Mesenchymal Stem Cells Inhibit Dendritic Cells Maturation To Alleviate Acute Lung Injury Via Jagged-1 Induced By Hepatocyte Growth Factor

Part Ⅰ Highexpressing HGF in mesenchymal stem cells affect dendritic cells maturation and lung injury in acute lung injury miceObjective To investigate the effect of HGF-MSCs the maturation of dendritic cells and lung injury in ARDS mice.Methods 133 C57BL/6 mice were randomly divided into seven groups:(1)Control(2)ARDS(3)MSCs(4)HGF-MSC(5)NC-HGF-MSCs(6)sh HGF-MSCs(7)NC-sh HGF-MSCs.MSCs,NC-HGF-MSCs,HGF-MSCs,NC-sh HGF-MSCs and sh HGF-MSCs are injected into mice through caudal vein after dividing,then 5mg/kg LPS is injected through airway to induce ARDS mice model.Mortality of the mice was assessed until 7 days after LPS challenge.Following steps were carried out and corresponding data were recorded until 12 hours after LPS challenge:(1)murine sepsis score(MSS)before execution;(2)obtain the bronchoalveolar lavage fluid(BALF),and measure the total protein(TP),total cell count(TCC),inflammatory cytokines IL-1β and IL-6 in BALF;(3)calculate lung wet weight to body weight ratio(LWW/BW);take partial lung tissues for HE and Masson staining,and calculate relevant Smith score and Ashcroft score;(4)prepare single cell suspension of lung tissue,measure the maturity of dendritic cells in lung and carry out correlation analysis between the maturity of dendritic cells and Smith score;detect the activation of T cells in single cell suspension of lung tissue.Results In vivo experiments,there was no significant difference of mortality at 7 days in ARDS and MSCs groups.But HGF-MSCs group showed a significant downward trend in mortality compared with ARDS groups(P < 0.05).Mice injected with MSCs and HGF-MSCs had a significant lowmurine sepsis score(MSS)compared with ARDS mice(P<0.05).The Smith score depended on HE staining showed that lung injury was significantly alleviated by HGF-MSCs compared with MSCs group(P<0.05).Compared with ARDS mice,HGF-MSCs significantly down-regulated LWW/BW,and TP,TCC and proinflammatory factor IL-1β and IL-6 in BALF(P < 0.05),which indicates HGF-MSCs can reduce the pulmonary edema and elevated alveolar epithelial permeability induced by LPS.HGF-MSCs significantly decreasing the Ashcroft score indicated the ability of HGF-MSCs to alleviate pulmonary fibrosis(P<0.05).Compared with MSCs,HGF-MSCs can also significantly reduce the maturation of DCs and the activation of T cells in lung(P<0.05).Further correlation analysis verified that the maturation of DCs in lung was positively correlated with lung injury.Conclusion Highexpressing HGF can improve the ability of MSCs to inhibite DCs maturation in lung and alleviate lung injury.Part Ⅱ Mesenchymal stem cells affect dendritic cells maturation via Jagged-1induced by hepatocyte growth factorObjective To investigate the role of autocrine hepatocyte growth factor(HGF)and Jagged-1 in maturation of dendritic cells(DCs)induced by mesenchymal stem cells(MSCs)and the related mechanisms.Methods Monocytes were induced from mouse bone marrow into immature DCs and then purified by magnetic activated cell sorting or percoll density gradient centrifugation.Purified immature DCs are co-cultured with MSCs for 48 hours,and stimulated by 50ng/ml LPS at the same time.Then detect the change of costimulatory molecules(CD40,CD86),phagocytic function and ability to stimulate lymphocyte proliferation of DCs by flow cytometry.Second,purified immature DCs,companied by 50ng/ml LPS,were co-cultured for 48 hours with HGF-MSCs,whose HGF expression level had been up-regulated,or sh HGF-MSCs,whose HGF expression level had been down-regulated.The change of costimulatory molecules(CD40,CD86)was detected.Third,purified immature DCs companied by 50ng/ml LPS were co-cultured with MSCs,recombinant mouse Jagged-1 or Jagged-1 monoclonal antibody MHJ1-29 for 48 hours.Then the change of costimulatory molecules(CD40,CD86)was detected again.The expression of c-Met,p-Met,Akt,p Akt and Jagged-1 of MSCs whose expression level of HGF had been regulated were detected by Western blot.The expression of Jagged-1 of MSCs that had been managed by Akt inhibitor MK2206 was also detected.Results In vitro experiments,LPS can stimulate the maturation of immature im DCs.Meanwhile,MSCs can significantly reduce the percentage of CD40 and CD86,improve phagocytic function,and reduce the ability to stimulate lymphocyte proliferation of DCs(P<0.05),which indicats MSCs can inhibit the maturation of im DCs even if LPS exists.MSCs with HGF up-regulated can further inhibit the maturation of im DCs,because MSCs with HGF up-regulated significantly reduced the percentage of CD40 and CD86 on DCs(P<0.05).Rm Jagged-1,the same as MSCs,can significantly reduce the maturation of im DCs when LPS exists.However,MHJ1-29 which reacts with mouse Jagged-1 did the opposite.Upon further discovery,There was no difference in the total c-met protein and Akt protein expression between the MSCs group,whose HGF had been regulated,but HGF-MSCs significantly express more p-Met,p-Akt and Jagged-1 protein.And MSCs expressed more Jagged-1 after managed by Akt inhibitor MK2206,which indicates HGF promotes Jagged-1 expression of MSCs through Akt signal.Conclusion hepatocyte growth factor secreted by mesenchymal stem cells can inhibit dendritic cells maturation via Jagged-1 induced by Akt signaling.