BACKGROUND: Mitochondrial transfer is becoming recognized as an important immunomodulatory mechanism used by mesenchymal stem cells (MSCs) to influence immune cells. While effects on T cells and macrophages have been documented, the influence on B cells remains unexplored. This study investigates the modulation of B lymphocyte fate by MSC-mediated mitochondrial transfer. METHODS: MSCs labelled with MitoTracker dyes or derived from mito::mKate2 transgenic mice were co-cultured with splenocytes. Flow cytometry assessed mitochondrial transfer, reactive oxygen species (ROS) levels, apoptosis and mitophagy. Glucose uptake was measured using the 2-NBDG assay. RNA sequencing analysed gene expression changes in CD19+ mitochondria recipients and nonrecipients. Pathway analysis identified affected processes. In an LPS-induced inflammation model, mito::mKate2 MSCs were administered, and B cells from different organs were analysed for mitochondrial uptake and phenotypic changes. MSC-derived mitochondria were also isolated to confirm uptake by FACS-sorted CD19+ cells. RESULTS: MSCs transferred mitochondria to CD19+ cells, though less than to other immune cells. Transfer correlated with ROS levels and mitophagy induction. Mitochondria were preferentially acquired by activated B cells, as indicated by increased CD69 expression and glycolytic activity. Bidirectional transfer occurred, with immune cells exchanging dysfunctional mitochondria for functional ones. CD19+ recipients exhibited increased viability, proliferation and altered gene expression, with upregulated cell division genes and downregulated antigen presentation genes. In vivo, mitochondrial acquisition reduced B cell activation and inflammatory cytokine production. Pre-sorted B cells also acquired isolated mitochondria, exhibiting a similar anti-inflammatory phenotype. CONCLUSIONS: These findings highlight mitochondrial trafficking as a key MSC-immune cell interaction mechanism with immunomodulatory therapeutic potential.

1st Faculty of Medicine Charles University Prague Czech Republic

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Department of Physiology Faculty of Science Charles University Prague Czech Republic

Laboratory of Molecular Therapy Institute of Biotechnology Czech Academy of Sciences Prague West Czech Republic

School of Pharmacy and Medical Science Griffith University Southport Queensland Australia

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Dong LF, Rohlena J, Zobalova R, et al. Mitochondria on the move: horizontal mitochondrial transfer in disease and health. J Cell Biol. 2023;222(3):e202211044. doi: 10.1083/JCB.202211044/213873 PubMed DOI PMC

Yan W, Diao S, Fan Z. The role and mechanism of mitochondrial functions and energy metabolism in the function regulation of the mesenchymal stem cells. Stem Cell Res Ther. 2021;12(1):140. doi: 10.1186/s13287-021-02194-z PubMed DOI PMC

Huang T, Zhang T, Gao J. Targeted mitochondrial delivery: a therapeutic new era for disease treatment. J Control Release. 2022;343:89‐106. doi: 10.1016/J.JCONREL.2022.01.025 PubMed DOI

Paliwal S, Chaudhuri R, Agrawal A, Mohanty S. Human tissue‐specific MSCs demonstrate differential mitochondria transfer abilities that may determine their regenerative abilities. Stem Cell Res Ther. 2018;9(1):1‐9. doi: 10.1186/S13287-018-1012-0 PubMed DOI PMC

Lee H, Jeong OY, Park HJ, et al. Promising therapeutic effects of embryonic stem cells‐origin mesenchymal stem cells in experimental pulmonary fibrosis models: immunomodulatory and anti‐apoptotic mechanisms. Immune Netw. 2023;23(6):e45. doi: 10.4110/IN.2023.23.E45 PubMed DOI PMC

Liu Z, Sun Y, Qi Z, Cao L, Ding S. Mitochondrial transfer/transplantation: an emerging therapeutic approach for multiple diseases. Cell Biosci. 2022;12(1):66. doi: 10.1186/S13578-022-00805-7 PubMed DOI PMC

