Digoxin Attenuates Acute Cardiac Allograft Rejection by Antagonizing RORγt Activity

Th17 responses have been suggested to participate in the pathogenesis of acute allograft rejection. RORγt is the master transcription factor that controls Th17 cell differentiation and expansion. However, little is known about the effect that antagonizing RORγt activity may have on acute cardiac allograft rejection.A model of heterotopic murine cardiac transplantation with total allomismatch (BALB/c to B6 mice) was used. Digoxin, which was recently identified as a specific antagonist of RORγt, was injected intraperitoneally daily (40 μg) starting 1 day after cardiac transplantation. The severity of rejection was determined by histology. The mRNA expression levels of cytokines and transcription factors in the grafts were measured by quantitative real-time PCR. The proportion and number of T-cell subpopulations in the allografts and spleens were analyzed by flow cytometry. In vitro, the effect of digoxin on allogeneic responses and the interleukin (IL)-6-mediated conversion of regulatory T cells (Treg) into Th17 cells were investigated.Treatment with digoxin significantly prolonged cardiac allograft survival compared with dimethyl sulfoxide treatment (mean survival time, 16.5±2.2 versus 8.1±0.7 days; P<0.01). Treatment with digoxin also markedly suppressed the mRNA expression levels of IL-17A, IL-17F, and granulocyte-macrophage colony-stimulating factor, reduced the number of Th17 cells, and induced Treg expansion in the allografts. In vitro, treatment with digoxin did not inhibit the proliferation of T cells in a mixed lymphocyte reaction, but it did inhibit the IL-6-mediated conversion of Tregs into Th17 cells.RORγt may be a promising therapeutic target to attenuate acute cardiac allograft rejection. Digoxin therefore provides a molecular basis for the design of novel immunosuppressive agents.