Mesenchymal Stem Cells Induce Dendritic Cell Immune Tolerance Via Paracrine Hepatocyte Growth Factor To Alleviate Acute Lung Injury

Part I Mesenchymal stem cells induce immune tolerance of mature dendritic cells in LPS-induced acute lung injuryObjective To evaluate the effect of bone marrow-derived mesenchymal stem cell transplantation on pulmonary dendritic cell function in lipopolysaccharide(LPS)-induced acute lung injury mice.Methods In vitro experiments,monocytes were isolated from mouse bone marrow,and induced into immature DCs under GM-CSF and IL-4,and then purified by CD1 lc magnetic beads;Immature DCs were stimulated at different concentrations of LPS(0,10,50,100,200,500 and 1000 ng/mL)for different duration(24 and 48 hours).The cell phenotype was then analyzed by flow cytometry to determine the optimal concentration and minimum duration of LPS stimulation for DCs maturation Then,the mature DCs were co-cultured with MSCs through Transwell,and the DCs phenotype was detected by flow cytometry.The effect of MSC on the function of DCs after co-culture was detected as follows:the PD-L1 expression of DCs,which indirectly reflected the inhibitory ability of DCregs on T cell proliferation,and the effect of DCs on lymphocyte proliferation after co-culture was also detected.In animal experiments,the ALI mouse model was established by injecting LPS into the airway at different times,and the number of pulmonary classic DCs(CD11c+CD11b+DCs)and maturity(MHC?+cDCs,CD86+cDCs)and their correlation with lung injury score were detected after 0,1,2,4,6,12 and 24 hours after LPS injection.According to that,the appropriate interval was selected to give MSCs transplantation to ALI mice for treatment.After 24h,the subsequent indicators were detected,and flow cytometry was used to detect the number and maturity of lung DCs,as well as the activation ratio of lung CD4+T cells(The ratio of CD44+CD4+T and CD4+CD69+cells).Histopathological examination was performed with H&E staining and lung injury score was performed to evaluate the severity of lung injury.The lung wet weight/weight ratio(LWW/BW)was measured to evaluate the degree of pulmonary edema.DCregs obtained from mesenchymal stem cell culture for 3 days were diluted with PBS to 150uL by tail vein injection to treat LPS-induced ALI.Lung histopathology evalution,lung T cell number and activation ratio were performed to evaluate the effect of DCregs on ALI.ALI mice were transplanted with CFSE-labeled DCs and then treated with MSCs.Flow cytometry was used to detect the CFSE+CD11c+DCs in the lungs and blood,so as to clarify the effect of MSCs on the number of lung-DCs.Results In vitro experiments,LPS can stimulate the maturation of immature DCs in a certain range of concentration dependence.50 ng/mL LPS significantly increased the proportion of mature DCs(P<0.05),and the level of maturity was not significantly changed when the concentration was further increased compared with that of 50 ng/mL(P>0.05).DCs was stimulated by 50 ng/mL LPS at Oh to 24h,and DCs maturity was significantly improved(P<0.05).The stimulation time was 24h to 48h,DCs maturity was not significantly increased(P>0.05).Compared with mDCs group,the percentage of MHCII,CD86 and CD40 positive DCs in MSC-DC group was significantly reduced(P<0.05),the percentage of PD-L1 positive cells expressing immune tolerance molecule was significantly increased(P<0.05),and the percentage of lymphocyte proliferation was significantly reduced(P<0.05).In animal experiments,compared with the Con group,the amount of lung DCs increased significantly 2 hours after LPS was injected into the airway,which gradually increased with time,but stopped after 12 hours.DCs maturity increased significantly after 2 hours,and the lung DCs was dominated by mature DCs subgroups(P<0.05),the degree of lung pathological damage was significant,which were aggravated over time(P<0.05).Correlation analysis