Digoxin Attenuates Acute Cardiac Allograft Rejection by Antagonizing RORγt Activity

Background. Th17 responses have been suggested to participate in the pathogenesis of acute allograft rejection. ROR gamma t is the master transcription factor that controls Th17 cell differentiation and expansion. However, little is known about the effect that antagonizing ROR gamma t activity may have on acute cardiac allograft rejection. Methods. 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 ROR gamma t, was injected intraperitoneally daily (40 Kg) 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. Results. 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. Conclusions. ROR gamma t 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.