Tacrolimus Maintains the Balance of Neutrophil Extracellular Traps by Inducing DNA Methylation of Neutrophils to Reduce Immune Rejection

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.