Abstract
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-) β1 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 μg/ml) and anti-CD28 (2 μg/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% O2. Based on LPS (100 ng/ml) and hypoxia conditions (5% O2) as stimuli, MSCs were set as direct coculture or indirect coculture 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 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-γ, IL-17A, IL-21, TGF-β1, 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-β1, 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-β1 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-β1 modulating skewed Treg/Th17 induced by LPS-hypoxia challenge, providing evidence when proposing future T lymphocyte-targeted cell therapy in a specific condition.
Highlights
Regulatory T cell (Treg)/T helper (Th) 17 skewing is important in the development of acute respiratory distress syndrome (ARDS)
To figure out the impact of hypoxia, LPS, or combined stimulation on Treg and Th17 generation, CD4+ T cells with RAW264.7 cells added as antigenpresenting cells were cultured in vitro in the absence of or with LPS (10, 100, and 1000 ng/ml, respectively), hypoxia (5% O2), or combined stimulation for 48 hours
We found that TGFβ1 blocking by neutralization in mesenchymal stem cell- (MSC-)treated CD4+ T cells or blocking transforming growth factor- (TGF)-β1-mediated signaling by ALK5 inhibitor caused a reversal effect of MSCs on restoring Treg/Th17 imbalance induced by LPS and hypoxia challenge (P < 0:05, Figure 3), suggesting that MSCs improved Treg/Th17 exposed to LPS and hypoxia stimulation by increased TGF-β1 secretion
Summary
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) β1 on CD4+ T cells are environment-sensitive and lack discussion in hypoxic and inflammatory conditions. 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% O2. Based on LPS (100 ng/ml) and hypoxia conditions (5% O2) as stimuli, MSCs were set as direct coculture or indirect coculture by transwell system. These findings suggested miR-155 suppression of CD4+ T cells mediated MSC-secreted TGF-β1 modulating skewed Treg/Th17 induced by LPS-hypoxia challenge, providing evidence when proposing future T lymphocyte-targeted cell therapy in a specific condition.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.