| ObjectiveAcute graft-versus-host disease(aGVHD)is an important cause of early non-recurrent death after allogeneic hematopoietic stem cell transplantation(Allo-HSCT).It is particularly important to explore indicators that can predict the occurrence of aGVHD early and therapeutic targets for aGVHD intervention.In the early stage,our research group detected and analyzed peripheral blood plasma and mononuclear cells of transplant patients at different time points by metabonomics and transcriptome,and found that lysophosphatidylcholine(LysoPC),a choline metabolite,significantly increased in plasma of aGVHD patients on day 15 after transplantation.High expression of choline kinase α(CHKA)gene was observed in mononuclear cells.As a key enzyme in the choline metabolic pathway,CHKA can phosphorylate choline to phosphocholine,and then form phosphatidylcholine under the action of phosphocholine cytidyl transferase and phosphocholine transferase(PCT),which is a key component of cell membrane.Therefore,CHKA plays an important role in cell synthesis.In addition,phosphatidylcholine forms LysoPC in response to phospholipase A1(PLA1)and arachidonic acid(AA)in response to phospholipase A2(PLA2).Proliferation and differentiation of T cells stimulated by antigen is the main cause of aGVHD.Therefore,we speculated that the high expression of CHKA in mononuclear cells and the increase of choline metabolites in plasma of aGVHD patients may be related to the proliferation and differentiation of T cells.However,the expression of CHKA in T cells under aGVHD state is still unknown.And the role of CHKA in the proliferation and differentiation of T cells in aGVHD state has not been reported at home and abroad.In this study,a mouse aGVHD model was established to explore the relationship between CHKA and proliferation and differentiation of acute graft-versus-host disease T cells and its regulatory mechanism,providing new ideas for early prevention and treatment of aGVHD.MethodsThe model adopted in this study was a completely incompatible allogeneic bone marrow transplantation model,with BALB/C mice as recipients,C57BL/6 mice and C57BL/6 background systemic CHKA gene knockout heterozygous mice as donors.The groups were as follows:(1)BMT group:BALB/C mice irradiated with lethal dose received bone marrow cells removed from C57BL/6 source(TCD-BM 5×106);(2)aGVHD group:BALB/C mice irradiated with lethal dose received TCD-BM(5×106)and spleen cells(2×107)from C57BL/6;(3)CK37-aGVHD group:in the background of aGVHD mice,CK37(CHKA inhibitor)was injected intraperitoneally for 2.5mg/kg/day to 21 days from the first day after transplantation;(4)aGVHDspleen-CHKA+/-group:BALB/C mice irradiated with lethal dose received TCD-BM from C57BL/6(5×106)and spleen cells from CHKA knockout heterozygous mice(C57BL/6 as background)(2×107).1.Analyze the relationship between the expression of CHKA in T cells and aGVHD occurrence1)BMT model and aGVHD model were established in mice and the changes of white blood cells and body weight of the two groups were monitored regularly.The state of mice was observed to evaluate the occurrence time of aGVHD2)Flow cytometry was used to analyze the changes of T cell subsets in the two groups of mice at different time points,and the characteristics of T cell proliferation and differentiation before and after aGVHD was analyzed.Luminex method was used to detect the changes of cytokines in the two groups of mice at different time points3)Plasma metabolomics was used to analyze the changes of choline metabolites.The spleen T cells of the two groups were sorted by magnetic beads at different time points,and the expression of CHKA and its downstream enzymes in the two groups were explored by real-time quantitative PCR(QRT-PCR)and western-blot2.To explore the effects of CK37 drug intervention in mouse aGVHD model on mouse survival,aGVHD occurrence and T cell proliferation and differentiation1)Mouse aGVHD model was established,CK37 drug concentration gradient was set,and the drug concentration that could improve the survival of mice aGVHD was searched2)The appropriate CK37 concentration was selected to establish the CK37-aGVHD model,and the changes of white blood cells and body weight at different time points in the CK37-aGVHD group and aGVHD group were monitored regularly,and the occurrence of aGVHD in mice was evaluated after CK37 intervention3)Flow cytometry was used to analyze the changes of T cell subsets at different time points in the two groups,Luminex method was used to detect the changes of cytokines before and during the occurrence of aGVHD4)Plasma metabolomics was used to analyze the changes of choline metabolites.QRTPCR and western-blot were used to