Background/Purpose:   Fibroblast-like synoviocyte (FLS) is expected to be a novel therapeutic target for rheumatoid arthritis (RA) because of their contribution to pathogenesis. FLS expresses matrix metalloproteinases, chemokines and cytokines in a response to various cytokines in affected joints, which leads bone and cartridge destruction, inflammatory cell recruitment and angiogenesis in pannus. The objective of this study is to investigate the transcriptional organization of stimuli responses and to clarify a character of RA-FLS.

Methods:   Synovium were obtained from RA (n = 21) and osteoarthritis (OA) patients (n = 23) who underwent joint replacement surgery with informed consent. Minced synovium were treated with collagenase and cultured in Dulbecco modified Eagle’s medium supplemented with 10% fetal bovine serum. At passage 2, FLS were isolated with removal of contaminating CD14 positive cells by magnetic-activated cell sorting. Purified FLS were stimulated with cytokines (TNF-α, IL-1β, IL-6/sIL-6R, IL-17A, IL-18, IFN-γ, IFN-α, TGF-β1 and combination of all 8 cytokines which simulated the mixture of stimuli in the joint) for 10 and 24 hours. Total RNA was extracted and libraries for RNA-sequence were prepared using TruSeq Stranded mRNA Library Prep kit (Illumina), and paired-end sequencing was performed using HiSeq 2500 (Illumina).

Results:   The overview of transcriptomes with multi-dimensional scaling plot revealed that there were conserved transcriptome differences between RA and OA-FLS for each stimulatory condition, although the fluctuation vector with each stimuli were similar between RA and OA-FLS. Through the pathway analysis, 12 transcriptional regulators and those downstream genes were supposed to be significantly upregulated in RA-FLS compared to OA-FLS. In particular, E2F transcription factor 6 (E2F6), heat shock factor protein 1 (HSF1), Lysine (K)-specific demethylase 5B (KDM5B), nuclear protein 1 (NUPR1), tumor protein P53 (TP53) were expected to affect the downstream gene expression in stimulatory conditions compared with non-stimulatory condition. Among 1468 downstream genes of these 5 transcriptional regulators, 26 genes related to RA genome-wide association study (GWAS) were detected. Notably, colony stimulating factor 2 (CSF2), cyclin-dependent kinase 2 (CDK2) and flap structure-specific endonuclease 1 (FEN1) were significantly differentially expressed gene not only in RA-FLS compared with OA-FLS, but also in cytokine-stimulated FLS compared with non-stimulatory condition. Furthermore, through the correlation analysis, CSF2 expression which was induced by certain cytokines (IL-1β, TNF-α, all cytokine mixture) was highly correlated with particular transcription factors (i.e. JUN, FOX, JUND).

Conclusion:   CSF2 was specifically expressed in RA-FLS and significantly expressed in the presence of stimulation. Although CSF2 locus was raised in RA GWAS, the expression quantitative trait loci (eQTL) effect for CSF2 gene has yet to be determined. Through comprehensive analysis, the mechanism for CSF2 expression and the pathway of RA-FLS specific transcription is expected to be elucidated.

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-comprehensive-analysis-for-the-transcriptional-organization-of-stimuli-responses-in-fibroblast-like-synoviocytes-from-rheumatoid-arthritis-patients/