Overexpression of TGFβ1 in murine mesenchymal stem cells improves lung inflammation by impacting the Th17/Treg balance in LPS-induced ARDS mice

Abstract

<jats:title>Abstract</jats:title> <jats:p>Background: T helper 17 cells (Th17) /regulatory T cells (Treg), as subtypes of CD4 + T cells, play an important role in the inflammatory response of acute respiratory distress syndrome (ARDS). However, there is still a lack of effective methods to regulate the differentiation balance of Th17/Treg. It was proven that mesenchymal stem cells (MSCs) could regulate the differentiation of CD4 + T cells, but the mechanism is still unclear. TGFβ1, a paracrine cytokine of MSCs, could also regulate the differentiation of Th17/Treg but is lowly expressed in MSCs. Therefore, mouse MSCs (mMSCs) overexpressing TGFβ1 were constructed by lentivirus transfection and intratracheally transplanted into LPS-induced ARDS mice in our study. The aim of this study was to evaluate the therapeutic effects of mMSCs overexpressing TGFβ1 on inflammation and immunoregulation by impacting the Th17/Treg balance in LPS-induced ARDS mice. Methods: mMSCs overexpressing TGFβ1 were constructed using lentiviral vectors. Then, mouse bone-marrow-derived MSCs (mBM-MSC) and mBM-MSC-TGFβ1 (mBM-MSC overexpressing TGFβ1) were transplanted intratracheally into ARDS mice induced by lipopolysaccharide. At 3 d and 7 d after transplantation, the mice were sacrificed, and the homing of the mMSCs was assayed by ex vivo optical imaging. The relative numbers of Th17 and Treg in the lungs and spleens of mice were detected by FCM. IL-17A and IL-10 levels in the lungs of mice were analysed by western blot. Permeability and inflammatory cytokines were evaluated by analysing the protein concentration of BALF using ELISA. Histopathology of the lungs was assessed by haematoxylin and eosin staining and lung injury scoring. Alveolar lung fibrosis was assessed by Masson’s trichrome staining and Ashcroft scoring. The mortality of ARDS mice was followed until 7 days after transplantation. Results: The transduction efficiencies mediated by the lentiviral vectors ranged from 82.3-88.6%. Overexpressing TGF-β1 inhibited the proliferation of mMSCs during days 5-7 ( p &lt;0.05) but had no effect on mMSCs differentiation or migration ( p &gt;0.05). Compared to that in the LPS+mBM-MSC-NC group mice, engraftment of mMSCs overexpressing TGFβ1 led to much more differentiation of T cells into Th17 or Treg ( p &lt;0.05), improved permeability of injured lungs ( p &lt;0.05) and ameliorative histopathology of lung tissue in ARDS mice ( p &lt;0.05). Moreover, IL-17A content was also decreased while IL-10 content was increased in the LPS+mBM-MSC-TGFβ1 group compared with those in the LPS+mBM-MSC-NC group ( p &lt;0.05). Finally, mMSCs overexpressing TGFβ1 did not aggravate lung fibrosis in ARDS mice ( p &gt;0.05). Conclusion: MSCs overexpressing TGFβ1 could regulate lung inflammation and attenuate lung injuries by modulating the imbalance of Th17/Treg in the lungs of ARDS mice.</jats:p>

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

“…By establishing an in vitro coculture system with LPShypoxia stimulation on CD4 + T cells, we found that MSC coculture restored skewed Treg/Th17 and significantly augmented the anti-inflammatory cytokine secretion. We have proved that MSCs overexpressing TGF-β1 could regulate lung inflammation and attenuate lung injuries by modulating the imbalance of Th17/Treg in the lungs of ARDS mice [12]. Establishing an in vitro cell coculture model with LPS and hypoxia challenge would promote our knowledge in the interaction between Treg and Th17 cell skewing and MSCs in a specific condition.…”

“…Coculture System. Mouse bone marrow MSCs were purchased from Cyagen Biosciences Inc. (Guangzhou, China), which has been identified by detecting cell surface markers with ≥90% positivity for expression of cell surface antigens CD29, CD44, and CD105 and ≤5% positivity for expression of cell surface antigens CD45 and CD34 [12], and cells were 2…”

“…Accordingly, Tregs, characterized by forkhead box P3 (Foxp3), contribute to the resolution of the hyperinflammatory responses by regulating responses mediated by T helper cells. MSCs have been shown regulating Treg/ Th17 balance in Th17 dominance multiple disease models such as systemic lupus erythematosus and ARDS [11,12]. However, Treg/Th17 skewing upon the hypoxic and inflammatory environment in vitro along with effects of MSCs remains not completely discussed.…”

“…Meanwhile, TGF-β1 is a critical and initiative factor for both Th17 and Treg development by inducing the expression of the transcription factors retinoic-acid-receptor-related orphan receptor γt (RORγt) and Foxp3, respectively [17,18]. In an ARDS mouse model, MSCs overexpressing TGF-β1 could regulate lung inflammation and attenuate lung injuries by modulating the imbalance of Th17/Treg in the lungs [12]. Thus, MSC-generated TGF-β1 could theoretically restore skewed Treg/Th17 in inflammatory or hypoxia condition.…”

Vitamin D has an effect on airway inflammation and Th17/Treg balance in asthmatic mice

“…11,13 Therefore, the balance of the Th17/Treg is critical in the inflammatory response to ameliorate lung inflammation by a uniform balance of Th17/Treg. 14,15 A deficiency of vitamin D can impede the downregulation of Th17 cells and the upregulation of Treg cells, thus unbalancing the proportion of Th17/Treg. 16 Currently, no investigation has determined whether vitamin D can regulate the airway inflammation of asthmatic mice by regulating the balance of Th17/Treg.…”