目的:先前的研究阐明卡培他滨(CAP)作为一种具有公认免疫抑制作用的抗肿瘤药物。然而,这些影响的复杂机制仍有待阐明。在这项研究中,我们旨在阐明CAP发挥其免疫抑制作用以减少同种异体移植排斥反应的分子途径.
方法:将男性BALB/c供体的心脏移植到男性C57BL/6受体中,并用CAP处理7天。使用一系列技术评估了这些心脏移植的排斥反应,包括H&E染色,免疫组织化学,RNA测序,LS-MS/MS,和流式细胞术。体外,原始CD4+T细胞被分离并在Th1条件培养基中培养,不同的处理,流式细胞术,LS-MS/MS用于描述胸苷合酶(TYMS)在Th1分化过程中的作用。
结果:CAP治疗显著减轻了急性同种异体移植物排斥反应,并通过减少移植物损伤提高了移植物存活,T细胞浸润,和循环促炎细胞因子的水平。此外,它减少了CD4+T细胞增殖和脾脏中Th1细胞的存在。RNA-seq显示TYMS,CAP的目标,移植后脾细胞强烈增加。体外,TYMS及其代谢产品dTMP在Th0和Th1中差别表达,并在激活CD4+T细胞和Th1分化后必需。TYMS特异性抑制剂,雷替曲塞,和卡培他滨的代谢物,5-氟尿嘧啶,可以抑制Th1的增殖和分化。最后,联合使用CAP和常用的免疫抑制剂雷帕霉素可以诱导同种异体移植物的长期存活。
结论:CAP经历代谢转化以干扰嘧啶代谢,它靶向TYMS介导的Th1分化,从而在减轻小鼠模型中急性心脏同种异体移植排斥反应中发挥重要作用。
OBJECTIVE: Previous studies elucidated that capecitabine (CAP) works as an anti-tumor agent with putative immunosuppressive effects. However, the intricate mechanisms underpinning these effects remain to be elucidated. In this study, we aimed to unravel the molecular pathways by which CAP exerts its immunosuppressive effects to reduce allograft rejection.
METHODS: Hearts were transplanted from male BALB/c donors to male C57BL/6 recipients and treated with CAP for seven days. The rejection of these heart transplants was assessed using a range of techniques, including H&E staining, immunohistochemistry, RNA sequencing, LS-MS/MS, and flow cytometry. In vitro, naïve CD4+ T cells were isolated and cultured under Th1 condition medium with varying treatments, flow cytometry, LS-MS/MS were employed to delineate the role of thymidine synthase (TYMS) during Th1 differentiation.
RESULTS: CAP treatment significantly mitigated acute allograft rejection and enhanced graft survival by reducing graft damage, T cell infiltration, and levels of circulating pro-inflammatory cytokines. Additionally, it curtailed CD4+ T cell proliferation and the presence of Th1 cells in the spleen. RNA-seq showed that TYMS, the target of CAP, was robustly increased post-transplantation in splenocytes. In vitro, TYMS and its metabolic product dTMP were differentially expressed in Th0 and Th1, and were required after activation of CD4+ T cell and Th1 differentiation. TYMS-specific inhibitor, raltitrexed, and the metabolite of capecitabine, 5-fluorouracil, could inhibit the proliferation and differentiation of Th1. Finally, the combined use of CAP and the commonly used immunosuppressant rapamycin can induce long-term survival of allograft.
CONCLUSIONS: CAP undergoes metabolism conversion to interfere pyrimidine metabolism, which targets TYMS-mediated differentiation of Th1, thereby playing a significant role in mitigating acute cardiac allograft rejection in murine models.