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

Abstract

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<0.05) but had no effect on mMSCs differentiation or migration (p>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<0.05), improved permeability of injured lungs (p<0.05) and ameliorative histopathology of lung tissue in ARDS mice (p<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<0.05). Finally, mMSCs overexpressing TGFβ1 did not aggravate lung fibrosis in ARDS mice (p>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.

Immunosuppressive effect of mesenchymal stem cells on lung and gut CD8+ T cells in lipopolysaccharide‐induced acute lung injury in mice

“…The immunomodulatory effects of MSCs in the respiratory system involve T-cell differentiation. 32 In this study, the therapeutic effects of MSCs were found to be mediated by reduced infiltration of Ly6C + CD8 + T cells, in line with the lower IFNγ level in the LPS/ MSC group, since excessive IFNγ synthesized by large numbers of CD8 + T cells can aggravate tissue injury. 50 Moreover, enhanced macrophage phagocytosis in MSC-treated mice contributed to repair of injured tissue, which is consistent with a prior report.…”

“…The anti-inflammatory properties of MSCs have been described. 32,47 In this study, MSCs ameliorated inflammatory response in BALF, as indicated by decreased levels of IFNγ, TNFα, IL-1α, IL-1β and IL-6. Our results are consistent with the previous studies on the anti-inflammatory effects of MSCs in ALI.…”

“…The anti‐inflammatory properties of MSCs have been described 32,47 . In this study, MSCs ameliorated inflammatory response in BALF, as indicated by decreased levels of IFN‐γ, TNF‐α, IL‐1α, IL‐1β and IL‐6.…”

“…Modulation of the mucosal immune response shows promise for alleviating the pulmonary and intestinal inflammatory injury induced by LPS. The immunomodulatory effects of MSCs in the respiratory system involve T‐cell differentiation 32 . In this study, the therapeutic effects of MSCs were found to be mediated by reduced infiltration of Ly6C + CD8 + T cells, in line with the lower IFN‐γ level in the LPS/MSC group, since excessive IFN‐γ synthesized by large numbers of CD8 + T cells can aggravate tissue injury 50 .…”

“…Another set of mice was sacrificed. The lungs were washed three times with 800 µl ice‐cold PBS through a tracheal cannula as described previously, 32 and the bronchoalveolar lavage fluid (BALF) was collected and centrifuged at 800 g for 10 min at 4°C. The supernatant was stored at −80°C for subsequent assay of cytokines and protein concentration, and the cell pellet was resuspended in 200 μl for cell counting and cell smear generation.…”