PDF(Pubmed)
Abstract：
Immune rejection is a significant concern in organ transplantation, as it can lead to damage to and failure of the transplanted organ. To prevent or treat immune rejection, transplant recipients are commonly administered immunosuppressive drugs. Tacrolimus (FK506) is a widely used immunosuppressive drug in organ transplantation. The excessive formation of neutrophil extracellular traps (NETs) can contribute to inflammation and tissue damage. Although NETs play an antimicrobial role, their overproduction can be harmful. To investigate the mechanism by which FK506 suppresses immune rejection, we utilized HL-60 cells, which were differentiated into neutrophils using DMSO and induced to form NETs with phorbol myristate acetate (PMA), a very efficient and frequently used drug for inducing NET formation. By comparing pre- and post-treatment with FK506, we examined whether FK506 affects the formation of NETs. Various experimental techniques were employed, including confocal imaging for visualizing cell NETs, qPCR and Western blotting for gene and protein expression analyses, ELISAs for protein content detection, and LC-MS/MS for methylation detection. In our study, we discovered that FK506 can enhance DNA methylation, which likely contributes to the reduction in NETs. Genes and proteins related to methylation, namely, DNMT3B and TET3, exhibited significant correlations with methylation. Consistent changes in both genes and proteins suggest that DNMT3B and TET3 are key factors that are influenced by FK506, resulting in enhanced DNA methylation and the potential inhibition of PMA-induced NET production. In summary, we have identified a novel mechanism by which FK506 inhibits NET production through the enhancement of DNA methylation. This finding highlights a new aspect of FK506\'s immunosuppressive effect. Our results provide valuable insights for clinical research, immunosuppression, and organ preservation strategies.
摘要：
免疫排斥是器官移植中的一个重要问题，因为它可能导致移植器官的损伤和衰竭。为了预防或治疗免疫排斥，移植受者通常服用免疫抑制药物。他克莫司(FK506)是一种广泛应用于器官移植的免疫抑制剂。中性粒细胞胞外陷阱（NETs）的过度形成可导致炎症和组织损伤。虽然NET发挥抗菌作用，他们的生产过剩可能是有害的。探讨FK506抑制免疫排斥反应的机制,我们利用HL-60细胞，使用DMSO将其分化为嗜中性粒细胞，并用佛波醇肉豆蔻酸酯乙酸酯（PMA）诱导形成NETs，一种非常有效且经常使用的诱导NET形成的药物。通过用FK506比较治疗前和后处理,我们检查了FK506是否影响NETs的形成。采用了各种实验技术，包括用于可视化细胞NET的共聚焦成像，qPCR和蛋白质印迹用于基因和蛋白质表达分析，用于蛋白质含量检测的ELISA，和LC-MS/MS用于甲基化检测。在我们的研究中,我们发现FK506可以增强DNA甲基化，这可能有助于减少NET。与甲基化相关的基因和蛋白质，即,DNMT3B和TET3与甲基化显著相关。基因和蛋白质的一致变化表明DNMT3B和TET3是受FK506影响的关键因素，导致DNA甲基化增强和PMA诱导的NET产生的潜在抑制。总之,我们已经确定了FK506通过增强DNA甲基化抑制NET产生的新机制。这一发现突出了FK506免疫抑制作用的一个新方面。我们的结果为临床研究提供了有价值的见解，免疫抑制,和器官保存策略。
