The Role And Mechanism Of Heme Oxygenase-1 In Acute Graft Versus Host Disease

Part I:The expression and clinical value of HO-1 in acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantationObjective:The current study aimed to investigate the expression of heme oxygenase-1(HO-1)in patients with acute leukemia(AL)received allogeneic hematopoietic stem cell transplantation(allo-HSCT)and analysis the clinical relevance with acute graft-versus-host disease(aGVHD).Methods:We collected samples and clinical data of 174 patients with AL received allo-HSCT.Real-time PCR was utilized to detect HO-1 expression in patients before and after allo-HSCT.Immune subsets in peripheral blood were detected by flow cytometry in 80 patients(40 aGVHD patients and 40 non-aGVHD patients).The correlations of HO-1 gene expression and aGVHD,relapse and overall survival after transplantation were analyzed.We performed Kaplan-Meier analysis to predict the value of HO-1 expression levels before and after transplantation in relapse,aGVHD development and overall survival.Results:HO-1 expression was significantly decreased in patients after transplantation than which before transplantation.The relative HO-1 expression was significantly reduced in aGVHD patients than those who haven't underwent aGVHD after transplantation.After transplantation,the relative expression rate of HO-1 was positively correlated with the percentage of CD3+CD25+Treg cells and CD4/CD8 ratio.ROC curve demonstrated that HO-1 could predict the occurrence of aGVHD after transplantation.The area under the ROC curve(AUC)was 0.860(0.796?0.924)(P<0.01).Moreover,HO-1 expression in patients without complete remission was higher than which in patients with complete remission.HO-1 expression before conditioning in relapse patients was significantly higher than which in patients during the same period without relapse(P<0.0001).Patients with high HO-1 expression before transplantation had a high relapse and poor survival.Conclusions:After transplantation,HO-1 expression was negatively correlated with the development of aGVHD,low HO-1 expression was associated with high occurrence of aGVHD.HO-1 expression had a predictive value on aGVHD development.HO-1 expression level was related to the tumor progression.However,patients with high HO-1 expression before conditioning had a high relapse rate and poor survival.Part II:The role of HO-1 in allo-reactive T cell responsesObjective:To investigate the difference of HO-1 expression level between syngeneic and allogeneic transplanted mice,to assess the influence of HO-1 expression in T cell activation,to explore the function of HO-1 in T cell immune responses by upregulation or inhibition of HO-1 expression.Methods:1.In the current study,syngeneic and allogeneic mouse transplantation models were established by using BALB/c(H2-Kd)mice as recipients and BALB/c and C57BL/6(H2-Kb)mice as donors.BALB/c recipients received lethal irradiation with 750cGy by X-Ray and were injected intravenously with 1×107 bone marrow(BM)cells and 5×106 splenocytes(SP)from BALB/c or C57BL/6 mice respectively.Lethally irradiation was separated into 2 steps by 4 hours to reduce the gastrointestinal toxicity.The spleen,liver,intestine and lungs of the two groups mice were isolated two weeks after transplantation,and the expression of HO-1 gene in GVHD target organs were detected by RT-PCR,DC cells,T cells,NK cells,macrophages and granulocytes in the spleen of the two groups were isolated by magnetic beads or sorted by flow cytometry,and HO-1 expression in different immune subsets was compared between the two groups by RT-PCR.2.Splenic T cells from B6 mice were treated with or without 10?g/mL ConA.BM derived DCs from BALB/c mice were cultured in the presence with 10ng/mL GM-CSF and 10ng/mL IL-4.7 days later,DC cells were stimulated with or without 10?g/mL LPS.RT-PCR was performed to detect the HO-1 expression in T cells and DCs.3.BM derived DCs from BALB/c mice were cultured in the presence with lOng/mL GM-CSF and lOng/mL IL-4.DCs were used as stimulators and irradiated by X-ray to suppress proliferation.Splenic T cells from B6 WT and HO-1 KO mice were isolated and labeled with CFSE.2×104 DCs were co-cultured with 1×105 WT T cells and 1×105 HO-1 KO T cells respectively.Proliferation of allo-reactive T cells were assessed by FACS.4.Peripheral blood mononuclear cells from 2 healthy volunteers were collected and used as effector cells and stimulator cells respectively.Stimulator cells were irradiated with X-ray to suppress proliferation.CFSE labeled 1×105 effectors and 1×105 stimulators were co-cultured in 96-well plate.Then,HO-1 inhibitor ZNPP and HO-1 agonist COPP were added at different concentrations.After 72 hours,cell proliferation,activation and apoptosis were detected by flow cytometry.In the same way,cells from healthy donor were used as stimulators,and cells from patients with and without aGVHD 4 weeks after transplantation were used as effectors.Then,HO-1 inhibitor ZNPP and HO-1 agonist COPP were added at different concentrations respectively.Cell proliferation was detected by flow cytometry after 72 hours.Results:1.The HO-1 gene expression level in recipients with allogeneic transplantation was significantly decreased than whose received syngeneic transplantation.The level of HO-1 gene in CD4+T cells in recipients with allogeneic