Mesenchymal Stem Cells Affects The Lung Inflammation And Immune Function Via TGF?1 Paracrine Impacting Th17/Treg Balance In LPS-induced ARDS Mice

Background The imbalanced inflammatory responses are the basic pathophysiological process in the early phase of acute respiratory distress syndrome(ARDS).It plays an important role that balancing the differentiation of pro-inflammatory cells and anti-inflammatory cells for the treatment of ARDS.CD4~+T cells as important effector cells of adaptive immunity,Th17/Treg,the subgroup of which,makes a difference in the dysregulated inflammatory response in the early phase of ARDS.Previous studies have shown that mesenchymal stem cells(MSCs)could effectively improve the inflammatory response of ARDS,and inhibited the differentiation of Th17 and Treg.However,the mechanisms of which are still unknown.Transforming growth factor?1(TGF?1),which could also be secreted by MSCs,is one of the main regulatory cytokines of Th17/Treg differentiation.Thus,it is speculated that regulating the paracrine of TGF?1 could improve the therapeutic effects of MSCs for ARDS with attenuated inflammation in injured lungs,and the mechanism of which was mainly due to the regulation on the imbalance of Th17/Treg.In this study,overexpression of TGF?1 in mBM-MSC was constructed by gene transfection and then transplanted into LPS-induced ARDS mice intratracheally or intravenously.The pathology of injured lungs,expression of CD4~+T cell subsets and inflammatory factors were detected,respectively.The purpose of this study was to investigate the effect of MSCs on Th17/Treg differentiation and then the local and systematic immune functions of ARDS mice through its paracrine of TGF?1.Part ? Effects of overexpressing TGF?1 on the proliferation,differentiation and migration of murine bone?marrow?derived mesenchymal stem cellsObjectives To evaluate the effects of TGF?1 overexpression by lenti-virus transfection on the proliferation,differentiation,and migration of murine bone-marrow-derived MSCs(mBM-MSC).Methods The plasmid carrying TGF?1 was transfected into mBM-MSC and the transfection efficiency of mBM-MSC was identified by fluorescence microscopy,and flow cytometry analysis(FCM)for the percentage of green fluorescence protein(GFP)positive cells.The m RNA and protein levels in mBM-MSC-TGF?1(mBM-MSC overexpressing TGF?1)were detected by q RT-PCR,enzyme-linked immunosorbent assay(ELISA)and Western blot,respectively.The multipotential differentiation abilities of mBM-MSC were evaluated by differentiation kit.The proliferation of mBM-MSC was analyzed by cell counting kit-8 and the migration abilities were determined by scratch assay and transwell assay.Results It was showed that the transduction efficiencies of TGF?1 mediated by lentiviral vectors were 82.3-88.6%.TGF?1 m RNA expression,TGF?1 protein in mBM-MSC and the secreted TGF?1 protein in the supernatant of mBM-MSC was also significantly elevated in the mBM-MSC-TGF?1 group than in the mBM-MSC-NC(mBM-MSC normal control)group(p<0.0001).Besides,there were no significant differences in the multi-differentiation abilities among the groups.Moreover,overexpressing TGF?1 significantly inhibited the proliferation of mBM-MSC(p<0.05)during day 3-7 but not the migration of mBM-MSC(p>0.05).Conclusions Lentiviral vector transduction can facilitate sustained and efficient gene modification of overexpressing TGF?1 in mBM-MSC,which inhibited the proliferation but did not affect differentiation or migration of mBM-MSC.Part ? Overexpression of TGF?1 in murine bone?marrow?derived mesenchymal stem cells improve the lung inflammation via impacting Th17/Treg balance in LPS-induced ARDS miceObjectives To evaluate the effect of overexpressing TGF?1 in mBM-MSC on improving the lung inflammation and its therapeutic potential in