Inhibition of DRP1–FIS1–Mediated mitochondrial fission as a novel strategy to prevent gvhd

Abstract Graft-versus-host disease (GVHD) remains a leading cause of non-relapse mortality following hematopoietic stem cell transplantation (HSCT). GVHD is primarily driven by the activation of donor T cells by host-derived antigen-presenting cells (APCs), a process that is initiated by tissue damage induced by pre-HSCT cytotoxic conditioning. The severity of GVHD has been closely linked to the release of damage-associated molecular patterns (DAMPs), particularly mitochondrial DAMPs (mtDAMPs), released from intestinal epithelial cells (IECs). Our previous work has demonstrated that cytotoxic conditioning promotes excessive mitochondrial fission in IECs, mediated by activation and interaction of dynamin-related protein 1 (DRP1) and Fission Protein 1 (FIS1) resulting in extracellular release of mtDAMPS and exacerbation of GVHD. In this study, we tested the efficacy of P110, a novel peptide inhibitor of the DRP1–FIS1 interaction, as a therapeutic strategy for reducing conditioning mediated extracellular release of dysfunctional mitochondria in attenuating APC activation, and preventing GVHD without broadly suppressing immune function. Using the Caco-2 cell line as a model of intestinal epithelium, we observed that P110 treatment following irradiation preserved mitochondrial morphology and significantly reduced the levels of circulating cell-free mitochondria. In vivo, lethally irradiated Balb/c mice exhibited profound mitochondrial fragmentation in IECs by electron microscopy, which was markedly alleviated by P110 administration. Flow cytometric analysis of platelet-depleted plasma revealed a significant reduction in extracellular mitochondria 24 hours after irradiation in P110-treated mice compared to controls. In a B10.D2 to Balb/c minor mismatch GVHD model, P110 treatment significantly reduced clinical GVHD severity. Immune profiling of spleen and mesenteric lymph nodes showed a reduced frequency of CD8⁺ T cells and an increased proportion of naïve CD4⁺ and CD8⁺ T cells, along with a significant decrease in effector memory T cells in P110-treated mice. Additionally, activation markers CD25 and CD69 were downregulated in both CD4⁺ and CD8⁺ T cells, while expression of the exhaustion marker PD-1 was elevated. Together, these findings suggest that inhibition of DRP1–FIS1 interaction by P110 represents a promising therapeutic strategy to mitigate GVHD.