Mechanisms Of Endometrial Regenerative Cells In Regulating Immune Mediated Ulcerative Colitis Treatment

Objective:To separate and identify endometrial regeneration cells(ERC)from menstrual blood,to observe programmed death-ligand 1(PD-L1)expression on the cell surface of ERC.To build the animal model of ulcerative colitis(UC).To explore ERC distribution in animal model of UC in vivo.To explore the development and progression mechanism of UC controlled by ERC and the role of PD-L1 on the surface of ERC in the treatment of UC.Methods:1)ERC were collected from menstrual blood after informed consent.Mononuclear cells were separated by standard Ficoll method.ERC cell surface antigen by flow cytometry.2)Build BALB/c UC model by drinking the different concentrations of dextran-sulfate-sodium(DSS)method.During the building of experimental animals,to observe the clinical,disease activity index(DAI)and histopathological changes of laboratory mice,to select the best concentration of DSS and evaluate animal model.3)For in vivo tracking of administered ERC,cells were isolated and labeled with PKH26(Paul Karl Horan 26)red fluorescent.ERC labeled with PKH26 were applied to animal model of UC.The distribution of ERC in animal models of UC were assessed by luorescence microscope method.4)In vivo experiment,experimental animals were divided into four groups((1)normal control group,(2)UC model group,(3)ERC treatment group,(4)PD-L1 pretreated ERC treatment group).ERC or anti-PD-L1 m Ab-pretreated ERC were injected(1×106/0.2ml/mouse,i.v.)into mice on day 2,5,and 8 following colitis induction.Colonic and splenic tissues were collected on day 15 post-DSS-induction.Clinical signs,DAI,pathological changes,cytokine profiles and cell populations were evaluated.Moreover,ERC or ERC by preincubated anti-PD-L1 m Ab were co-culturing with splenocytes from normal BALB/c mice.In addition,ERC were co-culturing with splenocytes using transwell.Following by stimulated with diferent factors,splenocytes phenotypes in coculturing were analyzed using flow cytometry.Results:1)ERC express PD-L1,CD90,CD105.ERC not express CD34,CD45.There was a basal level of PD-L1 expression on ERC,and following incubation with various concentrations of recombinant mouse IFN-?,a significant increase of PD-L1expression was induced by the treatment with 0.5 ng/ml of IFN-?,and further increased with 5 ng/ml,in a dose-dependent manner.2)DSS-induced mice developed severe colitis,characterized by body-weight loss,bloody diarrhea,mucosal ulceration and colon shortening,as well as pathologic changes of lamina propria cell infiltrations of neutrophils,macrophages,lymphocytes,crypt distortion,lymphoplasmacytosis,and hyperplastic muscularis mucosae.With the concentration increased and the time extension,the above performance present severity gradually.3)PKH26-positive ERC were detected by fluorescence microscopy in the intestine(injured tissue)and the spleen(lymphoid organ)of ERC-treated mice.Moreover,after 48 hours the labeled ERC were aslo found in the lung > liver > spleen > colon tissue,kidney not observed fluorescence.The fluorescence intensity of colon tissue in model group is greater than the normal group.4)ERC attenuated colitis with significantly reduced DAI,decreased levels of intra-colon pro-inflammatory cytokines(TNF-?,IFN-?),increased expressions of intra-colon anti-inflammatory cytokines(IL-10,TGF-?),decreased populations of CD3~+CD4~+T cells,CD3~+CD8~+T cells,CD11c~+MHC-II~+DCs and F4/80~+Macrophages,increased the population of CD4~+CD25~+Foxp3~+Tregs and F4/80~+CD206~+Macrophages.Blockade of PD-L1 on ERC with monoclonal antibody seriously abrogated the therapy-induced immune tolerance.In vitro ERC-mediated suppression of immune also mainly depended on PD-L1 function and cell contact between immune cells and ERC.Conclusion 1)ERC separated from the menstrual blood is a kind of regeneration cells similar to mesenchymal stem cells.PD-L1 expression in cultured ERC was up-regulated following IFN-? stimulation.2)3% DSS is optimal concentration for mouse model of UC.3)ERC can migrate into intestinal inflammation site of UC mice model.4)ERC can effectively control the occurrence and development of the experimental UC.ERC play its immunosuppressive effects by inhibiting the maturation of dendritic cells and T cell proliferation,enhancing the generation of Tregs and F4/80~+CD206~+Macrophages.PD-L1 on ERC plays an important role in the effective treatment of ERC for colitis.