Mesenchymal Stem Cells Alleviate Inflammatory Bowel Disease Via Inducing Tr1 Cells

BackgroundInflammatory bowel disease(IBD)is a non-specific autoimmune condition involving the gastrointestinal tract,comprising ulcerative colitis(UC)and Crohn’s disease(CD).The main clinical manifestations of IBD are abdominal pain,diarrhea,purulent and bloody stool,accompanied by fatigue,weight loss,malnutrition and so on.IBD is a lifelong chronic disease with increased risk of tumorigenesis,and is difficult to cure.The incidence of IBD has been rising in recent years,which becomes a serious threat to human health.Although the exact aetiology remains unknown,the disorders of intestinal mucosal barrier and intestinal flora in susceptible people,leading to the destruction of immune tolerance,resulting in imbalance of mucosal immune system,is considered as the pathogenesis of IBD.Under normal conditions,the pro-inflammatory and anti-inflammatory factors of the immune system are in a dynamic balance.However,excessive pro-inflammatory responses and insufficient anti-inflammatory responses promote the progress of IBD.Type 1 regulatory T cells(Tr1 cells)are a subset of regulatory CD4~+T cell.Unlike the well-known FOXP3~+Treg cells,Tr1 cells are FOXP3-negative.Tr1 cells are characterized by the high secretion of IL-10 and strong immunosuppressive effect in an IL-10-dependent manner.Although the number of Tr1 cells in peripheral blood is very low,Tr1 cells possess robust immunosuppressive potential in the immune modulating network.Tr1 cells act as guardians of gastrointestinal tract,not only are the main source of IL-10 in gastrointestinal tract,but also promote intestinal barrier function by secreting IL-22,and induce goblet cell differentiation.In IBD patients,Tr1 cells are insufficient or defective.In animal models of IBD,infusion of exogenous Tr1 cells can alleviate the symptoms of colitis.However,the alloantigen specificity of Tr1 cells makes it difficult to achieve allotransplantation.Therefore,Tr1cells may be induced by other ways to rebalance the immune status of IBD.Mesenchymal stem cells(MSCs)are adherent fibroblast-like cells which can differentiate into osteoblasts,adipocytes,and chondroblasts under specific conditions.There are various names according to the different tissue sources,such as bone marrow derived MSCs,adipose derived MSCs,umbilical cord derived MSCs,and so on.MSCs impede the function of various immune cells,including T cells,B cells,macrophages,natural killer(NK)cells,dendritic cells(DCs)and neutrophils,and enhance regulatory function of immune cells,such as FOXP3~+Treg cells,regulatory B cells,IL10-producing DCs,and M2-macrophages.Human umbilical cord-derived MSCs(hUCMSCs)adopted in the study are easily accessible,show lower immunogenicity and possess a stronger immunosuppressive capacity compared to MSCs from bone marrow and adipose tissue.MSC therapy is effective and safe for IBD in several clinical trials,however,the precise mechanism involved remains unclear.We hypothesize that Tr1 cells are important target cells of MSCs,and MSCs enhanced the function of Tr1 cells to control inflammation and reduce the severity of IBD.MethodsUmbilical cords of healthy term infants were collected and hUCMSCs were extracted.The cells were confirmed through morphology,cell surface phenotype by flow cytometry and induced differentiation.Acute colitis in BALB/c mice were induced by enema of 2,4,6-trinitrobenzene sulfonic acid(TNBS),and hUCMSCs were injected intraperitoneally later.The mortality,score for disease activity index(DAI score),weight change,colon shortening,macroscopic score and histological score were used to evaluate effect.To study the optimal dose of hUCMSCs,mice were randomly divided into five groups:control,TNBS,5×10~5hUCMSCs,1×10~6hUCMSCs and 2×10~6hUCMSCs.On day 3,mice were sacrificed,and tissues were collected.Colons were stained with HE,and Tr1 cells,Th1 cells,Th2 cells and Th17 cells were detected by flow cytometry.Subsequently,hUCMSCs with optimal dose were adopted to treat mouse colitis.Tissues were taken on day 3 and day 6,and the duration of action of hUCMSCs was assessed.Magnetic beads method(MACS)was used to sort CD4~+T cells and naive CD4~+T cells,and IL10 capture assay was applied to isolated Tr1 cells by flow cytometry.The sorting strategy of Tr1 cells was Aqua~-CD4~+CD44~+IL10~+.To explore the involved mechanism,firstly,a series of co-culture systems was established.Indomethacin(a specific inhibitor for COX-2/PGE2),NS398(a nonspecific inhibitor for COX-2/PGE2),and 1-MT(an inhibitor of IDO),were added into the hUCMSCs/splenocytes co-culture system.Apoptosis and proliferation of Tr1 cells co-cultured with hUCMSCs were detected by flow cytometry.To determine whether the function of Tr1 cells was affected by hUCMSCs,the inhibitory effect on proliferation of T cells was evaluated by flow cytometry.To determine the role of IDO on the regulation of Tr1 cells by hUCMSCs,we transfected the si RNA targeting IDO into hUCMSCs,and validated the knockdown effect by q PCR and Western-Blot.IDO knockdown hUCMSCs were injected into TNBS-induced colitis mice.ResultsThe hUCMSCs grew in spindle and polygonal shape,and the cell surface phenotype was identified using flow cytometry as follows:positive for CD29,CD44,CD73,CD90,and CD105(≥95%);negative for CD34 and CD45(≤2%).As for the differentiation ability,hUCMSC adipogenesis,chondrogenesis and osteogenesis were induced using specific stimuli.The therapeutic effects of 3 different doses of hUCMSCs on TNBS-induced colitis in mice were different.2×10~6hUCMSCs/mice was the most effective dose,as the mortality,weight loss,colon shortening,DAI scores,macroscopic scores and histological scores of the mice in the group were significantly improved.The effect of1×10~6hUCMSCs/mice was moderate,and 5×10~5hUCMSCs/mice was the weakest.In addition,hUCMSCs attenuated colitis throughout acute and convalescent stages.To determine whether Tr1 cells are involved in the therapeutic effect of hUCMSCs in IBD,the proportion of Tr1 cells was determined via flow cytometry.We found that Tr1 cell proportion was increased in mouse spleen and MLN,while the proportion of Th1 cells and Th17 cells was decreased.The proportion of Tr1 cells was negatively correlated with the histological score,suggesting that Tr1 cells might be the target cells of hUCMSCs.hUCMSCs could increase the proportion of Tr1 cells in splenocytes and CD4~+T cells,and 1-MT reversed the increase in Tr1 cell proportions induced by hUCMSCs.Interestingly,hUCMSCs could not induce Tr1 cells from naive CD4~+T cells.hUCMSCs could enhance the proliferation and inhibit the apoptosis of Tr1 cells,and enhance the immunosuppression of Tr1 cells on T cells.Knockdown of IDO by si RNA in hUCMSCs reduced the anti-inflammatory effect in the treatment of colitis and reversed the increase in Tr1 cell proportion.ConclusionhUCMSCs alleviate TNBS-induced colitis in a dose-dependent manner.The proportion of Tr1 cells is increased by hUCMSCs in the whole process,and negatively correlated with histological score of colitis.Tr1 cells are the target cells of hUCMSCs:hUCMSCs not only increase the number but also enhance the immunosuppressive function.hUCMSCs upregulates Tr1 cells to inhibit inflammatory responses and alleviate the symptoms of IBD via IDO.In addition,hUCMSCs can inhibit apoptosis and promote proliferation of Tr1 cells.