showed that the number and maturity of lung DCs were positively correlated with the lung injury score in ALI(P<0.05).Compared with the Con group,the proportion of pulmonary cDCs(CD11c+CD11b+DCs)in the ALI group was increased,and the proportions of MHCII+cDCs and CD86+cDCs were significantly increased(P<0.05).The proportion of lung cDCs and mature MHCII+cDCs decreased in ALI mice after MSCs treatment(P<0.05),so MSCs inhibited lung recruitment of DCs and reduced the proportion of mature DCs in ALI mice.The proportion of CD4+T cells and CD69+in CD4+T cells in the lungs of the ALI group was increased relative to that of the con group(P<0.05).The percentages of CD69+in CD4+T cells in the lung were significantly decreased after treatment with MSCs(P<0.05),while the number of CD4+T cells in the lungs decreased after MSC treatment,but there was no statistical difference.Compared with Con group,ALI mice had aggravated lung pathological injury,significantly increased lung injury score and LWW/BW(P<0.05),and the above indicators were significantly reduced after mesenchymal stem cell treatment(P<0.05).Similarly,after treatment with DCregs,ALI mice showed less pulmonary lesions,decreased lung injury scores,reduced percentages of CD44+CD4+T and CD4+CD69+T cells(P<0.05).MSCs inhibited DCs migration from blood to lung in ALI mice.After the occurrence of ALI for 24h,the proportion of CFSE+CD11c+DCs in peripheral blood and the proportion of CFSE+CD11c+DCs migrating to lung tissues increased significantly(P<0.05),and both decreased after MSC treatment(P<0.05).Conclusion MSCs could induce the differentiation of mature DCs into immune tolerant DCregs,inhibit DCs recruitment to the lungs of ALI,inhibit T cell proliferation and activation,and alleviate lung injury.Part II Mesenchymal stem cells induce differentiation of mature dendritic cells into regulatory dendritic cells via the paracrine HGF-activated Akt pathwayObjective To investigate the role of paracrine hepatocyte growth factor and Akt pathway in immune tolerance of dendritic cells induced by MSCs and the related mechanisms.Methods The mature DCs was induced for 72 hours with different recombinant HGF(rhHGF)concentrations(0,10,50,100,150 and 200 ng/mL),and then the DCs phenotype was detected by flow cytometry to determine the optimal concentration of rhHGF-induced DCregs.After 72h of co-culture of mature DCs with MSCs or rhHGF through Transwell,flow cytometry was used to detect the phenotype of DCs,the phagocytic function of DCs,the proliferation ability of DCs stimulated lymphocytes,and the expression of PD-Lland IL-27.The secretion of DCs inflammatory cytokines cytokines IL-10,IL-12 and TGF-? was detected by ELISA.Expression of protein c-Met and phosphorylation of c-Met in HGF downstream pathway in DCs were detected by Western blot.Lentivirus vectors with high expression of HGF were constructed and transferred to mouse bone marrow derived MSCs.HGF shRNA lentiviral vectors were also used to down-regulate HGF RNA expression in MSCs.The efficiency of transfection MSCs with EGFP was evaluated by fluorescence microscopy and flow cytometry.The mRNA levels of HGF in MSCs were detected by qRT-PCR.HGF protein levels in MSCs were detected by Western blot.The paracrine HGF levels of MSCs were detected by ELISA.MSCs with high and low expression of HGF were co-cultured with mature DCs by Transwell for 72 hours.Flow cytometry was used to detect the phenotype of DCs,the phagocytic function of DCs and the proliferation ability of DCs stimulated lymphocytes.ELISA was used to detect the secretion of inflammatory cytokines IL-10,IL-12 and TGF-? by DCs.The expression of Akt and phosphorylated Akt in the downstream c-Met pathway in DCs was detected by Western blot.After blocking the induction of DCregs by rhHGF with Akt inhibitor MK2206,flow cytometry was performed to detect the DCs phenotype,DCs phagocytic function,and the proliferation