LAUSR

Background In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) play an important role in joint destruction. We have shown a disease-specific DNA methylation pattern in RA patient-derived FLSs (RA FLSs). In 2009, active demethylation enzymes, Ten-Eleven translocation (TET) 1/2/3, were demonstrated. However, little is known about the role of the TET protein family in RA FLSs Objectives The aim of this study was to examine the role of the TET protein family in the pathological process of RA from an in vivo perspective with a mouse model, and from an in vitro perspective using human synovial tissue. Methods K/BxN serum-induced arthritis was induced in Wild-type (WT) and TET3 heterozygous-deficient (TET3+/-) C57BL/6 mice. Synovial tissues were obtained from patients with RA and Osteoarthritis (OA) who had received joint replacement surgery. FLSs were transfected with siRNA and knocked down (KD). Gene expression was determined by qPCR and protein expression by western blot and immunostaining. 5-hydroxymethylcytosine (5hmC) was determined by dot blot and hMeDIP assay. Cell migration were assessed using a scratch assay. Results TET3 was expressed in particularly infiltrative synovial areas in K/BxN-WT mice. The arthritis score of K/BxN-TET3+/- was not increased after day 8. Histologically, synovial inflammation and proliferation, and bone destruction were significantly suppressed in the K/BxN-TET3+/- mice. In the RA synovial membrane, TET3 was more highly expressed in RA than in OA. In cultured FLSs, the expression of TET3 mRNA tended to be slightly higher in RA than in OA. After 2 h of stimulation with various cytokines, the expression level of TET3 mRNA was increased by stimulation with TNFα, interleukin (IL)-1, or IL-17. Assessment of the protein expression of TET3 after stimulation by TNFα using extracted intranuclear proteins of FLSs showed an increase in the expression level of TET3 protein over time. Similarly, the expression level of 5hmC was increased after stimulation by TNFα. Next, the function of TET3 in the FLSs was examined using TNF stimulation follwoing TET3-KD. A scratch assay of cell migration and invasion demonstrated that the TNFα-dependent migration and invasion of FLSs was completely inhibited by TET3-KD. The protein levels of CCL2 were strongly induced by TNFα stimulation, and inhibited by TET3-KD. In addition, flow cytometry of the expression of intercellular adhesion molecule-1 (ICAM-1) revealed that the expression of ICAM-1 was TNFα-dependent and was inhibited by TET3-KD. Assessment of gene expression changes showed that the induction of TNFα-dependent CCL2 and ICAM-1 mRNA expression was significantly inhibited by TET3-KD. Subsequently, although 5hmC level on the ICAM-1 promoter was increased by TNF stimulation, it was strongly inhibited by TET3-KD. Conclusions In vivo, the arthritis and bone erosion were significantly decreased in the TET3+/- mice. In vitro, TET3 was induced by inflammatory cytokine stimulation, and TET3 knockdown inhibited the cytokine-induced expression of CCL2 and ICAM-1 in RA FLSs. In addition, hydroxymethylation of the ICAM-1 promoter region was dependent on TET3. These results suggest that continuous exposure to inflammatory cytokines results in leaving an inflammatory memory in FLS in a TET3-dependent manner, thereby promoting pannus formation and increasing the probability of joint destruction. Disclosure of Interest A. Kawabe: None declared, K. Naknao: None declared, K. Sakata: None declared, K. Yamagata: None declared, S. Nakayamada: None declared, Y. Tanaka Grant/research support from: Y. Tanaka, has received consulting fees, speaking fees, and/or honoraria from Abbvie, Chugai, Daiichi-Sankyo, Bristol-Myers, Mitsubishi-Tanabe, Astellas, Takeda, Pfizer, Teijin, Asahi-kasei, YL Biologics, Sanofi, Janssen, Eli Lilly, GlaxoSmithKline and has received research grants from Mitsubishi-Tanabe, Takeda, Daiichi-Sankyo, Chugai, Bristol-Myers, MSD, Astellas, Abbvie, Eisai