After introducing biological DMARDs, increase of ccfDNA at 8 weeks was associated with improvement of disease activities. Compared with biomarkers reported, ccfDNA is able to predict the early therapeutic effects of biological DMARDs in RA patients.

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BackgroundEffects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts.MethodsUnder treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression.ResultsMTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high.ConclusionsWe present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1552-9) contains supplementary material, which is available to authorized users.

BackgroundDNA is fragmented and released into blood circulation as results of damage or death of cells from peripheral bloods as well as organ tissues, and circulating cell-free DNA (ccfDNA) has been recognized as biomarkers of several medical conditions [1]. In patients with rheumatoid arthritis (RA), serum concentration of ccfDNA has been reported to be elevated [2], however, the relation between ccfDNA and the pathogenesis of RA remains unclear.ObjectivesTo evaluate the correlation between ccfDNA in plasma and clinical disease activities in patients with RA.MethodsThe study group included 29 patients with RA who started biological DMARDs therapy. The concentration of ccfDNA in plasma was measured by quantitative real-time PCR at baseline to 24 weeks in every 4 weeks-period from 29 patients, and 21 healthy individuals. We also evaluated the correlation between ccfDNA and the clinical activities or the response for biological DMARDs, using disease activity score of 28 joints erythrocyte sedimentation rate (DAS28ESR) and the european league against rheumatism (EULAR) response criteria. In addition, synovial fluid samples of knee joint were collected from 14 patients with RA and12 with osteoarthritis (OA) to measure ccfDNAResultsThe concentration of ccfDNA in RA patients at baseline was higher than healthy controls (p=0.016).When evaluated the concentration of ccfDNA, it was increased until 8 weeks-period from the baseline, and then decreased after 12weeks-period. The average of DAS28ESR was improved in all patients enrolled. At 12weeks-period after treatment, 15 patients were good responders of the EULAR response criteria, 9 showed the moderate response and 5 showed no response. The concentration of ccfDNA in good responder was increased until 8weeks while those of moderate or no responder were not (P=0.042) (figure), and there was no significant difference between ccfDNA at baseline and other parameters in all patients. In joint fluid of RA patients, the concentration of ccfDNA was remarkably increased as compared to those from OA (P=0.00011).ConclusionsIncrease of ccfDNA at 8 weeks-period was associated with improvement of disease activity. Compared with the biomarkers reported before, ccfDNA is possible to predict the early therapeutic effects of biological DMARDs in RA patients.ReferencesJiajia Y, Guohao G, Shaoqing J. Recent Advances in Clinical Applications of Circulating Cell-free DNA Integrity. Lab medicine 2014;45:6–12Zhong XY, Muhlenen IV, Li Y, Kang A, Gupta AK, Tyndall A, et al. Increased Concentrations of Antibody-Bound circulatory cell-free DNA in rheumatoid arthritis. Clin Chem 2007;53:1609–1614Disclosure of InterestNone declared

Endogenous DNA is released into the bloodstream as cell-free DNA (cfDNA) following cell death and is associated with various pathological conditions. However, their association with therapeutic drugs against rheumatoid arthritis (RA) remains unknown. Therefore, we investigated the significance of cfDNA in RA treated with tocilizumab and tumor necrosis factor inhibitor (TNF-I). Biological DMARDs (bDMARDs), including tocilizumab and TNF-I, were administered to 77 and 59 RA patients, respectively. Plasma cfDNA levels were measured at weeks 0, 4, and 12 by quantitative polymerase chain reaction. Disease activity was evaluated at the same time point using DAS28ESR. cfDNA levels from RA synovial cells treated with tocilizumab or etanercept for 24 h were measured. Human toll-like receptor 9 (hTLR9)-expressing HEK293 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon NF-κB activation, were stimulated by cfDNA from RA patients, and subsequently, SEAP levels were determined. NF-κB translocation was evaluated by immunofluorescence staining with or without tocilizumab. The DAS28ESR significantly improved in both bDMARD groups at week 12. However, plasma cfDNA levels significantly decreased in the tocilizumab group at week 12 compared to that in week 0. cfDNA levels correlated with DAS28ESR in biological treatment-naïve patients administered tocilizumab. cfDNA levels in synovial cells were significantly suppressed by tocilizumab treatment and unaltered with etanercept. HEK293 cells released SEAP upon cfDNA stimulation, and the observed NF-κB nuclear translocation was suppressed by tocilizumab. Tocilizumab suppressed inflammation via the TLR9 pathway by decreasing cfDNA levels. Regulation of cfDNA may be a therapeutic target for RA.