capacity of DCs stimulated lymphocytes.ELISA was used to detect the secretion of inflammatory cytokines IL-10,IL-12 and TGF-? by DCs.Results When the concentration of rhHGF gradually increased from 0 ng/mL to 50 ng/mL,the expression of MHCII,CD86 and CD40 in DCs were significantly decreased(P<0.05),but higher concentration did not significantly change the DC phenotype.Both MSCs and rhHGF could induce mDCs differentiation into DCregs with low expression of MHCII,CD86 and CD40(P<0.05),which did not change after LPS stimulation.Compared with mDCs,MSCs or rhHGF-induced DCs had stronger phagocytic function(P<0.05),had a weaker ability to stimulate lymphocyte proliferation(P<0.05),expressed more PD-Lland IL-27(P<0.05),secreted more anti-inflammatory factors IL-10 and TGF-? cytokines,and less pro-inflammatory factor IL-12(P<0.05).There was no significant change in the expression of total c-Met protein in DCregs,but the expression of phosphorylated c-Met protein was significantly increased.Lentivirus mediated MSCs with high expression and low expression HGF.The transfection efficiency was as high as 93%after transfection by fluorescence microscope and flow cytometry.The expression of HGF mRNA in the HGF-MSC group was about 12 times of that in the NC-HGF-MSC group,and that in the shHGF-MSC group was 1/3 of that in the NC-shHGF-MSC group.The expression of HGF protein and the amount of HGF secreted by DCs cells in the HGF-MSC group were higher than those in the control group,while those in the shHGF-MSC group were lower than those in the control group(all P<0.05).The proportion of CD86 and MHCII positive DCs was lower in the HGF-MSC group than in the NC-shHGF-MSC group,and higher in the shHGF-MSC group than in the NC-shHGF-MSC group(all P<0.05).Compared with DCregs induced by NC-HGF-MSCs,HGF-MSCs induced DCregs had stronger phagocytic function(P<0.05),weaker ability to stimulate lymphocyte proliferation(P<0.05),more secretion of anti-inflammatory factors IL-10 and TGF-? cytokines,and less pro-inflammatory factor IL-12(P<0.05).While compared with DCregs induced by NC-shHGF-MSCs,DCregs induced by shHGF-MSCs had weaker phagocytic function(P<0.05),stronger ability to stimulate lymphocyte proliferation(P<0.05),less secretion of anti-inflammatory factors IL-10 and TGF-? cytokines,and more inflammatory factor IL-12(P<0.05).There was no difference in the total Akt protein expression between the NC-HGF-MSC group and the HGF-MSC group,the NC-shHGF-MSC group and the shHGF-MSC group,but the phosphorylated Akt protein expression was lower in the NC-HGF-MSC group than in the HGF-MSC group,and higher in the NC-shHGF-MSC group than in the shHGF-MSC group.After restraining the Akt pathway with Akt inhibitor MK2206,the percentage of MHCII and CD40 positive cells in DCregs induced by rhHGF increased significantly(P<0.05).Compared with rhHGF-DC,DCs of rhHGF-DC+MK2206 group had weaker phagocytic function,stronger ability to stimulate lymphocyte proliferation,less secretion of anti-inflammatory factors IL-10 and TGF-? cytokines,and more inflammatory factor IL-12(all P<0.05).Conclusion MSCs induce mDCs to differentiate into immune-tolerant DCregs via paracrine HGF and activated Akt pathway.Part III Highexpressing HGF in mesenchymal stem cells regulates the immune function of lung dendritic cells in LPS-induced ALI miceObjective To evaluate the effects of Highexpressing HGF in MSCs on immune function of lung dendritic cells in lipopolysaccharide(LPS)-induced ALI mice.Methods C57BL/6 mice labeled with or without CFSE were randomly divided into Con,ALI,MSC,NC-HGF-MSC,HGF-MSC,NC-shHGF-MSC and shHGF-MSC groups.LPS was injected into the airway to induce the ALI mouse model.MSCs with different HGF gene modifications were given for treatment later.To evaluate the role of Akt in the HGF regulation of lung DCs function in ALI mice,C57BL/6 mice were randomly divided into Con group,ALI group,MSC