MITOCHONDRIAL TRANSPLANTATION INTO HUMAN MONOCYTES PROMOTES IMMUNE RESOLUTION IN INFLAMMATORY BOWEL DISEASE

Abstract INTRODUCTION Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, affects over 2.4 million individuals in the United States and remains a major global health challenge. Current therapies primarily suppress inflammation but do not correct underlying immune dysfunction, leading to resistance, limited durability, and safety concerns. Monocytes are key regulators of intestinal inflammation and epithelial repair, but in IBD their transition to pro-resolving macrophages is impaired due to mitochondrial dysfunction, dysregulated IL-10 signaling, and persistent TNF-α production. Innovative strategies that restore monocyte function are urgently needed. METHODS Fresh mitochondria were isolated from HEK293T cells, quality-checked by Seahorse oxidative phosphorylation (OXPHOS) assays, and transplanted into human CD14+ monocytes (1 × 105 cells, 24 h, 37 °C). Uptake was assessed by flow cytometry, confocal imaging, and citrate synthase (CS) activity. THP1-Dual™ reporter monocytes were used to test activation of NF-κB and IRF pathways. Bioenergetics were measured using Seahorse mitochondrial stress tests at 1- and 6-day cultures. Cytokine secretion in LPS-stimulated monocytes was quantified using LEGENDplex assays, and phagocytosis was measured with pHrodo™ E. coli uptake. In vivo efficacy was tested in C57BL/6 mice with DSS-induced colitis treated with intravenous MITO or PBS. RESULTS We observed that MITO transplantation increased monocyte CS activity in a dose-dependent manner and confirmed uptake without loss of viability. THP1-Dual™ assays showed no activation of NF-κB or IRF, confirming biocompatibility. MITO-transplanted monocytes (MTM) demonstrated significantly enhanced basal, ATP-linked, and maximal respiration at both 1 day and 6 days, indicating durable OXPHOS augmentation. In an IBD-like environment, MTM exhibited a profound cytokine shift with ∼10-fold reduction in TNF-α, ∼20-fold increase in IL-10, and elevated IL-2 compared to LPS-stimulated controls. MTM also showed significantly enhanced OXPHOS-dependent phagocytic activity. In DSS-colitis mice, MITO therapy preserved epithelial architecture, maintained crypt integrity, reduced mucosal inflammation, and lowered histological damage scores compared with PBS controls. CONCLUSION In conclusion, we observed that mitochondrial transplantation into monocytes is feasible, biocompatible, and reprograms immune metabolism toward a pro-resolving phenotype. By enhancing OXPHOS, shifting cytokine balance, and improving microbial clearance, MITO therapy addresses a root defect in IBD pathogenesis. In vivo, mitochondrial therapy preserved epithelial integrity in experimental colitis, supporting its potential as a first-in-class, non-steroidal, cell-targeted therapy for IBD.