The Study Of 4-OI Improving Mice AGVHD After HSCT

Objective:The development of acute graft-versus-host disease(aGVHD)is a common and serious complication after allogeneic hematopoietic stem cell transplantation(allo-HSCT),affecting the efficacy of transplantation therapy and limiting the use of allo-HSCT.The development of aGVHD is driven by a strong immune attack by T cells in the donor graft against normal tissue of the recipient.After activation of naive/resting T cells in the transplant recipient by allogeneic antigens,they proliferate and differentiate into allo-reactive T cells,causing target organ damage.Allo-reactive T cells have higher glycolytic activity compared to their non-allo-reactive counterparts.The activation and function of allogeneic T cells can be reduced by inhibiting glucose metabolizing enzymes.Our previous study found that 4-OI(a cell-permeable derivative of endogenous itaconic acid)can reduce T cell growth and activation by inhibiting aerobic glycolysis.The main objective of this work was to create an aGVHD mouse model and evaluate the effect of 4-OI on aGVHD after allo-HSCT in order to provide new possibilities for the development of new therapeutic medicines to prevent and treat aGVHD.Method:In vivo experiments,Splenocytes and bone marrow cells obtained from C57BL/6 mice(H-2Kb)were transplanted into lethally irradiated(600 c Gy)BALB/c mice(H-2Kd)to establish an aGVHD model.orbital blood collection to detect the percentage of H-2Kb cells in the recipient mice.A daily dose of 4-OI(50 mg/kg)or a vehicle was given(control was configured according to the 4-OI formulation,where 4-OI was replaced with PBS).After 4 weeks,we observed the survival status and evaluated the severity of aGVHD by clinical score and pathology score.Effect of 4-OI on donor T cell proliferation by flow cytometry.We used C57BL/6 background B6.FVB-ptprca L2G85 mice donor T cells expressing luciferase linked to theβ-actin promoter and used Bioluminescence imaging(BLI)to measure the expansion of donor T cells.The effects of 4-OI on cell metabolism were examined by seahorse.Detection of 4-OI effects on cytokines in mice using CBA kit.Results:Mice demonstrated weight loss,decreased mobility,arching back,and shrugging hair from day 2 after allo-HSCT.Peripheral blood cell flow results showed that the percentage of CD45~+H2-Kb~+cells in peripheral blood cells was greater than 80%,confirming the successful establishment of aGVHD model.In aGVHD disease model mice,4-OI treatment reduced aGVHD sign manifestations and clinical scores,and improved survival rates.BLI imaging showed that 4-OI treatment resulted in a significant decrease in proliferation of donor T cells.In the 4-OI treatment group,the glycolysis rate of T cells from spleen of recipient mice was significantly decreased,the activity of GAPDH enzyme in mouse spleen and peripheral blood T cells was significantly reduced.And when we detected the cytokines of the serum,the levels of TNF-α,IFN-γ,MCP-1,IL-12p70 and IL-6 from 4-OI treatment group were also greatly reduced compared to the control group.Conclusion:We demonstrated that 4-OI,as an immune inflammatory regulator,could delay the development of aGVHD after allo-HSCT in mice by inhibiting T cell proliferation and inflammatory factor secretion.These results offered novel insights and possibilities to inhibit the development of aGVHD through metabolic reprogramming and highlight the importance of targeting aerobic glycolysis,in the treatment of immune diseases.