Mukkala AN, Jerkic M, Khan Z, Szaszi K, Kapus A, Rotstein O. Therapeutic effects of mesenchymal stromal cells require mitochondrial transfer and quality control. Int J Mol Sci. 2023;24(21):15788. doi: 10.3390/IJMS242115788 PubMed DOI PMC

Liu D, Gao Y, Liu J, et al. Intercellular mitochondrial transfer as a means of tissue revitalization. Signal Transduct Target Ther. 2021;6(1):1‐18. doi: 10.1038/s41392-020-00440-z PubMed DOI PMC

Zampieri LX, Silva‐Almeida C, Rondeau JD, Sonveaux P. Mitochondrial transfer in cancer: a comprehensive review. Int J Mol Sci. 2021;22(6):3245. doi: 10.3390/IJMS22063245 PubMed DOI PMC

Morrison TJ, Jackson M v, Cunningham EK, et al. Mesenchymal stromal cells modulate macrophages in clinically relevant lung injury models by extracellular vesicle mitochondrial transfer. Am J Respir Crit Care Med. 2017;196(10):1275‐1286. doi: 10.1164/rccm.201701-0170OC PubMed DOI PMC

Jackson MV, Morrison TJ, Doherty DF, et al. Mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS. Stem Cells. 2016;34(8):2210‐2223. doi: 10.1002/STEM.2372 PubMed DOI PMC

Yuan Y, Yuan L, Li L, et al. Mitochondrial transfer from mesenchymal stem cells to macrophages restricts inflammation and alleviates kidney injury in diabetic nephropathy mice via PGC‐1α activation. Stem Cells. 2021;39(7):913‐928. doi: 10.1002/STEM.3375 PubMed DOI

Piekarska K, Urban‐Wójciuk Z, Kurkowiak M, et al. Mesenchymal stem cells transfer mitochondria to allogeneic Tregs in an HLA‐dependent manner improving their immunosuppressive activity. Nat Commun. 2022;13(1):856. doi: 10.1038/s41467-022-28338-0 PubMed DOI PMC

Do JS, Zwick D, Kenyon JD, et al. Mesenchymal stromal cell mitochondrial transfer to human induced T‐regulatory cells mediates FOXP3 stability. Sci Rep. 2021;11(1):10676. doi: 10.1038/S41598-021-90115-8 PubMed DOI PMC

Court AC, Le‐Gatt A, Luz‐Crawford P, et al. Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response. EMBO Rep. 2020;21(2):e48052. doi: 10.15252/EMBR.201948052 PubMed DOI PMC

Luz‐Crawford P, Hernandez J, Djouad F, et al. Mesenchymal stem cell repression of Th17 cells is triggered by mitochondrial transfer. Stem Cell Res Ther. 2019;10(1):1‐13. doi: 10.1186/S13287-019-1307-9 PubMed DOI PMC

Akhter W, Nakhle J, Vaillant L, et al. Transfer of mesenchymal stem cell mitochondria to CD4+ T cells contributes to repress Th1 differentiation by downregulating T‐bet expression. Stem Cell Res Ther. 2023;14(1):1‐16. doi: 10.1186/S13287-022-03219-X PubMed DOI PMC

Irwin RM, Thomas MA, Fahey MJ, Mayán MD, Smyth JW, Delco ML. Connexin 43 regulates intercellular mitochondrial transfer from human mesenchymal stromal cells to chondrocytes. PubMed DOI PMC

Yarosz EL, Chang CH. The role of reactive oxygen species in regulating T cell‐mediated immunity and disease. Immune Netw. 2018;18(1):e14. doi: 10.4110/IN.2018.18.E14 PubMed DOI PMC

Zhang H, Wang L, Chu Y. Reactive oxygen species: the signal regulator of B cell. Free Radic Biol Med. 2019;142:16‐22. doi: 10.1016/J.FREERADBIOMED.2019.06.004 PubMed DOI

Han D, Zheng X, Wang X, Jin T, Cui L, Chen Z. Mesenchymal stem/stromal cell‐mediated mitochondrial transfer and the therapeutic potential in treatment of neurological diseases. Stem Cells Int. 2020;2020:8838046. doi: 10.1155/2020/8838046 PubMed DOI PMC

