Mesenchymal Stem Cell-Secreted TGF-β1 Restores Treg/Th17 Skewing Induced by Lipopolysaccharide and Hypoxia Challenge via miR-155 Suppression

Background. Regulatory T cell (Treg)/T helper (Th) 17 skewing is important in the development of acute respiratory distress syndrome (ARDS). Immunomodulatory effects of mesenchymal stem cell- (MSC-) secreted transforming growth factor- (TGF-) beta 1 on CD4(+) T cells are environment-sensitive and lack discussion in hypoxic and inflammatory conditions. Methods. Mouse splenic CD4(+) T cells were precoated with anti-CD3 (5 mu g/ml) and anti-CD28 (2 mu g/ml) overnight. 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, and 1000 ng/ml or/and at hypoxia condition of 5% O-2. Based on LPS (100 ng/ml) and hypoxia conditions (5% O-2) as stimuli, MSCs were set as direct coculture or indirect coculture by transwell system. Anti-TGF-beta 1 neutralization antibody was added to explore the role of TGF-beta 1 among the soluble factors secreted by MSCs; miR-155 overexpression of CD4(+) T cells was performed by transfection, and then, cells were added to the MSC-CD4(+) T cell coculture system in hypoxic- and LPS-stimulated condition. After 48 hours, cells or supernatants were collected for detection of frequency of Treg and Th17 subsets, CD4(+) T cell apoptosis and proliferation capacity assay by flow cytometry, secretion of INF-gamma, IL-17A, IL-21, TGF-beta 1, and IL-10 by ELISA, and levels of miR-155, Rorc, Foxp3, and Ptpn2 mRNA expression of CD4(+) T cells by RT-PCR. Results. MSCs could restore skewed Treg/Th17 induced by LPS and hypoxia compared to groups without MSCs with increased secretion of TGF-beta 1, IL-10, and IL-17A (P < 0.05) and attenuate the increased expression of miR-155 in CD4(+) T cells via cell-to-cell contact mechanism while TGF-beta 1 neutralization significantly inhibited the effects of MSCs restoring 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 MSC-secreted TGF-beta 1 modulating skewed Treg/Th17 induced by LPS-hypoxia challenge, providing evidence when proposing future T lymphocyte-targeted cell therapy in a specific condition.

Automated summary

This study found that under hypoxic and inflammatory conditions, the transforming growth factor-β1 (TGF-β1) secreted by mesenchymal stem cells (MSCs) can regulate the Treg/Th17 skewing induced by lipopolysaccharide (LPS) and hypoxia by inhibiting the expression of miR-155. These findings provide evidence for future T lymphocyte-targeted cell therapy in specific conditions.