*Purpose: FTY720, a S1PR agonist has been shown to protect kidneys from IRI. However, systemic use is limited due to adverse side effects. Dendritic cells (DCs) have the ability of regulate innate and adaptive responses. In the current study we tested if FTY720 could induce mitochondria biogenesis to induce regulatory DCs phenotype to use as cellular therapy to limit the off-target effects associated with systemic drug administration in kidney IRI.

*Methods: Renal injury was assessed by plasma creatinine (PCr; mg/dl). 8-wk old C57BL/6 WT mice were used for generating highly pure DCs from bone marrow precursors. Cells were treated with cocktail of GM-CSF ± FTY720. On day 7 DCs were treated with 100ng/ml LPS or Veh to induce activation. All four groups (Veh/Veh, Veh/LPS, FTY/Veh and FTY/LPS) were analyzed using real-time PCR for cytokines and mitochondria: nuclear DNA ratio, facs (for activation (MHCII) and co-stimulation (CD40/80/86) markers, and mitochondrial function (mass (MitoTrackerGreen), potential (CMXRos), oxidation (MitoSOX)) and Seahorse flux analyzer for MitoStress. For in vivo studies, Veh/LPS and FTY/LPS BMDCs (0.5X10⁶, i.v.) were transferred 24 hrs prior to or 4hrs after IRI.

*Results: FTY720-DCs have higher oxygen consumption rate (38.18 vs 107.16; p=0.01), ATP production (45.29 vs 101.12; p=0.01) and spare respiratory capacity (163.67 vs 314.82) compared to vehicle DCs. FTY/LPS-DCs have significantly lower expression of MHCII and CD40/80/86 compared to Veh/LPS-DCs. FTY/LPS-DCs also have higher mtDNA/ncDNA ratio, significantly higher MitoSox (MFI, 800±50.1 vs 152±10.3; p=0.001) and MitoTracker Green (515±25.1 vs 1000±16.1; p=0.001) signal compared to Veh/LPS-DCs. FTY/LPS-DCs have higher gene expression for IL10, S1Pr1 and PGC1α compared to Veh/LPS-DC. Furthermore, transfer of FTY/LPS-DCs to WT mice 24hrs prior (PCr, 0.17±0.02 vs 1.5±0.17, p=0.001) or 4hrs after IRI (PCr, 0.53±0.08 vs 1.3±0.12, p=0.01) significantly protects the kidneys from IRI compared to Veh/LPS-DCs treated mice. FTY-DCs dependent protection from IRI is abolished in absence of a spleen or if FTY-DCs mitochondria is functionally disrupted by treatment with rotenone/Antimycin A prior to transfer. Lastly, FTY-DC dependent protection is maintained in allogeneic transfers (Balb/c FTY-DC into C57BL/6).

*Conclusions: FTY720 induces regulatory phenotype in DCs by modulating mitochondrial biogenesis and adoptive transfer of FTY720-DCs either prior to or after ischemic injury protects kidneys from IRI. FTY720-DCs donate mitochondria to splenic marginal zone CD169⁺ macrophages to induce an increase in Tregs numbers by producing CCL22.

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