimmune disease and leads to progressive disability and joint destruction. RA management has improved dramatically but a significant fraction of patients remains refractory to treatment or relapses shortly after drug discontinuation. While the available therapeutic modalities are directed primarily against cytokine networks and immune cell populations, the role of fibroblast-like synoviocyte (FLS) stromal cells in RA pathogenesis is increasingly appreciated. RA FLS exhibit an epigenetically imprinted aggressive phenotype and have emerged as key players in cartilage destruction and maintenance of chronic inflammation in synovium. This work aimed at defining the role selected groups of epigenetic regulatory proteins and endoplasmic reticulum (ER) stress play in controlling FLS inflammatory activation. Regulation of inflammatory molecule production is a multi-layered process in which epigenetic regulatory proteins participate by marking chromosomic regions destined to be expressed. We analysed the role of two families of epigenetic proteins, namely BET proteins and histone deacetylases (HDACs), responsible for the reading and erasing of histone acetylation marks, respectively, in the regulation of FLS activation. Pharmacological inhibition of members of both families led to strong anti-inflammatory effects. BET protein inhibitor prevented cytokine and tissue-destructive enzyme expression in FLS exposed to cytokines and TLR ligands, while simultaneously promoting the expression of anti-inflammatory, anti-proliferative and tissue-protective molecules. Similarly, the pan-specific HDAC inhibitor (HDACi) ITF2357 prevented expression of key genes involved in RA pathogenesis in stimulated FLS. Analysis of the molecular events associated with ITF2357 administration highlighted the role of mRNA stability regulation in the anti-inflammatory activity of HDACi. The protein tristertaprolin was identified as a crucial mediator of this effect. To better understand the contributions of individual HDAC family members to pan-specific HDACi effects we exposed FLS to inhibitors selective for HDAC3/6, HDAC8, and HDAC1/2. Results from these experiments point towards HDCA3 as a key controller of inflammatory gene expression in FLS and identify another HDCAi mechanism of action resulting from STAT1 hypoactivation. The ER stress signature is a feature of the RA synovium but the effects of ER stress on FLS physiology are not well-studied. Here, we have demonstrated for the first time that stressed FLS become poised for augmented expression of inflammatory mediators in response to additional triggers. Synergistic action observed between ER stress and IL1β, TNF, and TLR ligands was determined to be a general feature of fibroblastic but not myeloid cells and depended primarily on cytokine mRNA stabilisation. To better assess the possible impact of ER stress in vivo, we performed transcriptomic analysis of acute and chronic ER stress. ER stress alone has similar effects at both short and long timepoints and prolonged ER stress maintained its overall pro-inflammatory properties. However, chronic ER stress potently prevented expression of interferon-responsive genes following stimulation with LPS but not other inflammatory triggers. Lastly, we have uncovered the capacity of ER stress to coordinately regulate the expression of epigenetic regulatory proteins potentially contributing to the stabilisation of the pathologic RA FLS phenotype.