transplantation was significantly reduced than recipients with syngeneic transplantation.2.The expression of HO-1 in activated T cells was significantly decreased after ConA stimulation,whilst LPS stimulation did not change the expression of HO-1 in DC cells.3.The murine mixed lymphocyte reaction assay revealed that HO-1 deficiency in T cells significantly promoted the allo-reactive T cell proliferation in both CD4+T cells and CD8+T cells.4.The human mixed lymphocyte reaction assay also indicated that the activation and proliferation of both CD4+T cells and CD8+T cells were significantly increased after treatment with HO-1 inhibitor ZNPP in vitro.ZNPP could inhibit the apoptosis of CD4+T cells and CD8+T cells.The percentage of CD4+T cells was reduced after treated with HO-1 agonist COPP in vitro,especially in patients without aGVHD,and the difference showed statistically significance.(P<0.05).Conclusions:The expression of HO-1 gene in recipients with allogeneic transplantation was significantly decreased than those received syngeneic transplantation.Further studies suggested that the expression level of HO-1 in CD4+T cells in allogeneic transplanted mice showed a significant reduction than which in syngeneic transplanted mice.The expression of HO-1 gene was significantly decreased in activated T cells after ConA stimulation.However,LPS have no effect on HO-1 expression in DCs.Genetic depletion of HO-1,HO-1 agonist and inhibitor suggested that HO-1 markedly inhibited T cell proliferation in both murine and human studies.Part III:The role and mechanism of HO-1 in aGVHDObjective:To investigate the role and mechanism of HO-1 in murine aGVHD.Methods:1.BALB/c(H2-Kd)mice were used as recipients.C57BL/6(H2-Kb)and HO-1-/-mice were used as donors.BALB/c recipients received lethal irradiation with 750cGy by X-Ray which was separated into 2 steps by 4 hours apart to reduce the gastrointestinal toxicity.And then recipients were injected intravenously with 1×107 bone marrow(BM)cells and 5×106 splenocytes(SP)from C57BL/6 or HO-1-/-mice respectively.2.C57BL/6(H2-Kb)and HO-1-/-mice were used as recipients.BALB/c(H2-Kd)mice were used as donors.Recipients received lethal irradiation with 900cGy by X-Ray which was separated into 2 steps by 4 hours apart to reduce the gastrointestinal toxicity.1×107 bone marrow cells and 1×107 whole spleen cells from donors were injected into recipients by tail vein 4 hours post systemic lethal radiation.3.BALB/c(H2-Kd)mice were used as recipients and received lethal irradiation with 750cGy by X-Ray.1×107 bone marrow cells from CD45.1 C57BL/6(H2_Kb)mice together with 1×106 splenic T cells from CD45.2 C57BL/6(H2-Kb)mice or HO-1 knockout mice were injected into BALB/c(H2-Kd)recipients by via tail vein 4 hours after total body irradiation.4.The survival and weight of mice were assessed after transplantation in the two groups,GVHD score was recorded,and pathological analysis was conducted on GVHD target organs including spleen,liver,skin and small intestine.Flow cytometry was used to detect the immune subsets including T cells,activated T cells,DC cells,macrophages,MDSC and Treg cells,as well as the level of cytokines secreted by T cells in the spleen,liver,lung and small intestine of the two groups 14 days after transplantation.Results:1.In the donor deficiency study,we found that HO-1 knockout could significantly promote the development of aGVHD after transplantation:(1)aGVHD related death were significantly accelerated,(2)weight loss were markedly increased,(3)the clinical score of GVHD was substantially increased.Flow cytometry analysis showed HO-1 knockout could promote the infiltration of T cells in target organs,activation of T cells and the implantation of stem cells.Donor HO-1 depletion could promote the distribution of macrophages and DCs in GVHD target organs and promote the activation of DCs,whilst inhibit the infiltration of Treg cells in GVHD target organs.In addition,the production of cytokines in serum and GVHD target organs in HO-1 knockout group were also significantly increased.2.In the host deficiency study,we uncovered that HO-1 deficiency significantly augmented the development of aGVHD after transplantation.Host HO-1 deficiency promoted the distribution of macrophages,DC cells and neutrophils in GVHD target organs.Host HO-1 deficiency promoted the activation of T cells and inhibited the infiltration of Treg cells in aGVHD target organs.3.In the donor T cell HO-1 deficiency study,we observed that compared with the control group,inflammatory cytokines including IFN-y and TNF-a in both CD4+and CD8+T cells in HO-1 knockout T cell transplanted mice were higher than in the control group.Moreover,the activation of T cells were significantly increased and Treg cells were significantly decreased in GVHD target organs in T cell.Conclusions:Deficiency of HO-1 in host and donor transplantation models suggested that HO-1 depletion significantly promote the development of aGVHD,enhance the infiltration of macrophages,DC cells and production of cytokines in GVHD target organs,augmented the proliferation and activation of T cells and DC cells,inhibited the proliferation of Treg cells leading to the damage of GVHD target organs.HO-1 depletion in donor T cells further demonstrated that HO-1 deficiency aggravated the development of aGVHD by regulating T cells functions directly.