Intravenous Administration Of Human Mesenchymal Stem Cells In Experimental Autoimmune Myasthenia Gravis Animals

Background:Myasthenia gravis (MG) is a T-cell dependent, acetylcholine receptor (AchR) antibody-mediated complex autoimmune neuromuscular disease, which is associated with the imbalanced regulation of humoral and cellular AChR-specific immune responses. Blocking or loss of AChR leads to an impaired neuromuscular transmission, resulting in muscle weakness and fatigue. The pathogenesis of MG involves the hyperactivity of various immune cells, activation of complement system, deficiency of immunomodulation and impairment of immune homeostasis. It has been suggested that a dysfunction of stromal progenitors, endothelial progenitors and bone marrow microenvironment usually occurs in autoimmune diseases. Mesenchymal stem cells (MSC) are resident of the microenvironment, and also, they are able to give rise to other cellular constituents of the microenvironment including reticular cells, adipocytes and osteoblasts. MSC could express various molecules such as intercellular adhesion molecule (ICAM)-1, ICAM-2, lymphocyte function-associated antigen (LFA) 3, fibronectin, lamin and collagen, which are involved in the process of immune reaction. It has been reported that MSC are able to suppress T lymphocytes activation and proliferation in mixed lymphocyte reaction experiment. The underlying mechanisms relate with their ability to secrete bioactive cytokines in addition to their direct physical contact with T lymphocytes. Furthermore, MSC are capable of modulating the functions of other immune cells such as natural killer cells, dendritic cells and B cells. Therefore, MSC can be potentially used for immunomodulation and correction of impaired immune homeostasis in diseases like MG.Objective:This study was designed to determine the potential of human MSC (hMSC) in improving the functional deficits of experimental autoimmune myasthenia gravis (EAMG) and the related mechanisms.Methods:EAMG animal models were established by subcutaneous injection of synthetic analogue of acetylcholine receptor (AchR), then, hMSC were intravenously delivered into these mice repeatedly. We studied the proliferation level of AChR-specific T cells both in vivo and in vitro. Also, we investigated the level of Treg cell (CD4+CD25+) and the expression of foxp3 on CD4 T cells by immunofluorescent flow cytometry. Real-time PCR was used to test the levels of cytokines and costimulatory factors. The homing of hUC-MSC was studied by in vivo live imaging and immunofluorescent microscopy. ELISA and ELISPOT methods were used to test AchR antibodies both in vivo and in Results:we used two administration methods. Method one was 1×106 hMSC administrated after the first immunity. Method two was intravenous administration of 1 X 107 hMSC began on day 1,3,5 after the second immunity. we found that 1 X 106 hMSC administrated after the first immunity could specifically home to spleen tissue and hMSC treatment significantly improved the functional deficits of EAMG mice. In addition, AchR antibody level was dramatically decreased in cell-treated group when compared with untreated control at 10 days after the second cell injection. Moreover, both in vivo and in vitro mixed lymphocyte proliferation assays revealed that hMSC could definitely inhibit the proliferation of AchR-specific lymphocyte. We found that hMSC enhance foxp3 expression on CD4+T and rise Treg cells number. In addition, hMSC diminish LFA-1/ICAM-1 on CD4+T in vitro/vivo and affect mRNA expression of IP-10 and TNF-a/IL-10 on CD4+T.Conclusion:Our study demonstrated that hMSC treatment began at early stage was therapeutically useful in autoimmune myasthenia gravis mice, and the underlying mechanism may relate with their immunomodulatory potential.