Conclusion
Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.
Methods
To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus.
Results
Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus.