Silencing MiR-146a Targeting B Cells Ameliorates The Experimental Autoimmune Myasthenia Gravis

Objective:To observe the therapeutic effect of antagomiR-146a by silencing miR-146a in B cells of experimental autoimmune myasthenia gravis (EAMG), further more to explore of the possible immunomodulatory mechanisms involved.Methods:First to induce EAMG, C57BL/6mice were immunized subcutaneously with R97-116peptide in CFA at day0,30and60. Then30EAMG mice with more than1.5clinical score were chose at day70and enrolled randomly into the following three groups:(1) AntagomiR-146a group:AntagomiR-146a at20mg/kg dose dissolved in200μL PBS solution was used for treatment.(2) NC group:AntagomiR negative control (NC) at the same dose dissolved in200μL PBS solution was used for treatment.(3)PBS group:200μL PBS solution was used for treatment. The EAMG mice above all were administered with respective treatment drug via the caudal vein for continuous three days. Clinical assessment and myasthenic score of the mice in each group were finished every other day. At the same time the serum specimens were collected to detect the secretion level of anti-R97-116total IgG and its subtypes by ELISA. At day10after the treatment, the mice were sacrificed and spleen cells were collected. Lymphocyte proliferative response was measured with CFSE, frequency of B cell subsets were detected by flow cytometry, while the expression of miR-146a and its possible target genes TRAF6, IRAK1in mouse B cells were detected by qPCR.Results:(1) Treatment with AntagomiR-146a effectively reduced the average scores of myasthenic symptoms, as well as the levels of serum anti-R97-116antibodies such as IgG, IgG1and IgG2b, rather then IgM, when compared with NC and PBS groups.(2) Low proliferative response were observed in co-cultured lymphocytes from antagomiR-146a group with T-AChR and R97-116, while the lymphocytes from the NC group and PBS group cultured with the same antigen stimulation presented high proliferative responses.(3) B1cells, memory B cells and plasma cells were significantly reduced in mice from antagomiR-146a group compared with the NC group and PBS group, the same as the expression of CD40, CD80and CD86on B cells.(4) The expression of miR-146a in B cells was significantly reduced in antagomiR-146a group compared with the NC group and PBS group.(5) The expression differences of TRAF6and IRAK1in mouse B cells from three groups were not statistically significant.Conclusion:(1) AntagomiR-146a could effectively silence the expression of miR-146a in B cells, and then effectively alleviate myasthenic symptoms in mice with ongoing EAMG, with the impaired expression and class switch of serum anti-R97-116antibodies.The therapeutic effect was associated with reduced antigen-specific lymphocyte proliferation, decreased number of B1cells and obstacle of B cells differentiation into memory B cells and plasma cells.(2) TRAF6and IRAK1were not the targets for the miR146a-induced immunomodulation in B cells of EAMG Abnormal expression of miR-146a might act on other potential targets to control the mechanisms of EAMG/MG...