MiR-146a engineered extracellular vesicles derived from mesenchymal stromal cells more potently attenuate ischaemia–reperfusion injury in lung transplantation

Abstract

Background: The limited donor lung pool for lung transplantation (LTx) is largely due to concerns over ischaemia–reperfusion injury (IRI), a major cause of primary graft dysfunction (PGD). NLRP3 inflammasome activation is known to play a pivotal role in the onset of IRI. While human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hucMSC-EVs) have shown potential in reducing acute lung injury, their effects on NLRP3 activation in the context of LTx remain unclear.

Methods: In this study, engineered hucMSC-EVs were delivered via nebulisation to mitigate IRI in rat LTx models. We utilised both a rat orthotopic LTx model and a cell cold preservation reperfusion model to evaluate the therapeutic efficacy of hucMSC-EVs. Bulk-RNA sequencing, single-cell sequencing analysis, immunofluorescence and Western blot techniques were employed to assess NLRP3 inflammasome activation and inflammation.

Results: Nebulised hucMSC-EVs were efficiently internalised by alveolar macrophages (AMs), significantly reducing lung injury and improving oxygenation in the LTx models. Mechanistically, the engineered hucMSC-EVs, which enhance the expression of miR-146a, can more effectively suppress the activation of the NLRP3 inflammasome by targeting the IRAK1/TRAF6/NF-κB pathway, resulting in decreased levels of IL-1β, IL-18 and other inflammatory cytokines. These findings highlight the potential of miR-146a-modified EVs in modulating innate immune responses to alleviate IRI.

Conclusion: Our results demonstrate that nebulised delivery of engineered hucMSC-EVs effectively mitigates IRI in LTx by inhibiting NLRP3 inflammasome activation. This innovative approach presents a promising strategy for enhancing donor lung preservation and improving post-transplant outcomes in LTx.

Keywords

extracellular vesicles / ischaemia–reperfusion injury / lung transplantation / miR-146a

References

[1] Reeb J, Keshavjee S, Cypel M. Expanding the lung donor pool. Curr Opin Organ Transplant. 2015; 20(5): 498-505.

[2] Miñambres E, Coll E, Duerto J, et al. Effect of an intensive lung donor-management protocol on lung transplantation outcomes. J Heart Lung Transplant. 2014; 33(2): 178-184.

[3] Munshi L, Keshavjee S, Cypel M. Donor management and lung preservation for lung transplantation. Lancet Respir Med. 2013; 1(4): 318-328.

[4] Capuzzimati M, Hough O, Liu M. Cell death and ischemia-reperfusion injury in lung transplantation. J Heart Lung Transplant. 2022; 41(8): 1003-1013.

[5] Shepherd HM, Gauthier JM, Li W, Krupnick AS, Gelman AE, Kreisel D. Innate immunity in lung transplantation. J Heart Lung Transplant. 2021; 40(7): 562-568.

[6] Laubach VE, Sharma AK. Mechanisms of lung ischemia-reperfusion injury. Curr Opin Organ Transplant. 2016; 21(3): 246-252.

[7] Cullen Sean P, Kearney Conor J, Clancy Danielle M, Martin Seamus J. Diverse activators of the NLRP3 inflammasome promote IL-1β secretion by triggering necrosis. Cell Rep. 2015; 11(10): 1535-1548.

[8] Fan EKY, Fan J. Regulation of alveolar macrophage death in acute lung inflammation. Respir Res. 2018; 19(1): 50.

[9] Arora S, Dev K, Agarwal B, Das P, Syed MA. Macrophages: their role, activation and polarization in pulmonary diseases. Immunobiology. 2018; 223(4-5): 383-396.

[10] Cantu E, Lederer DJ, Meyer K, et al. Gene set enrichment analysis identifies key innate immune pathways in primary graft dysfunction after lung transplantation. Am J Transplant. 2013; 13(7): 1898-1904.

[11] Zhou Y, Yamamoto Y, Xiao Z, Ochiya T. The immunomodulatory functions of mesenchymal stromal/stem cells mediated via paracrine activity. J Clin Med. 2019; 8(7): 1025.

[12] Joo HS, Suh JH, Lee HJ, Bang ES, Lee JM. Current knowledge and future perspectives on mesenchymal stem cell-derived exosomes as a new therapeutic agent. Int J Mol Sci. 2020; 21(3): 727.

[13] Yuan X, Li T, Shi L, Miao J, Guo Y, Chen Y. Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3. Mol Med. 2021; 27(1): 91.

[14] Stone ML, Zhao Y. Robert Smith J, et al. Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death. Respir Res. 2017; 18(1): 212.

[15] Hsu CC, Chen SY, Ko PY, et al. MicroRNA-146a gene transfer ameliorates senescence and senescence-associated secretory phenotypes in tendinopathic tenocytes. Aging. 2024; 16(3): 2702-2714.

[16] Liang C, Zou T, Zhang M, et al. MicroRNA-146a switches microglial phenotypes to resist the pathological processes and cognitive degradation of Alzheimer's disease. Theranostics. 2021; 11(9): 4103-4121.

[17] Li X, Liao J, Su X, et al. Human urine-derived stem cells protect against renal ischemia/reperfusion injury in a rat model via exosomal miR-146a-5p which targets IRAK1. Theranostics. 2020; 10(21): 9561-9578.

[18] Saba R, Sorensen DL, Booth SA. MicroRNA-146a: a dominant, negative regulator of the innate immune response. Front Immunol. 2014; 5: 578.

[19] Wong A, Duong A, Wilson G, et al. Ischemia-reperfusion responses in human lung transplants at the single-cell resolution. Am J Transplant. 2024; 24(12): 2199-2211.

[20] Chen S, Yuan M, Chen H, et al. MiR-34a-5p suppresses cutaneous squamous cell carcinoma progression by targeting SIRT6. Arch Dermatol Res. 2024; 316(6): 299.

[21] Zhu J, Fan Y, Yang S, et al. Oral delivery of miR-146a-5p overexpression plasmid-loaded Pickering double emulsion modulates intestinal inflammation and the gut microbe. Int J Biol Macromol. 2024; 261(Pt 2): 129733.

[22] