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A1.68 An Acetyl-Histone MiMetic blocks inflammatory activation of rheumatoid arthritis fibroblast-like synoviocytes
  1. P Kabala1,2,
  2. A M Grabiec1,2,
  3. N Smithers3,
  4. J Witherington3,
  5. P P Tak3,
  6. R K Prinjha3,
  7. K A Reedquist1,2
  1. 1Department of Experimental Immunology, Academic Medical Center, University of Amsterdam
  2. 2Amsterdam Rheumatology and Immunology Center (AMC campus), Amsterdam, The Netherlands
  3. 3GlaxoSmithKline, Stevenage, UL

Abstract

Background and Objectives Genetic backgrounds do not completely explain the etiology of rheumatoid arthritis (RA), and increasing attention has turned to the potential contributions of epigenetic mechanisms. Fibroblast-like synoviocytes (FLS) isolated from affected joints of RA patients maintain an aggressive phenotype in vitro, indicating potential alteration of epigenetic regulatory processes, and changes in global and gene-specific promoter DNA methylation and histone modification are observed in RA FLS. I-BET compounds, acetyl histone mimetics which interfere with reading of acetylated histones by BET family bromodomain proteins BRD2-4, prevent LPS-induced inflammatory gene expression in murine bone marrow-derived macrophages in vitro, as well as in vivo models of endotoxic shock. This study was undertaken to assess the potential of I-BET to influence the inflammatory activation of RA FLS.

Methods RA FLS were treated with IL-1β or TNF in the presence or absence of increasing concentrations of I-BET, and IL-6 and IL-8 production measured by ELISA. Cellular viability was assessed using MTT assay and cell death ELISA kits. Activation of intracellular signalling pathways was examined by immunoblotting. Total RNA was extracted and mRNA expression of genes regulated by IL-1β or TNF in RA FLS was analysed using a low density quantitative PCR custom array.

Results I-BET reduced RA FLS (n = 6) production of IL-6 protein in response to IL-1β (1 μM, 70% reduction, P < 0.001) and TNF (50%, P < 0.001). IL-8 production in response to IL-1β (>75% reduction, P < 0.001) and TNF (>70%, P < 0.001) was also inhibited. Similar effects were observed on IL-6 and IL-8 mRNA expression. Inhibition of IL-6 and IL-8 production was maintained when I-BET treatment was delayed 1-4 hours post-stimulation. I-BET had no effect on cell viability or survival, and failed to modulate IL-1β or TNF–induced activation of MAPK or NF-κB signalling pathways. Low density qPCR array analysis of 26 genes induced by IL-1β in RA FLS (n = 3) demonstrated that I-BET reduced induction of 17 genes by >50%, including TNF, MMP-1, MMP-3, SELE, VCAM-1, CXCL-6, CXCL-9 and CXCL-11.

Conclusions Our results demonstrate that disrupting the recruitment of BET family proteins to acetylated histones efficiently blocks RA FLS production of a range of inflammatory mediators in response to IL-1β and TNF. This study provides initial evidence that targeting interactions between epigenetic modifications, such as histone acetylation, and the proteins which interpret these modifications, may have therapeutic potential in the treatment of RA.

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