Correlation between the frequency of Th17 cell and the expression of microRNA-206 in patients with dermatomyositis

Abstract

It was reported that IL-17 had been detected in the inflammatory infiltrates of patients with DM (dermatomyositis). In this study, we investigated the frequency of Th17 cells and the expression of microRNA-206 (miR-206) in DM patients. Firstly, we observed that the frequency of Th17 cells and the expression of transcription factors were increased significantly in the PBMCs of DM patients. Secondly, we found that there was a positive correlation between the percentages of Th17 cells and serum level of CK in DM patients. And the serum concentrations of IL-6, IL-1β, TGF-β, and IL-23, the important cytokines of Th17 differentiation, were increased in DM patients. It was predicted that Krüppel-like factor 4 (KLF4) is one of the multiple targets of miR-206. We detected the expression of miR-206 in DM patients, and it was decreased in the serum and PBMCs of DM patients. The augmented expression of KLF is accompanied by the attenuated expression of miR-206. Furthermore, a negative correlation between the percentages of Th17 cells and the expression of miR-206 in DM patients has been found. Taken together, these findings suggest the attenuated expression of miR-206, and the augmented frequency of Th17 cells in DM patients.

Figure 1

Th17 cells in the peripheral blood from DM patients. PBMCs from DM patients and healthy controls were incubated with PMA/ionomycin, stained for cell surface molecules CD3 and CD8 as well as intracellular IL-17, and analyzed by flow cytometry. (a) Representative dot plots from DM patient and a control subject are shown. Values correspond to the percentage of Th17 lymphocytes. We used isotype controls to determine the positive cells, and all the values are gated on the CD3+CD8− cells. (b) Percentages of Th17 lymphocytes were compared between DM patients and control subjects. (c) Levels of IL-17 mRNA in PBMCs pretreated with PMA/ionomycin from DM patients and controls. (d) The levels of RORC mRNA in PBMCs were detected by real-time PCR from DM patients and healthy controls. (e) The levels of KLF4 mRNA in PBMCs were detected by real-time PCR from DM patients and healthy controls. (f) The correlation between the percentages of Th17 lymphocytes and the expression of KLF4 mRNA in PBMCs from DM patients. Each data point represents an individual subject; horizontal lines show the mean. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

The correlation between the percentages of Th17 lymphocytes in PBMCs and LDH (left)/CK (right) in DM patients.

Figure 3

Serum levels of proinflammatory cytokines are increased in DM patients compared with healthy controls. (a) Serum levels of IL-6 were determined by ELISA in serum samples from DM patients and controls. (b) Serum levels of IL-1β were determined by ELISA in serum samples from DM patients and controls. (c) Serum levels of TGF-β were determined by ELISA in serum samples from DM patients and controls. (d) Serum levels of IL-23 were determined by ELISA in serum samples from DM patients and controls. Horizontal lines show the mean. **P < 0.01; ns: no significant differences.

Figure 4

Levels of miR-206 were decreased in DM patients compared with healthy controls. (a) Serum levels of miR-206 were determined by real-time PCR in serum samples from DM patients and controls. (b) The level of miR-206 in PBMCs was detected by real-time PCR from DM patients and controls. (c) The correlation between the expression of miR-206 and the expression of KLF4 mRNA in DM patients. (d) The correlation between the percentages of Th17 lymphocytes in PBMCs and the expression of miR-206 in DM patients. Horizontal lines show the mean. *P < 0.05.