Vallabhaneni KC, Haller H, Dumler I. Vascular smooth muscle cells initiate proliferation of mesenchymal stem cells by mitochondrial transfer via tunneling nanotubes. Stem Cells Dev. 2012;21(17):3104‐3113. doi: 10.1089/SCD.2011.0691 PubMed DOI PMC

Osteikoetxea‐Molnár A, Szabó‐Meleg E, Tóth EA, et al. The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes. Cell Mol Life Sci. 2016;73(23):4531‐4545. doi: 10.1007/S00018-016-2233-Y PubMed DOI PMC

Zamora R, Korff S, Mi Q, et al. A computational analysis of dynamic, multi‐organ inflammatory crosstalk induced by endotoxin in mice. PLoS Comput Biol. 2018;14(11):e1006582. doi: 10.1371/JOURNAL.PCBI.1006582 PubMed DOI PMC

Zhang Z, Wang L, Li F, et al. Therapeutic effects of human umbilical cord mesenchymal stem cell on sepsis‐associated encephalopathy in mice by regulating PI3K/AKT pathway. J Integr Neurosci. 2022;21(1):38. doi: 10.31083/J.JIN2101038/1757-448X-21-1-038.PDF PubMed DOI

Li H, Dai H, Li J. Immunomodulatory properties of mesenchymal stromal/stem cells: the link with metabolism. J Adv Res. 2023;45:15‐29. doi: 10.1016/J.JARE.2022.05.012 PubMed DOI PMC

Xu Y, Shen J, Ran Z. Emerging views of mitophagy in immunity and autoimmune diseases. Autophagy. 2020;16(1):3‐17. doi: 10.1080/15548627.2019.1603547 PubMed DOI PMC

Malekpour K, Hazrati A, Soudi S, Hashemi SM. Mechanisms behind therapeutic potentials of mesenchymal stem cell mitochondria transfer/delivery. J Control Release. 2023;354:755‐769. doi: 10.1016/J.JCONREL.2023.01.059 PubMed DOI

Tan AS, Baty JW, Dong LF, et al. Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metab. 2015;21(1):81‐94. doi: 10.1016/J.CMET.2014.12.003 PubMed DOI

Esteban‐Martínez L, Sierra‐Filardi E, McGreal RS, et al. Programmed mitophagy is essential for the glycolytic switch during cell differentiation. EMBO J. 2017;36(12):1688‐1706. doi: 10.15252/EMBJ.201695916 PubMed DOI PMC

Plotnikov EY, Khryapenkova TG, Galkina SI, Sukhikh GT, Zorov DB. Cytoplasm and organelle transfer between mesenchymal multipotent stromal cells and renal tubular cells in co‐culture. Exp Cell Res. 2010;316(15):2447‐2455. doi: 10.1016/J.YEXCR.2010.06.009 PubMed DOI

Soto‐Heredero G, de Gómez las Heras MM, Gabandé‐Rodríguez E, Oller J, Mittelbrunn M. Glycolysis—a key player in the inflammatory response. FEBS J. 2020;287(16):3350. doi: 10.1111/FEBS.15327 PubMed DOI PMC

Islam A, Shen X, Hiroi T, Moss J, Vaughan M, Levine SJ. The Brefeldin A‐inhibited guanine nucleotide‐exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome‐like vesicles. J Biol Chem. 2007;282(13):9591‐9599. doi: 10.1074/JBC.M607122200 PubMed DOI

Nylander S, Kalies I. Brefeldin a, but not monensin, completely blocks CD69 expression on mouse lymphocytes:: efficacy of inhibitors of protein secretion in protocols for intracellular cytokine staining by flow cytometry. J Immunol Methods. 1999;224(1–2):69‐76. doi: 10.1016/S0022-1759(99)00010-1 PubMed DOI

Morris RL, Hollenbeck PJ. The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth. J Cell Sci. 1993;104(3):917‐927. doi: 10.1242/JCS.104.3.917 PubMed DOI

