The effects of miR-10a on the immune function of splenic CD4 + CD25 + Treg cells in septic mice

Abstract

Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis. Methods Sepsis mouse model was established by cecal ligation and puncture(CLP). Balb/c mice of clean grade were sacrificed 1, 3, 5, and 7 days after operation. Blood as well as spleen samples were harvested at given intervals. The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads. Cells were cultured, and phenotypes were analyzed by flow cytometry. The miR-10a expressed in Treg cells were detected by Real-time PCR. After administration of LV-mmu-miR-10a-5p-inhibition, the immunosuppressive function have been detected. Statistical analyses were performed using one-way analysis of variance (SPSS19.0, Chicago, USA) test followed by Dunnett-t test to compare among three or more groups or by Student’s t-test to compare between two groups. Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)% in normal group, and the increase in percentage of Tregs in spleen has been observed in septic mice (P<0.05). The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P<0.05). The expression of miR-10a was significantly elevated on CLP 1-7 day (P<0.05). After down-regulation of miR-10a in septic mice, the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P<0.05), the MFI of Foxp3+Treg was increased in septic mice compared with control group (P<0.05). The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P<0.05).After down-regulation of miR-10a in septic mice, the CD4+T cell proliferative activity was significantly suppressed compared with control group (P<0.05). Conclusions Treg plays a critical role in immunosuppression in septic mice. Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg. Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy . Key words: Regulatory T cells; Sepsis; MicroRNA-10a; Forkhead/winged helix transcription factor p3; Immune function; Effector T cell; Proliferative activity; Mean fluorescence intensity