TCZ Modulates the Production of Ccfdna Derived from RA Synovial Cells

Naonori Hashimoto¹, Kohsuke Yoshida¹, Teppei Hashimoto², Ayako Nakai¹, Kenta Kaneshiro¹, Kohjin Suzuki¹, Yoshiko Kawasaki², Nao Shibanuma³, Natsuko Nakagawa⁴, Yoshitada Sakai⁵ and Akira Hashiramoto⁶, ¹Department of Biophysics, Kobe University Graduate School of Health Sciences, Kobe, Japan, ²Department of Rheumatology, Kobe Kaisei Hospital, Kobe, Japan, ³Departmant of Orthopaedic Surgery, Kobe Kaisei Hospital, Kobe, Japan, ⁴Department of Orthopaedic Surgery, Konan-Kakogawa Hospital, Kakogawa, Japan, ⁵Division of Rehabilitation Medicine, Kobe University Graduate School of Medicine, Kobe, Japan, ⁶Department of Biophysics, Department of Biophysics, Kobe University Graduate School of Health Sciences, Kobe, Japan

Meeting: 2016 ACR/ARHP Annual Meeting

Date of first publication: September 28, 2016

Keywords: Biomarkers, DNA, Rheumatoid arthritis (RA), synovial cells, synovial fluid and tocilizumab

Session Information

Date: Sunday, November 13, 2016

Title: Rheumatoid Arthritis – Human Etiology and Pathogenesis - Poster I

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose: DNA is fragmented and released into blood circulation as a circulating cell-free DNA (ccfDNA) because of damage or death of cells. Although the lower concentration of ccfDNA is present in peripheral bloods from healthy individuals, significantly higher amount of those are detected in cancer patients or pregnant women (1). Thus, ccfDNA has recently been recognized as biomarkers of several medical conditions. In this study, we measured the amount of ccfDNA in peripheral bloods from RA patients and healthy controls, and in synovial fluid samples of knee joint from RA patients and osteoarthritis (OA) patients. Moreover, we investigated the mechanism of ccfDNA production and found that TCZ inhibited the production of ccfDNA derived from RA synovial cells.

Methods: The amount of ccfDNA was measured by qPCR in peripheral bloods from 29 patients with RA, and 21 healthy controls., and in synovial fluid of knee joint from 13 patients with RA, and 12 patients with OA. By using human synovial cells, culture-supernatants were collected in each steps of cellular-confluency; 40% to 120%. In addition, after stimulated with interleukin(IL)6/soluble IL6 receptor(sIL6R) or TNFα, synovial cells were further treated with and without tocilizumab(TCZ: 100μg/mL) or etanercept(ETN: 10μg/mL) to collect supernatants. When examining ccfDNA in supernatants by qPCR, we amplified both short and long DNA fragments using ALU115-bp primer and ALU247-bp primer to distinguish apoptotic fragments from non-apoptotic fragments, respectively. Viabilities of synovial cells were also examined by WST-8 assay.

Results: The amount of ccfDNA in RA patients was significantly increased as compared to healthy controls, and to OA patients. In supernatants, amounts of ccfDNA (ALU115 and ALU247) increased before 100% confluency cultured-state, and decreased after 100% confluency. Amounts of ccfDNA (ALU115 and ALU247) were significantly suppressed by TCZ treatment, while those with ETN were not changed. Notably, cellular viabilities showed no significant difference between non-treated and biological DMARDs-treated groups, and the ratio of ALU247 to ALU115 was significantly reduced by TCZ.

Conclusion: Results clearly showed that ccfDNA was a diagnostic biomarker for RA in both peripheral blood and joint fluid, and suggesting that ccfDNA in peripheral blood was derived from synovial tissue. Another source of ccfDNA was considered to be physiological cell division because the amount of ccfDNA was increased by cell proliferation and decreased in the growth inhibition by cell-to-cell contact. Thus it appeared that TCZ inhibit the physiological cell division and the ratio of ALU247 to ALU115 could represent the therapeutic response for TCZ, since ALU115 represents the total amount of ccfDNA and ALU247 specifically represents those released by cell division. References: (1) T B Hao, et al. 2014 Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer. British Journal of Cancer 111(8):1482-9

Disclosure: N. Hashimoto, None; K. Yoshida, None; T. Hashimoto, None; A. Nakai, None; K. Kaneshiro, None; K. Suzuki, None; Y. Kawasaki, None; N. Shibanuma, None; N. Nakagawa, None; Y. Sakai, None; A. Hashiramoto, None.

To cite this abstract in AMA style:

Hashimoto N, Yoshida K, Hashimoto T, Nakai A, Kaneshiro K, Suzuki K, Kawasaki Y, Shibanuma N, Nakagawa N, Sakai Y, Hashiramoto A. TCZ Modulates the Production of Ccfdna Derived from RA Synovial Cells [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/tcz-modulates-the-production-of-ccfdna-derived-from-ra-synovial-cells/. Accessed .

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