Background Rheumatoid arthritis (RA) and other types of inflammatory arthritis show a characteristic pattern of joint involvement. E.g. in RA there is a predilection for the small joints of hands and feet, whereas in spondyloarthropathy, single large joints are characteristically involved. We have recently shown that synovial fibroblasts (SF), which drive joint destruction in RA, display site-specific transcriptomes and functions, shaping unique microenvironments in different joints.
Objectives To analyze the role of transcripts expressed in SF at specific joint locations in defining location-specific functions of SF, relevant to the pathogenesis of RA.
Methods SF were isolated from hand, elbow, shoulder, feet, knee and hip joints of RA and OA patients undergoing joint replacement surgery and from knees of nonarthritic subjects with arthralgia. Transcriptomes and epigenomes of SF were determined by RNA-seq (Illumina HiSeq 2000, n=21), qPCR, ChIP-seq (H3K4me3, H3K27me3, H3K27ac, Illumina HiSeq 2500, n=7) and Infinium HumanMethylation450 BeadChip (n=12). Proliferative, adhesive and chemotactic properties of SF were studied by xCELLigence real time cell analysis and leukocyte chemotaxis towards supernatants of SF. The lncRNA HOTTIP was silenced in hand SF using LNA GapmeR oligos, followed by RNA-seq (n=2), pathway enrichment analysis (MetaCore, Thomson Reuters, FDR<0.05) and qPCR (n=5).
Results HOTTIP was the most differentially expressed transcript in distal (hand) vs. proximal (shoulder) SF. HOTTIP was specifically transcribed in SF from hand and feet SF, but absent from other joints, inferring distal-specific function to this lncRNA. Hand-specific HOTTIP expression coincided with the enrichment of activating histone marks H3K4me3 and H3K27ac, absence of repressive H3K27me3 and decreased DNA methylation at the HOTTIP promoter in hand SF. In contrast, the HOTTIP promoter displayed abundant DNA methylation and H3K27me3 in knee and shoulder SF. Silencing of HOTTIP led to downregulation of 3275 genes and upregulation of 4326 genes (log ratio >|0.5|, p<0.01, FDR<0.05). Distal-specific homeobox A13, a known HOTTIP target, was repressed in HOTTIP-silenced SF. Pathway enrichment analysis of genes repressed by HOTTIP silencing showed enrichment for pathways regulating cell adhesion, cell cycle, angiogenesis and inflammation, including NF-kB activation and IL-6 signalling. Meanwhile, upregulated genes were enriched in fewer pathways, e.g. IL17 and Notch signalling. 110 genes that were differentially expressed in hand vs. shoulder SF were also altered by HOTTIP silencing, e.g. TNFRSF1B and MAP3K14. Hand SF showed enhanced proliferative and chemotactic, but decreased adhesive properties compared to shoulder SF.
Conclusions The lncRNA HOTTIP, which is exclusively expressed in small, distal joints, via epigenetic mechanisms, is a regulator of inflammatory, proliferative and adhesive properties of SF. Such a functional specialization of arthritis relevant pathways in SF might represent an imprinted site-specific “risk” signature in SF, predisposing thereby to location-specific joint pathology, e.g. enhanced severity of hand arthritis in RA.
Disclosure of Interest M. Frank Bertoncelj Grant/research support from: euroTEAM, BTCure, IAR, Promedica, Georg und Berta Schwyzer Winiker Grant, E. Karouzakis Grant/research support from: BTCure, GSK, K. Klein Grant/research support from: BTCure, A. Bratus: None declared, C. Kolling: None declared, O. Distler Grant/research support from: Actelion, Bayer, Boehringer Ingelheim, Pfizer, Sanofi, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, ChemomAb, EpiPharm, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, Mepha, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Pfizer, Sanofi, Serodapharm, Sinoxa, Speakers bureau: AbbVie, iQone Healthcare, Mepha, A. Filer: None declared, R. Gay Grant/research support from: euroTEAM, BTCure, GSK, IAR, C. Buckley: None declared, S. Gay Grant/research support from: euroTEAM, BTCure, GSK, IAR, C. Ospelt Grant/research support from: euroTEAM, BTCure, CABMM, IAR, Promedica