Background Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease that affects synovial joints. A key characteristic of RA is hyperplasia of fibroblast-like synoviocytes (FLS). These FLS are known to develop a stable, auto-aggressive phenotype that augments tissue destruction. It is not known how this phenotype is stably maintained, however epigenetic changes have been implicated. Histone acetylation is one proposed epigenetic mechanism involved; a process controlled by histone deacetylases (HDACs). These enzymes condense the DNA through removal of acetyl groups from histones, leading to transcriptional repression. Recent studies have reported conflicting evidence of HDAC activity in RA synovium and FLS (1-3); however promising results were obtained using HDAC inhibitors in murine arthritis models and human juvenile arthritis.
Objectives Our aim is to determine the role of HDACs in regulating the auto-aggressive phenotype of RA FLS.
Methods Synovial biopsies obtained from RA and osteoarthritis (OA) patients were used to isolate FLS following digestion with collagenase-1. Real time-qPCR was used to assess the levels of HDAC1-11 in RA compared to OA FLS. Joint biopsies were co-stained with anti-HDAC1 and anti-fibroblast antibodies. HDAC1 and a non-targeting control (NTC) siRNA were transfected into FLS; HDAC1 knockdown (HDAC1KD) 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 migration and invasion were also assessed using a scratch assay and a matrigel invasion assay respectively. Cell proliferation was assessed using a tritiated thymidine assay and an Illumina BeadChip covering over 47,000 transcripts was used to analyse global gene expression.
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). HDAC1KD did not affect cell viability (n=6) (>95.5% viable HDACKD cells vs. >95% viable NTC cells). HDAC1KD reduced the number of FLS migrating (p=0.01, n=6) and invading the Matrigel (p=0.02, n=6) compared to the NTC. HDAC1KD also reduced the number of proliferating FLS compared to the NTC (p=0.04, n=6). Cluster analysis of the microarrays (n=3) revealed significant changes in genes involved in apoptosis (n=21, p≤0.009), migration (n=4, p≤0.008) and proliferation (n=9, p≤0.009).
Conclusions HDAC1 expression is increased in RA FLS compared to control. It significantly contributes to their ability to migrate, invade and proliferate in the RA joint possibly through up regulation of genes including VEGFA and FGF2 and down regulation of genes including TNFRSF19 and TNFSF15. Further work will determine if HDAC1KD in an arthritic mouse model can ameliorate disease.
Huber LC, et al. Histone deacetylase/acetylase activity in total synovial tissue derived from rheumatoid arthritis and osteoarthritis patients. Arthritis and Rheumatism. 2007;56(4):1087-93.
Kawabata T, et al. Increased activity and expression of histone deacetylase 1 in relation to tumor necrosis factor-alpha in synovial tissue of rheumatoid arthritis. Arthritis research & therapy. 2010;12(4):R133.
Horiuchi M, et al. Expression and Function of Histone Deacetylases in Rheumatoid Arthritis Synovial Fibroblasts. Journal of Rheumatology. 2009;36(8):1580-9.
Disclosure of Interest None declared