Study On The Effect Of IDO In MSCs On GVHD In Murine Allo-BMT

Over the last 30 years allogeneic bone marrow transplantation (Allo-BMT) has become an accepted curative therapy for a variety of malignant hematopoietic diseases, severe immunologic deficiency diseases and hematopoietic failure of bone marrow. To date, the chief obstacle to the deployment of this treatment in other than life-threatening conditions is graft-versus-host disease (GVHD), and in particular acute GVHD, which is most severe in patients receiving matched unrelated donor transplants. The immunosuppressive drugs and T-cell depletion is effective at controlling GVHD, but these regimens can lead to serious complications from infection and an increased risk of leukemia relapse after Allo-BMT. Many researchers have therefore sought to develop techniques to prevent GVHD that avoid the complications incurred by immunosuppression.Mesenchymal stem cells (MSCs) are multipotent precursors present in bone marrow, capable of differentiating into osteoblasts, adipocytes, and myoblasts. Some studies have confirmed MSCs could constitutively secrete a variety of cytokines that promote the homing or proliferation and differentiation of hematopoietic cells, and accordingly play important roles in promoting hematopoietic recovery. Moreover, MSCs are poor antigen-presenting cells and do not express MHC class II or co-stimulatory molecules, studies have also demonstrated MSCs could exhibit immunoregulatory properties in vitro or in vivo. Due to the distinct immunophenotype profile MSCs is concerned as a prophylaxis for GVHD, but the molecular mechanisms responsible for the immunosuppressive effects of MSCs have not been unequivocally identified.Indoleamine 2,3-dioxygenase(IDO) is the initial and rate-limiting enzyme of the kynurenine pathway of degradation of L-tryptophan. IDO functionally catabolizes conversion from tryptophan .Tryptophan is necessary to the survival and proliferation of T cells. In the absence of tryptophan, the T cells cycle is arrested at a mid-G1 point. Following stimulation with IFN-γ, it is induced in various types of cell lines and cell types such as fibroblasts, epithelial cells and macrophages. Recently, it has been implicated IDO synthesized by the macrophages of placenta plays an important role in the prevention of allogeneic fetal rejection. So in professional antigen-presenting cells (APCs) expression of IDO has recently been identified as a major immunosuppressive mechanisms that inhibit T-cell activation and proliferation to autoantigens and alloantigens.Basing on these, we tried to investigate the IDO mRNA and IDO protein expression in murine bone marrow-derived MSCs induced by IFN-γ, and the capacity of IDO activity in MSCs to induce allogeneic T cell proliferation; The in vivo effects of MSCs expressing IDO on acute graft-versus-host disease (aGVHD) in murine allogenic bone marrow transplantation (Allo-BMT) model and its underlying mechanisms were also investigated in this paper.PartⅠStudy on isolation and biological characteristics of mesenchymal stem cells from murine bone marrowObjective: To investigate the isolation, purification, expansion and biological characteristics of mesenchymal stem cells (MSCs) derived from murine bone marrow in vitro.Methods: MSCs were isolated from murine bone marrow and cultured in IMDM medium. The morphology of the cells was observed by Wright's staining and electron microscope. Cell cycle and immunophenotype were investigated by flow cytometry. Assays of adipogenic and osteogenic differentiation were performed in vitro, then, Von Kossa stain, Oil-redO staining and immunohistochemistr were used to indentified to the induced-cells.Results: The cells from murine bone marrow displayed a fibroblast-like morphology adhering to the culture plate. FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and I-ab were negative. Adipocyte, osteocyte and nerve-like cells differentiation were induced successfully.Conclusion: MSCs derived from murine bone marrow were successfully isolated and cultured in vitro. PartⅡStudy on the effect of IDO in MSCs on allogeneic T-lymphocyte proliferation Objective: To study the effect ofγ-interferon (IFN-γ) on the IDO mRNA and IDO protein expression of MSCs and the effect of IDO activity in MSCs on allogeneic T-lymphocyte