| Aplastic anemia(AA)is a serious hematological disease with high mortality and the main reason of death is uncontrollable bleeding and infection.The pathogenesis of acquired AA has not been fully elucidated,and the mainstream view is that AA is caused by abnormally activated T cells attacking bone marrow(BM)hematopoietic stem cells and other important BM cells,thus leading to normal BM cells replaced by fatty cells and BM failure.The evidence that immunosuppressive therapy combined with anti-thymocyte globulin and cyclosporine A(Cs A)can rescue most of the patients with AA further confirms the abnormal immune reaction in AA etiology.However,the serious side effects caused by long-term use of Cs A and relapse after the discontinuation of Cs A remain unresolved,so it is very impreative to develop new treatment strategies to supplement current therapeutics.As a normal metabolite of vitamin A in vivo,all-trans-retinoic acid(ATRA)displays immunoregulatory role and influences T cell differentiation in several studieas,so it may generate an effect in AA treatment.In this study,by employing T cell-mediated AA mouse model,we tested the potential of ATRA in the AA treatment and explored its possible mechanism by using several studying methods,such as flow cytometry,immunofluorescence,liquid chromatography-mass spectrometry,RNA-sequencing,chromatin immuneprecipitation sequencing,etc.,we found that ATRA was an effective agent to improve AA.ATRA reduced the infiltration of BM T cells,increased BM nucleated cell counts and hematopoietic stem cell counts,improved peripheral blood white cells and platelets.ATRA inhibited BM inflammatory hyperplastic angiogenesis and decreased the percentage of endothelial cells,protected important BM components,such as mesenchymal stem cells and megakaryocyte,and extended long-term survival of AA mice.ATRA inhibited in-vivo proliferation,activation,trafficking and effector function of T cells in AA mice,ATRA reduced Ki67,LFA-1,CD44,CXCR4 and Fasl expression on BM T cells,and decreased Fas expression of BM non-T cells.ATRA inhibited the metabolic process of T cells,increased the amount of betaine in BM T cells whereby ATRA cleared away the cellular and mitochondrial reactive oxygen spiecies in T cells and reduced palmitic amide which functions as an important energy source for activated T cells.ATRA maintained the proportion of BM regulatory T cells(Tregs)in AA mice to the similar level as TBI control,however,Tregs transplantation cannot improve the outcome of AA mice,indicating that maintaining the stability of Tregs was not the main mechanism that ATRA ameliorated AA mice.ATRA decreased the percentage of T helper(Th)1 cell and Th17 cell and increased the percentage of Th2 cell in BM of AA mice.ATRA reduced Th1 and Th17 cell-related pro-inflammatory cytokines and increased Th2-cell associated anti-inflammatory cytokines of plasma,therefore,ATRA rebalanced T cell substes which were disturbed by abnormal immune balance of AA and partially restored Th1/Th2 ratio.Further analysis revealed that ATRA inhibited expression of IFN-? and the Th1-cell key transcription factor-T-bet and promoted the positive regulator-Jun B of Th2 cell by targeting the NFAT1 pathway,thus shifting Th1 towards Th2 cell differentiation.In addition to downregulating Th17 cell mastor regulator-ROR?t,ATRA also activated RAR? signaling to upregulate Irf8 which is a negative regulator toward Th17 differentiation,and thus demonstrating powerful ability to inhibit Th17 cell differentiation.In conclusion,we confirmed the efficacy of ATRA in treating T cell-mediated acquired AA mice,and elucidated the mechanism of ATRA in restoring immune balance,and our study laid a solid foundation for further clinical application due to its economics and safety. |
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