AUGMENTED INFLAMMATORY RESPONSES IN AGING ARE DRIVEN BY CIRCULATING MTDNA AND AMELIORATED BY SENOLYTIC TREATMENT

Timm Heinbokel ⁰; Midas Seyda ^0,0; Markus Quante ⁰; Guangxiang Liu ⁰; Hirofumi Uehara ⁰; JeroenM. Schuitenmaker ⁰; Reza Abdi ⁰; ChristineS. Falk ⁰; Abdala Elkhal ⁰; StefanG. Tullius ⁰

⁰Division of Transplant Surgery and Transplant Surgery Research Laboratory, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, United States; ²Institute of Transplant Immunology, Hannover Medical School, Hannover, United States; ³Renal Division, Schuster Family Transplant Center, Brigham and Women's Hospital, Boston, MA, United States

Introduction: Older donor organs are increasingly utilized to close the gap between demand and supply. We submit that aging-associated kinetics of damage associated molecular patterns (DAMPs) such as mitochondrial DNA (mtDNA) drive the augmented susceptibility of older organs to ischemia and reperfusion injury (IRI).

Methods: Old and young mice underwent bilateral clamping of the renal pedicles (ischemic time, 22 min at controlled body temp.) representing an established model of IRI. MtDNA levels were tested by qPCR; Dendritic Cell (DC) and T cell activation was characterized by FACS. Old or young DCs were adoptively transferred into young recipients that subsequently received young or old cardiac allografts (Hx).

Results: DC maturation was associated with elevated levels of circulating mtDNA in naïve old mice. Young animals, in contrast, had no detectable mtDNA levels (n=5/group; p=0.016). Importantly, renal IRI induced the release of mtDNA into the circulation in an age-dependent fashion with elevated amounts in old animals (n=3-5/group by POD 2; p=0.019). At the same time, old animals showed an increased IFN-γ expression in splenic CD8⁺ T cells (n=4-5/group by POD 2; p=0.0001). DCs co-cultured with old plasma DNA demonstrated an up-regulation of the co-stimulatory molecules CD40 and CD80. Moreover, DCs showed a dose-dependent up-regulation of CD40 and augmented amounts of IL-6 in the presence of isolated mtDNA, while the addition of a TLR9 antagonist attenuated the up-regulation. Old DCs also promoted IFN-g and IL-17 responses of allogeneic T cells in vitro. In vivo, adoptive transfer of old but not young DCs prior to Hx reduced cardiac allograft survival (median graft survival: 11.5 d Y-Hx vs. 10 d O-Hx vs. 9 d O-Hx + O-DCs; Log-rank test: p<0.0001; n=8-10/group). Finally, treatment of old mice with either Navitoclax, a senolytic agent, or a TLR9 antagonist prior to IRI reduced IFN-γ expression in splenic CD8⁺ T cells (n=4-5/group by POD 2; p<0.05).

Conclusion: These results suggest that aging increases mtDNA levels, favoring DC activation which can orchestrate pro-inflammatory T cell responses affecting the survival of older grafts.