Pålsson‐McDermott EM, O'Neill LAJ. Targeting immunometabolism as an anti‐inflammatory strategy. Cell Res. 2020;30(4):300‐314. doi: 10.1038/s41422-020-0291-z PubMed DOI PMC

Konari N, Nagaishi K, Kikuchi S, Fujimiya M. Mitochondria transfer from mesenchymal stem cells structurally and functionally repairs renal proximal tubular epithelial cells in diabetic nephropathy in vivo. Sci Rep. 2019;9(1):5184. doi: 10.1038/S41598-019-40163-Y PubMed DOI PMC

Han H, Hu J, Yan Q, et al. Bone marrow‐derived mesenchymal stem cells rescue injured H9c2 cells via transferring intact mitochondria through tunneling nanotubes in an in vitro simulated ischemia/reperfusion model. Mol Med Rep. 2016;13(2):1517‐1524. doi: 10.3892/MMR.2015.4726 PubMed DOI PMC

Babenko VA, Silachev DN, Popkov VA, et al. Miro1 enhances mitochondria transfer from multipotent mesenchymal stem cells (MMSC) to neural cells and improves the efficacy of cell recovery. Molecules. 2018;23(3):687. doi: 10.3390/MOLECULES23030687 PubMed DOI PMC

Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone‐marrow‐derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med. 2012;18(5):759‐765. doi: 10.1038/nm.2736 PubMed DOI PMC

Huh JW, Kim WY, Park YY, et al. Anti‐inflammatory role of mesenchymal stem cells in an acute lung injury mouse model. Acute and Critical Care. 2018;33(3):154‐161. doi: 10.4266/ACC.2018.00619 PubMed DOI PMC

Feng YW, Wu C, Liang FY, et al. hUCMSCs mitigate LPS‐induced trained immunity in ischemic stroke. Front Immunol. 2020;11:1746. doi: 10.3389/FIMMU.2020.01746 PubMed DOI PMC

Hwang JW, Lee MJ, Chung TN, et al. The immune modulatory effects of mitochondrial transplantation on cecal slurry model in rat. Crit Care. 2021;25(1):20. doi: 10.1186/S13054-020-03436-X PubMed DOI PMC

Barrasso AP, Tong X, Poché RA. The mito::mKate2 mouse: a far‐red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo. Genesis. 2018;56(2):e23087. doi: 10.1002/DVG.23087 PubMed DOI PMC

Hajkova M, Javorkova E, Zajicova A, Trosan P, Holan V, Krulova M. A local application of mesenchymal stem cells and cyclosporine a attenuates immune response by a switch in macrophage phenotype. J Tissue Eng Regen Med. 2017;11(5):1456‐1465. doi: 10.1002/TERM.2044 PubMed DOI

Hajkova M, Hermankova B, Javorkova E, et al. Mesenchymal stem cells attenuate the adverse effects of immunosuppressive drugs on distinct T cell subopulations. Stem Cell Rev Rep. 2017;13(1):104‐115. doi: 10.1007/S12015-016-9703-3 PubMed DOI

Marvanova A, Kasik P, Elsnicova B, et al. Continuous short‐term acclimation to moderate cold elicits cardioprotection in rats, and alters β‐adrenergic signaling and immune status. Sci Rep. 2023;13(1):18287. doi: 10.1038/S41598-023-44205-4 PubMed DOI PMC

Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357‐360. doi: 10.1038/NMETH.3317 PubMed DOI PMC

Danecek P, Bonfield JK, Liddle J, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10(2):1‐4. doi: 10.1093/GIGASCIENCE/GIAB008 PubMed DOI PMC

Li H, Handsaker B, Wysoker A, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25(16):2078‐2079. doi: 10.1093/BIOINFORMATICS/BTP352 PubMed DOI PMC

Kleverov M, Zenkova D, Kamenev V, Sablina M, Artyomov MN, Sergushichev AA. Phantasus: web‐application for visual and interactive gene expression analysis. PubMed DOI PMC