Bone marrow mesenchymal stem cells suppress activated CD4+ T cells proliferation through TGF-beta and IL10 dependent of autophagy in pathological hypoxic microenvironment

Bone marrow mesenchymal stem cells (BMSCs) mediated immunomodulation by secreting certain bioactive cytokines has been recognized as a promising approach for disease treatment. However, microenvironmental oxygen tension affect immunomodulatory functions and activate autophagy in BMSCs. The mechanism governing BMSCs immunomodulation in hypoxia hasn't been expounded clearly. The aim of this study is to investigate the function of pathological hypoxia on immunomodulatory properties of bone marrow mesenchymal stem cells and its possible mechanism. BMSCs were cultured in either normoxia (21 % oxygen) or hypoxia (0.1 % oxygen) for 24 hours, then electron microscopy (EM) and immunofluorescence staining were used to detect the activation of autophagy. Besides autophagy-related markers were monitored by Western blotting. Atg5 siRNA induced autophagic inhibition. Additional, gene expression levels of Real-time fluorescence quantitative PCR and Western blot were used to detect BMSCs related cytokines. Both the proliferation and apoptosis of CD4+ T cell in co-culture were detected by flow cytometry. Exogenous anti-IL-10 antibody and anti-TGF-β1 antibody were used in co-cultured BMSCs-CM and CD4+ T cells, which enabled us to assess how autophagy affected BMSCs-mediated CD4+ T cell proliferation in low oxygen tension. Compared with normal BMSCs, Hypo-BMSCs enhanced the immunosuppressive effect of BMSCs on CD4+ T cell proliferation, while si-atg5 weakened the inhibition of Hypo-BMSCs. Furthermore, exogenous anti-TGF-β1 antibody and the addition of anti-TGF-β1 antibody reversed the immunosuppressive ability of Hypo-BMSCs. Our findings reveal that BMSCs possess significant immunosuppression on CD4+T cell through IL-10 and TGF-β1 dependent of autophagy in hypoxic microenvironment.