Background: Mesenchymal stem cells (MSCs) have been recognized as a promising tool for the treatment of various inflammatory disorders and autoimmune diseases. Stress conditions affect immune-mediated treatment and activate autophagy in MSCs. However, whether autophagy affects the MSC-mediated recruitment and differentiation of CD4+ T cells remains elusive.
Methods: MSCs were pretreated with 3-methyladenine (3-MA) and rapamycin to regulate autophagy, and then co-cultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of well-known chemokines were analyzed by quantitative real-time PCR. Enzyme-linked immunosorbent assays and western blot analysis were performed to detect C-X-C motif chemokine ligand 8 (CXCL8) and transforming growth factor (TGF)-β1 protein levels. An exogenous antibody and short hairpin RNA were used to regulate CXCL8 and TGF-β1 levels, which enabled us to evaluate how autophagy affected MSC-mediated CD4+ T cell migration and differentiation.
Results: 3-MA inhibited autophagy in MSCs, which was activated by rapamycin. Rapamycin increased the migration of CD4+ T cells, whereas 3-MA decreased their migration. Mechanistically, we found that autophagy strengthened CXCL8 secretion, and the addition of exogenous CXCL8 and an anti-CXCL8 antibody eliminated the difference of CD4+ T cell migration among groups. Further, the ratio of regulatory T (Treg) cells was increased in rapamycin-pretreated MSCs, but the ratio of T helper 1 (Th1) cells was decreased, while pretreatment of MSCs with 3-MA induced the opposite effect compared with the control group. TGF-β1 overexpression and knockdown using lentiviruses rectified the differences in the ratios of Treg and Th1 cells among the groups.
Conclusion: This study demonstrates that autophagy of mesenchymal stem cells mediates CD4+ T cell migration and differentiation through CXCL8 and TGF-β1, respectively. These results provide a potential new strategy for improving MSC-mediated therapy.
Keywords: Autophagy; CD4+ T cells; Immunomodulatory; Mesenchymal stem cells; Migration.