Altered expression of microRNAs in interleukin-17 producing T cells in rheumatoid arthritis patients +1Takuya Niimoto ; 1Tomoyuki Nakasa ; 1Masakazu Ishikawa; 1Osami Suzuki

1Nobuo Adachi; 1Masataka Deie; 1Mitsuo Ochi +1 Hiroshima University, Hiroshima, Japan

ochim@hiroshima-u.ac.jp ABSTRACT INTRODUCTION:

MicroRNA (miRNA)s are a family of non coding RNAs identified in organisms ranging from nematodes to humans. Several miRNAs exhibit a tissue-specific or developmental stage–specific expression pattern and have been reported to be associated with human diseases. The discovery of a new linage of CD4+effector T helper type17 cells (Th17cells) that selectively produce IL-17 has provided exciting new insights into immune regulation, host defense, and pathogenesis of autoimmune and other chronic inflammatory disorders including of rheumatoid arthritis (RA). In several studies, miRNAs play a role in RA pathogenesis. The purpose of this study is to identify miRNAs about differentiation of the IL -17 producing T cells, and to analyze their expression pattern in the RA patients. METHODS:

Human blood was collected from 5 healthy volunteers (31.8 ±1.1 years of age, mean ± SD). Isolation of human peripheral blood mononuclear cells (PBMC) and CD4+ T cells were isolated from PBMC using auto MACS(CD4+T cell Isolation kit, Miltenyi Biotec, ). Expansion of IL-17 producing T cells in human PBMC by IL -1 β, IL-2, IL -6, IL -23. After confirmation these cells production of IL -17 by real time PCR and ELISA, miRNA microarray was performed. Furthermore, real time PCR for identified miRNAs was performed in PBMC and synovium in RA (Table 1)and osteoarthritis (OA) patients. Moreover, immunohistochemistry of IL-17 in RA and OA synovium demonstrated that RA synovium have abundant Th17 cells.  RESULTS

As a result of microarray in expanded IL-17 producing T cells, six miRNAs were significantly up regulated in the differentiation of the IL-17 producing T cells. Six miRNAs were miR-26a (1.81 fold; p =0.029), 146a (1.43 fold; p =0.040), 146b (1.80 fold; p =0.040), 150 (1.61 fold; p =0.040), 155 (2.03 fold; p =0.007) and let-7a (2.12 fold; p =0.012). These miRNAs expression level in synovium and PBMC was significantly higher in RA patients（Fig.1, 2）. Especially, with intensely expression of IL-17 in PBMC and synovium, these miRNAs intensely expressed. DISCUSSION

Recently, a potential link between microRNA and several human diseases has been examined. In addition, the recent report that expression of microRNA-146 in RA patient's synovial tissue was performed, and the participation of some microRNA was suggested in the cause of the RA appearance of disease. Moreover, the discovery in mice of a new lineage of CD4+ effector T helper (Th) cells that selectively produce IL-17 has provided exciting new insights into immune regulation, host defence, and the pathogenesis of autoimmune and other chronic inflammatory disorders. A few studies suggest that IL-17may have a role in the pathogenesis of human RA as well. We thought whether became it to be a new treatment method to RA being able the control of microRNA related to IL-17.

In this research, first of all, we expanded the IL-17 producing T cells from healthy volunteer’s PBMC, and analyzed microRNAs by using the Microarray analysis. As a result, it was guessed that microRNA by which high expression of let-7a, miR-26a, 146a,b, 150, and 155 were seen, and these were related to IL-17. In the RA patients, the appearance of miR-146a.b and 155 have already been reported to be high. We wonder it is because these microRNA depends on the expression of IL-17 producing T cells as this reason. We did not isolate IL-17 producing T cells but expanded. In this point, there might be a limit of this research. However, after it had been concluded IL-17 producing T cells by real time PCR and ELISA, the expanded T cell was analyzed with miRNA microarray. As for the selected miRNAs, expression was intentionally seen from OA by RA in both PBMC and synovium. We are guessing whether these miRNAs related to the expression of IL-17 producing T cells. If the expression of IL-17 producing T cells were able to be suppressed by using these

miRNAs, we thought these mechanisms may be potential targets for the development of new RA therapies.

Table 1; RA patient list Peripheral blood was taken from 6 RA patients (67.3 ± 8.02 years of age, mean ± standard deviation (SD)). They were diagnosed according to the American Rheumatism Association Criteria for RA. Synovial tissues were obtained at the time of total knee arthroplasty on 4 RA patient and patient RA8 and RA9 were obtained at the time of synovectomy (67.7±10.4 years of age, mean ± SD).

Fig.1: miR-26a, 146a, 146b, 150, 155 expression in PBMC of healthy, OA, RA patients

Fig.2: miR-146a/b, miR-150, and miR-155 intensely expressed in RA synovium compared to OA synovium.

Poster No. 935 • 56th Annual Meeting of the Orthopaedic Research Society