proliferation. Methods: IDO mRNA and IDO protein expression of MSCs induced by IFN-γat various concentration (0,20,50,100,200 U/ml) were analyzed by RT-PCR method and Western blot method respectively ,and IDO activity was determined by high-performance liquid chromatography (HPLC); MLRs cultures were set up with mitomycin C–treated C57BL/ 6-derived splenocytes as stimulators and BALB/c-derived splenocytes as responder cells. In MSCs/MLRs coculture experiments, MLRs were performed on a layer of 1×105,5×104,1×104 or 5×103 C57BL/ 6-derived MSCs (MSCs :responder T cells =1:5,1:10,1:50,1:100 ) with or without 1-methyl-D-trytophan(1-MT). T-lymphocytes proliferation was determined by MTT assays and IDO activity in coculture supernatant was determined by HPLC. Results: IDO mRNA expression, IDO protein expression and IDO functional activity were found in MSCs induced by IFN-γ, and the amount of expression and IDO activity increased gradually with the concentration of IFN-γ; When cocultured with 1×10~5,5×10~4,1×10~4 MSCs (MSCs :responder T cells =1:5,1:10,1:50),T- lymphocytes proliferation decreased apparently and IDO activity increased obviously compared with control group(p<0.05);In parallel experiments, 1-MT was added. T-lymphocytes proliferation and IDO activity restored partially. While cocultured with 5×10~3 MSCs (MSCs :responder T cells =1:100)T- lymphocytes proliferation raised lightly and IDO activity had no change compared with control group(p>0.05) .Conclusion: IDO activity in MSCs is relation to T-lymphocyte proliferation, and MSCs may perform their immunosuppressive function by IDO activity in vitro.PartⅢEstablishment of acute graft-versus-host disease model in murine allogeneic bone marrow transplantationObjective: To establish murine acute graft-versus-host disease (aGVHD) model for the study of aGVHD in allogeneic bone marrow transplantation (Allo-BMT).Methods: Lethally irradiated BALB/C (female,H-2d) murine transplanted with 1×107 C57BL/6 (male,H-2b ) bone marrow cells and 5×106 peripheral splenocytes (bone marrow cells and peripheral splenocytes ratio was 2:1) acted as GVHD group. The following two groups were simultaneously used: blank control group, no bone marrow cells and peripheral splenocytes transplanted; Allo-BMT group, no peripheral splenocytes transplanted. Evaluated the degree of GVHD according to clinical and pathological data.Results: Typical aGVHD could not be induced in GVHD group. GVHD clinical scores of recipient in GVHD group was about 10, and all murine survived 6 to 24 days after transplantation. Clinical signs and pathological changes of aGVHD were seen in a relative localized period. Conclusion: Murine lethal aGVHD model was successfully established.PartⅣEffect of IDO in MSCs on aGVHD in murine allogeneic bone marrow TransplantationObjective: Study the in vivo effects of MSCs expressing IDO on acute graft-versus-host disease (aGVHD) in murine allogenic bone marrow transplantation (Allo-BMT) model and explore the mechanism responsible for the immunosuppressive effects of MSCs.Methods: Various Allo-BMT groups were established with lethally irradiated BALB/C (female, H-2d) murine and C57BL/6(male, H-2b) : GVHD group(Group A); Cotransplanted with large dose MSCs(Group B); Cotransplanted with low dose MSCs(Group C); Cotransplanted with large dose MSCs pre-deal by 1-MT(Group D). The degree of GVHD ,survival rates, histopathological changes and helper T-cell type l [Th1(IL-2,IFN-γ)] and type 2 [Th2(IL-4,IL-10)] cytokines level were observed in all the groups after BMT.Results: It showed that GVHD clinical scores of recipient in group B was about 4, and no histopathological changes were seen, and the survival rate was 100%. The degree of GVHD was evidently lessened and survival rates was significantly higher compare with group A (P<0.05); In group B, Th1 cytokines level decreased and Th2 cytokines level increased significantly compare with group A (P<0.05). But GVHD clinical scores , histopathological changes , survival rate and cytokines level were not obviously different among Group A, C and D (P>0.05).Conclusion: IDO activity in alloreactive MSCs shift the Thl/Th2 balance toward a Th2-production of type-2 cytokines dominant phenotype, then reduced the incidence of GVHD. We supposed that MSCs may perform their immunosuppressive function by IDO activity in vivo.