group,MSC+MK2206 group(MSC treatment+Akt inhibitor),rhHGF group,and rhHGF+MK2206 group.The mice were sacrificed 24 hours after the model was established,and the following indicators of the above groups were tested:the right upper lobe was retained for histopathological examination with HE staining and the lung injury score eveluation.The lung wet weight/body weight ratio(LWW/BW)was calculated to evaluate the degree of pulmonary edema.Lung and parbroncho-lymph node(PBLN)single-cell suspension were prepared,and the percentage of cDCs(CD11c+CDl lb+DCs or CD11c+CFSE+DCs)and maturity(MHCII+cDCs,CD86+cDCs)in lung or PBLN were detected by flow cytometry.Flow cytometry was also used to measure the number and activation of CD4+T cells(CD69+cells in CD4+T cells)in the lung.Alveolar lavage fluid was collected and the total number of cells was detected by flow cytometry.Results(1)At baseline,lung cDCs in the control group at 24 h expressed relatively low levels of CD86 and MHC II.In parallel to the accumulation of pulmonary cDCs in ALI mice,the expression of CD86 and MHC II on the surface of respiratory cDCs showed a significant increase.Notably,treatment with MSCs led to a marked reduction in CD86 or MHC II expression on pulmonary cDCs.Treatment with HGF-MSCs resulted in a significant reduction in CD86 on lung cDC compared to treatment with the empty vector control group but increased after treatment with shHGF-MSCs(all P<0.05).(2)Similarly,the proportion of CD69+cells in CD4+Tcells,CD4+Tcells in lung,and the total number of cells in BALF in the MSC group were lower in the ALI group.Treatment with HGF-MSCs resulted in a significant reduction in CD4 on lung cells or CD69 on lung CD4+T cells compared to treatment with the empty vector control group but increased after treatment with shHGF-MSCs(all P<0.05).The total number of cells in BALF in the HGF-MSC group were lower than those in the NC-HGF-MSC group,but there was no statistical difference.The total number of cells in BALF in the shHGF-MSC group were higher than those in the NC-shHGF-MSC group(all P<0.05).(3)Gross pathological results showed that LPS-induced ALI mice had hyperemia,edema and bleeding.The lung injury was significantly reduced after treatment of MSCs,NC-HGF-MSCs or NC-shHGF-MSCs,and the lung injury degree of mice was more significantly reduced after treatment of HGF-MSCs,and the lung injury degree of mice was significantly increased after treatment of shHGF-MSCs than treatment with the empty vector control group.HE staining lung tissue pathology showed that the degree of lung injury(edema,hemorrhage and inflammatory cell infiltration)in mice after treatment with MSCs,NC-HGF-MSCs or NC-shHGF-MSCs was significantly reduced compared with that in the ALI group.Lung tissue pathological injury score and LWW/BW were lower in MSC group than in ALI group,significantly lower in HGF-MSC group than in the empty vector control group,but higher in shHGF-MSC group(all P<0.05).(4)The migration of CD11c+CFSE+DCs to PBLN increased during ALI,and MSC therapy inhibited the migration of CD11c+CFSE+DCs(P<0.05).Compared with empty carrier group MSC,HGF-MSC promoted the migration of CD11c+CFSE+DCs,while shHGFM-SC weakened the migration of DC.It was further found that the expression level of CD86 in DCs promoted migration was decreased(P<0.05),indicating that HGF-MSC treatment p of HGF-MSC group romoted the migration of immunomodulatory DCregs.(5)The proportion of pulmonary cDCs and CD86+cDCs in rhHGF group was significantly lower than that in ALI group(all P<0.05).The pathological injury score of ALI lung tissue was significantly reduced after rhHGF treatment(P<0.05).Conclusion High expression of HGF can significantly enhance the immune regulation ability of MSCs to ALI lung DCs,reduce the number and maturity ratio of lung DCs,promote DCreg migration to PBLN,so as to alleviate excessive immune response and reduce lung injury.