Mesenchymal Stem Cells-Secreted TGF-β1 Restores Treg/Th17 Skewing Induced by Lipopolysaccharide and Hypoxia Challenge via miR-155 Suppression
Background: Regulatory T cells (Treg)/T helper (Th) 17 skewing plays a crucial role in development of acute respiratory distress (ARDS). Mesenchymal stem cells (MSCs)-secreted transforming growth factor (TGF)-β1 has remarkable immunomodulatory effects on CD4+T cells, being environment sensitive and remains lack of discussion in hypoxic and inflammatory condition of ARDS.Methods: Purified mice splenic CD4+ T cells were pre-coated with anti-CD3 (5ug/ml) and anti-CD28 (2ug/ml) overnight. RAW264.7
... vernight. RAW264.7 cells were added as antigen presenting cells (APCs). T cells with and without RAW264.7 cells were treated with various LPS concentrations of 0,10,100,1000ng/ml or/and at hypoxia condition of 5% O2. Based on LPS (100ng/ml) and hypoxia condition (5% O2) as stimuli, MSCs were set in direct coculture or indirect coculture methods by transwell system. Anti-TGF-β1 neutralization antibody was added to explore the role of TGF-β1 among the soluble factors secreted by MSCs; miR-155 overexpression of CD4+T cells were performed by transfection and then were added to the MSCs-CD4+T cells coculture system in hypoxic and LPS-stimulated condition. After 48 hours, cells or supernatant were collected for detection of frequency of Treg and Th17 subsets, CD4+T cells apoptosis and proliferation capacity assay by flowcytometry, secretions of INF-γ, IL-17A, IL-21, TGF-β1 and IL-10 by ELISA, levels of miR-155, Rorc, Foxp3 and Ptpn2 mRNA expression of CD4+T cells by RT-PCR.Results: MSCs could restore the skewed Treg/Th17 induced by LPS and hypoxia as compared to groups without MSCs with increased secretion of TGF-β1, IL-10 and IL-17A(P<0.05) and attenuate the increased expression of miR-155 in CD4+T cells, which was independent on cell-to-cell contact mechanism while TGF-β1 neutralization could significantly inhibit the effects of MSCs restoring the skewed Treg/Th17 and abolished its effect on miR-155 expression in CD4+T cells.Conclusions: These findings suggested miR-155 suppression of CD4+T cells mediated MSCs-secreted TGF-β1 modulating the skewed Treg/Th17 induced by LPS-hypoxia challenge, providing evidence when proposing future T lymphocyte-targeted cell therapy in ARDS.