Background Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease that affects synovial joints. A key characteristic of RA is hyperplasia of the synovial lining layer, which includes fibroblast-like synoviocytes (FLS). These FLS develop an aggressive phenotype that augments tissue destruction. It is not currently known how the phenotype of the FLS is stably maintained, however epigenetic changes have been implicated. Histone deacetylases (HDACs) are key enzymes that contribute to the epigenetic signature through changes in the acetylation status of histones. There are currently 11 HDACs, grouped into 4 classes (I, IIa, IIb and IV). These enzymes act to condense the DNA through removal of acetyl groups causing transcriptional repression. Recent studies have reported conflicting evidence of HDAC activity in RA synovium and FLS; however promising results were obtained using HDAC inhibitors in murine arthritis models and human juvenile arthritis. Further investigations into the specific role of individual HDACs in RA are required and may allow discovery of more specific therapeutic targets.
Objectives To determine the role of HDACs in maintaining the autoaggressive phenotype of RA FLS.
Methods Fresh synovial biopsies obtained from RA and osteoarthritis (OA) patients were used to isolate FLS following digestion with collagenase-1. Real time-qPCR (RT-qPCR) was used to assess the levels of HDAC1-11 in RA FLS compared to OA FLS. To determine the cellular localization of HDACs, sections from patient arthroscopies were co-stained with anti-HDACs and anti-fibroblast antibody. In addition, HDACs and a non-targeting control (NTC) were knocked down in FLS using siRNA transfection; this was confirmed by RT-qPCR and western blotting. Cell viability after knockdown was assessed by flow cytometry using annexin V/propidium iodide dual staining. Cell invasion after knock down was assessed by a matrigel invasion assay.
Results The mRNA levels of HDACs 1-11 are higher in RA compared to OA, with HDAC1 levels showing the greatest difference (4.3-fold higher). A 65% knockdown of HDAC1 (HDAC1KD) mRNA was achieved using siRNA compared to a non targeting control siRNA (NTCKD) (n=3 patients) and this did not affect cell viability (n=6) (>95.5% viable HDACKD cells vs. >95% viable NTCKD cells). Using the matrigel invasion assay we found that HDAC1KD reduced the number of FLS invading the Matrigel (p=0.056) compared to knockdown with the NTC (n=4).
Conclusions HDAC1 expression is increased in RA FLS compared to OA FLS. Knocking down HDAC1 in RA FLS does not affect cell viability but does reduce their ability to invade into Matrigel, suggesting that HDAC1 may contribute to the invasiveness of RA FLS. Further work will determine the effects of HDAC1 knockdown in FLS on proliferation, migration and the expressed genome.
Disclosure of Interest None Declared