lipopolysaccharide(LPS)-induced ARDS mice.Methods mBM-MSC,mBM-MSC-NC or mBM-MSC-TGF?1 were transplanted intratracheally into ARDS mice induced by LPS.The survival of the mice was assessed until 7d after LPS challenge.The homing of mBM-MSC were analyzed by labeling and tracking mBM-MSC with NIR815 dye.Th17 and Treg in the lungs were detected by FCM.Inflammatory cytokines and occludin in the lungs were analyzed by Western blot.The inflammation and permeability were evaluated by detecting the cytokine and protein measurements in bronchoalveolar lavage fluid using ELISA.Lung tissue injury and repair assessment were examined using hematoxylin and eosin staining,lung injury scoring,Masson's trichrome staining and fibrosis scoring,as well as the m RNA detection of collagen-I and ?-SMA in the lungs of mice by q RT-PCR.Results There was no significant difference in 7d survival after mBM-MSC transplantation(p>0.05)but a better survival could be found in the mice after MSCs overexpressing TGF?1 engraftment.Overexpression of TGF?1 did not increase the retention of mBM-MSC in the lung(p>0.05),but it led to an improvement of Th17/Treg balance(p<0.05),and alveolar permeability(p<0.05).Moreover,IL-17 A was also significantly decreased while IL-10 increased in the mice after mBM-MSC transplantation(p<0.05).The engraftment of mBM-MSC overexpressing TGF?1 led to attenuated histopathological impairment of the lung tissue and the lung injury scores(p<0.05).Additionally,mBM-MSC overexpressing TGF?1 inhibited lung fibrosis according to the deposition of collagen and the fibrosis score in the lungs(p<0.05).Conclusions mBM-MSC overexpressing TGF?1 could regulate lung inflammation and attenuated lung injuries via modulating the imbalance of Th17/Treg in the lungs of ARDS mice.Part ? mBM-MSC engraftment in ARDS mice: therapeutic efficacy of intratracheal vs intravenous administration on CD4+T cell differentiationObjective To compare the therapeutic efficacy of mBM-MSC engraftment intratracheally vs intravenously on CD4+T cell differentiation in the lungs and spleens of ARDS mice.Methods mBM-MSC,mBM-MSC-NC or mBM-MSC-TGF?1 were transplanted intratracheally into ARDS mice induced by LPS.Th17/Treg and Th1/Th2 in the lungs and spleens were detected by FCM.Total cell and neutrophils in BALF were counted under the microscope after the Wright's staining.The survival of the mice was assessed until 7d after LPS challenge.Results mBM-MSC engraftment could significantly increase the total number of CD4+T cells in the lungs of ARDS mice(p<0.05)and there was no difference between the two methods of transplantation(p>0.05).mBM-MSC-TGF?1 could further increase the total number of CD4+T cells in the spleen of ARDS mice(p<0.05),improve the neutrophils in BALF of ARDS mice,especially administrated intratracheally(p<0.05).mBM-MSC engraftment intratracheally could significantly reduce the Th17/Treg both in lungs and spleens(p<0.05)of ARDS mice,but intratracheal administration of mBMMSC-TGF?1 could only decrease the Th17/Treg in lungs(p<0.05)but not in spleens(p>0.05).mBM-MSC engraftment intravenously significantly reduced the Th17/Treg both in lungs and spleens at 3d(p<0.05)of ARDS mice,but intravenous administration of mBM-MSC-TGF?1 could only decrease the Th17/Treg in spleens(p<0.05)but not in lungs(p>0.05)at 3d.There were no significant differences of Th1/Th2 among the groups(p>0.05)neither in lungs nor in spleens no matter after the mBM-MSC transplantation intratracheally or intravenously.There was no significant difference in ARDS mice survival among the groups(p>0.05).Conclusion mBM-MSC and mBM-MSC overexpressing TGF?1 engraftment intratracheally could increase the total numbers of CD4+T and reduce the Th17/Treg in the lungs of ARDS mice,while the intravenous transplantation did the same effect in the spleen.