Tacrolimus Regulates MicroRNAs in Recipient Immune Cells after Experimental Cardiac Transplantation

Objectives: Tacrolimus is a potent therapeutic agent in the prevention of acute allograft rejection after heart transplantation. Detailed molecular mechanisms and modes of action exceeding calcineurin inhibition, however, partly still remain elusive. Manipulating regulatory, non-coding microRNAs (miRNAs) in transplantation may have substantial therapeutic impact via interference with multiple pathways but it is unclear if miRNAs are affected by tacrolimus. We here assessed the effect of tacrolimus treatment on miRNA modulation in rodent heart transplantation.

Methods: Heterotopic heart transplantations were performed in rats in a syngeneic (Lewis to Lewis) and an allogeneic high-responder strain combination (Brown Norway to Lewis) to induce acute cellular rejection. Allogeneic animals received either 6mg/kg tacrolimus or vehicle (control group) by oral gavage (n = 12/group). Hearts were harvested 6 days post transplantation (n = 6/group) and acute rejection was quantified via IFN-γ ELISPOT assays, mixed lymphocyte reactions and analysis of donor-specific antibodies. miRNA profiling was performed to determine differential expression. Graft survival was examined in n = 6/group.

Results: As expected, acute cellular rejection was more profound in controls in comparison to tacrolimus treatment as indicated by IFN-γ spot frequencies (2150 ± 634 vs. 34 ± 35, p < 0.0001), proliferating cells in mixed lymphocyte reactions (34.5 ± 16.9% vs. 0.1 ± 0.2%, n = 4 control animals, p < 0.0001) and donor-specific antibody production (mean fluorescence 411 ± 91 vs. 65 ± 30, p < 0.0001, respectively). Interestingly, miRNA analyses revealed differential expression of the miRNA-17–92 cluster. Whereas being upregulated in grafts and spleens in allogeneic in comparison to syngeneic transplantation, tacrolimus treatment alleviated changes of miRNA-17 and -19 to comparable levels as in syngeneic transplantation. 10 days of tacrolimus treatment resulted in a graft survival above 20 days whereas in none of the control animals graft survival exceeded 10 days.

Conclusion: miRNA-17 and -19 are overexpressed in acute cellular allograft rejection. By exerting pro-inflammatory effects and facilitating Th1-mediated immune responses they might significantly contribute to rejection. We demonstrate for the first time that tacrolimus treatment is targeting miRNA-17 and -19 which represents a novel mode of action of tacrolimus and is a promising new pathway for the development of novel immunosuppressive agents.