Preventative Effect of Glucolysis Inhibitor on Acute Graft Versus Host Disease in Mice after Allogenic Bone Marrow Transplantation

Background: T-cell activation plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Quiescent T cells utilize oxidative phosphorylation to generate ATP, whereas activated T cells utilize glycolysis, so using glycolysis inhibitor may be a metabolically regulator needed to control T cells inducing GVHD. Glucolysis inhibitor 3-Bromopyruvic acid (3-BrPA), a glucolysis inhibitor, can effectively induce multi-drug resistance leukemia cell lines apoptosis and enhance chemotherapy-induced cytotoxity to leukemia cells.

Objective: This study aims to investigate the effect of glucolysis 3-BrPA inhibitor on aGVHD in mice after allogenic bone marrow transplantation (allo-BMT) and its mechanism.

Methods: aGVHD model after allo-BMT was established by use of C57BL/6(H-2kb) mice as donors and BALB/c(H-2kd) mice as receptors. Bone marrow and/or spleen cells from donor mice were injected within 4h after total body irradiation (TBI) to receptor mice via tail vein. Drugs were administrated 1 h after the cells were injected as follows: (1) control: the TBI group without cells injection; (2) the BMT group with bone marrow cells injection; (3) the GVHD group with bone marrow and spleen cells injection; (4) the rap amycin group (RAPA, 20 nM/d×7d) with bone marrow and spleen cells injection; (5) the 3-BrPA group (3-BrPA, 50 nM/d×7d) with bone marrow and spleen cells injection; (6) the 3-BrPA(25 nM/d×7d) and rapamycin(10 nM/d) combination group with bone marrow and spleen cells injection. The transplanted mice were observed for symptoms of GVHD, survival time, survival rate and Thomas GVHD pathologic grade. H-2kb was determined in BALB/c(H-2kd) mice by flow cytometry (FCM) to confirm allogeneic chimeric rate, and serum level of cytokine was detected by protein microarray.

Results: Allogeneic chimeric rates of survived mice determined at day 21 after transplantation ranged from 95% to100%, confirming complete donor mouse implantation. All mice in TBI group died within 14 d. Median survival time of mice was respectively 9.1, 20, 17.1 and 24.5 days in GVHD group , rapamycin group, 3-BrPA group and the combination of 3-BrPA and rapamycin group. Rapamycin group, 3-BrPA group and the combination of 3-BrPA and rapamycin group had increased median survival time than GVHD group, and mice received the combination of rapamycin and 3-BrPA had better survival than rapamycin or 3-BrPA group(n=10 for each group, P<0.01). GVHD-related symptoms scores of all the drug treated groups on 7-day and 14-day after transplantation were decreased compared to GVHD group (F=15.006, P<0.001). The cytokine arrays showed that the levels of Th1-associated cytokines IFN-γ increased and the levels of Th2-associated cytokines IL-4 deceased in the groups with drug treated compared to the GVHD group.

Conclusions: In this study, the glycolysis inhibitor 3-BrPA has a significant inhibitory effect on GVHD, especially in combination with rapamycin. This effect might be achieved by regulating immune cells bias from Th1 cells towards Th2 cells.