explore whether there were differences in the expression of CHKA and its downstream enzymes in T cells of the two groups of mice at different time points3.The CHKA gene of donor spleen cells was knocked out to explore the effects of down-regulating the expression of donor T cells CHKA on the survival,occurrence of aGVHD and T cell proliferation and differentiation of aGVHD mice1)aGVHD and aGVHDspleen-CHKA +/-models were established,and body weight and leukocyte changes of mice in the aGVHD group and aGVHDspleen-CHKA +/-group were monitored regularly,and the effects of CHKA gene down-regulation in donor spleen cells on the survival and occurrence of aGVHD were evaluated2)Flow cytometry was used to analyze the changes of T cell subsets in the two groups at different time points.Luminex method was used to detect the changes of cytokines in the two groups at different time points3)Plasma metabolomics was used to analyze the changes of choline metabolites.QRTPCR and western-blot were used to explore whether there were differences in the expression of CHKA and its downstream enzymes in T cells of the two groups at different time points4.Explore the related pathways of CHKA intervention on T cell proliferation and differentiationThe common pathway of T cell proliferation and differentiation and CHKA regulation was found by reading literatures,and the expression of corresponding proteins in the pathway was detected by western-blot method5.Statistical analysisAll data were presented by mean±SEM.Student-t test was used for comparison of mean between two groups,univariate ANOVA was used for comparison of mean between multiple groups,Kaplan-Meier analysis was used for survival analysis,and log-rank test was used for comparison.P<0.05 was considered statistically significant.Results1.The expression of CHKA in T cells of mice in aGVHD group was significantly up-regulated on day 14 and day 21 after transplantation1)BMT and aGVHD models were successfully established in mice.The median time of aGVHD occurrence was 18 days(16-21 days),and the median survival time of aGVHD group was 33 days(23-39 days),which was significantly lower than that of BMT group.Bone marrow was fully implanted in mice on day 14 after transplantation.2)The proportion of CD3+T cells,CD8+T cells,Th1 cells and Th2 cells in aGVHD group was significantly higher than that in BMT group on day 14 and day 21 after transplantation,while the proportion of CD4+T cells and Treg cells in aGVHD group was significantly lower than that in BMT group.The proportion of Th17 cells in aGVHD group was significantly higher than that in BMT group on day 21 after transplantation.The expression of IFN-y,IL-10、IL-2,IL-4,TNFα,IL-6 and IL-5 in BMT group was significantly lower than that in aGVHD group on day 14 and day 21 after transplantation,and the expression of IL-17A was not significantly different between the two groups at the two time points.3)The production of choline metabolite AA in aGVHD group was significantly higher than that in BMT group on day 14 after transplantation,and the production of Lysopc16:0 and Lysopc18:0 in aGVHD group on day 21 after transplantation was significantly lower than that in BMT group.The expression of CHKA and PLA2 in mice T cells in aGVHD on day 14 and day 21 after transplantation was significantly higher than that in BMT group,and there was no significant difference in the expression of PL A1 and PCT.2.Inhibition of CHKA expression in mouse aGVHD model by CK37 can improve Treg differentiation and severity of aGVHD1)Continuous intraperitoneal injection of CK37 2.5mg/kg for 21 days can prolong the survival and decrease the GVHD score of aGVHD mice.The wbc count of CK37-aGVHD mice was significantly higher than that of aGVHD mice on day 21 after transplantation.2)On day 21 after transplantation,the proportion of Treg in CK37-aGVHD group was significantly higher than that in aGVHD group.On day 14 after transplantation,the expressions of IL-17A and TNFα in CK37-aGVHD group were significantly lower than those in aGVHD group.On day 21 after transplantation,the expression of IFN-y,IL-17A and TNFα in CK37-aGVHD group was significantly lower than that in aGVHD group,and the expression of IL-10 was significantly higher than that in aGVHD group.3)On day 14 and day 21 after transplantation,there was no significant difference in the expression of plasma choline metabolites between CK37-aGVHD group and aGVHD group.On day 14 and day 21 after transplantation,the expression of CHKA in CK37-aGVHD group was significantly lower than that in aGVHD group.On day 14 after transplantation,the expression of PLA2 gene in mice in CK37-aGVHD group was significantly lower than that in aGVHD