Introduction: Solid organ transplantation has seen a significant increase in the utilization of older organs. Here, we investigate how aging-associated kinetics of damage associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA) are driving the augmented susceptibility of older organs to ischemia and reperfusion injury (IRI) through dendritic cells (DCs) with subsequent inferior graft survival.
Methods: Old and young mice underwent bilateral clamping of the renal pedicles (22 min ischemic time at controlled normothermia). mtDNA, cytokine and senescence marker levels were tested by qPCR; DC and T cell activation were characterized by FACS. Old and young DCs were adoptively transferred into young recipients that subsequently received young or old cardiac allografts.
Results: DCs of old naïve mice showed higher frequencies and levels of maturation in parallel to increased baseline levels of mtDNA. Importantly, renal IRI induced a prominent release of mtDNA into the circulation of old animals (n=5/group after 48h; p=0.019) and increased IFN-γ expression in splenic CD8⁺ T cells (p=0.0001). Isolated DCs showed a dose-dependent up-regulation of CD40 with augmented amounts of IL-6 in the presence of mtDNA; the addition of a TLR9 antagonist attenuated this pro-inflammatory response of old DCs. In addition, old DCs promoted IFN-γ and IL-17 responses of allogeneic T cells in vitro. Of particular relevance, adoptive transfer of old but not young DCs prior to transplantation shortened cardiac allograft survival (log-rank test: p<0.0001; n=8-10/group).
Treatment with the senolytic agents Dasatinib (5mg/kg) and Quercetin (50mg/kg) not only reduced local expression of senescence marker p16 and p21 in kidneys, but also drastically reduced local and systemic mtDNA levels in old mice. In parallel, levels of IFN-γ and IL-17 and systemic frequencies of CD8⁺IFN-γ⁺ and CD4⁺IL-17⁺ T cells were reduced subsequent to the treatment with senolytics. These in-vitro effects translated into prolonged allograft survival of cardiac allografts procured from old donor mice that underwent pretreatment with senolytic agents, compared to allografts procured from old untreated donors (log-rank test: p<0.0280; n=6/group).
Conclusion: Our results suggest a pivotal role for mtDNA in driving allogeneic immune responses to old donor organs, critically affecting graft survival. Senolytic treatment reduced mtDNA release and associated inflammation with prolonged graft survival, thus representing a novel approach for increased utilization of marginal donor organs.