group,and there was no significant difference in the expression of PLA1 and PCT gene,while on day 21 after transplantation,the expression of PLA1 and PLA2 gene in mice in CK37-aGVHD group was significantly lower than that in mice in aGVHD group,and there was no significant difference in the expression of PCT.3.Knockout of CHKA gene in donor spleen cells can inhibit the proliferation and differentiation of CD4+T cells and Thl cells,promote the expression of Treg,and improve the progression of aGVHD in mice1)Knockout of CHKA gene in donor spleen cells can significantly prolong the survival time of aGVHD mice and reduce GVHD score.The wbc count of mice in aGVHDspleenCHKA+/-group was significantly higher than that in aGVHD mice on day 21 after transplantation.2)On day 14 after transplantation,the proportion of CD4+T cells in aGVHDspleenCHKA+/-group was significantly lower than that in aGVHD group.On day 21 after transplantation,the proportion of CD4+T cells and Thl cells in aGVHDspleen-CHKA+/-group was significantly lower than that in aGVHD group,and the proportion of Treg cells was significantly higher than that in aGVHD group.The secretion of IFN-γ,IL-2,IL-4,IL-17A and TNFa in aGVHDspleen-CHKA+/-group was significantly lower than that in aGVHD group on day 14 after transplantation.The secretion of IFN-y,IL-2,IL-17A and TNFa in aGVHDspleen-CHKA+/-group was significantly lower than that in aGVHD group,and the secretion of IL-10 was significantly higher than that in aGVHD group on day 21 after transplantation.3)On day 14 after transplantation,Lysopc18:0 expression of choline metabolite in aGVHDspleen-CHKA+/-group was significantly lower than that in aGVHD group.The expression of Lysopc18:0 and LysopC16:0 of choline metabolites in aGVHDspleen-CHKA+/group was significantly higher than that in aGVHD group.On day 14 and day 21 after transplantation,the expression of CHKA and PLA2 in T cells of aGVHDspleen-CHKA+/-group was significantly lower than that of aGVHD group,and there was no significant difference in the expression of PLA1 and PCT.4.CHKA may regulate the differentiation of T cell subsets under aGVHD by interfering PI3K/AKT pathway1)The expressions of AKT,P-AKT,Erk and P-Erk in T cells of aGVHD group were higher than those of BMT group on day 14 and day 21 after transplantation.2)On day 14 after transplantation,the expressions of AKT,P-AKT,Erk and P-Erk in T cells of CK37-aGVHD group were lower than those of aGVHD group;on day 21 after transplantation,the expressions of AKT,P-AKT and Erk were lower than those of aGVHD group,but the expression of P-Erk was higher than that of aGVHD mice.3)The expressions of Erk,P-Erk and P-AKT in aGVHDspleen-CHKA+/-group were lower than those in aGVHD group on day 14 and day 21 after transplantation,and the changes of AKT expression were not significant.Conclusion1.Before the occurrence of aGVHD,CD4+T cells began to show differentiation bias toward Th1 cells,the proportion of Treg cells decreased significantly,and the corresponding inflammatory cytokines began to secrete increased.The expression of CHKA in T cells was significantly up-regulated before and after aGVHD.The significant increase of plasma choline metabolites before aGVHD may be related to the up-regulation of CHKA expression in T cells.2.Inhibition of CHKA expression by CK37 can reduce the GVHD score of mice,prolong the survival of mice,and promote the differentiation of CD4+T cells into Treg cells,inhibit the secretion of inflammatory cytokines,and promote the production of IL-10.3.Knockout of CHKA gene in donor spleen cells can improve the severity of aGVHD in mice,and prolong the survival of mice.Moreover,it can promote the differentiation of CD4+T cells into Treg cells,inhibit the secretion of inflammatory cytokines,and promote the production of IL-10.4.CHKA may interfere with the differentiation of T cell subsets under aGVHD by regulating PI3K/AKT pathwayOur results suggest that the expression of CHKA in T cells is closely related to aGVHD.The expression of CHKA in T cells was up-regulated both before and after aGVHD.The increase of plasma choline metabolites before aGVHD may be related to the up-regulation of CHKA expression in T cells.Down-regulation of CHKA expression in donor T cells can promote the differentiation of CD4+T cells into Treg cells,inhibit the secretion of inflammatory cytokines,promote the production of IL-10,and improve the progression of aGVHD.CHKA may interfere with the differentiation of T cell subsets under aGVHD by regulating PI3K/AKT pathway.Down-regulation of CHKA expression in donor T cells may provide new ideas for early prevention and treatment of aGVHD. |
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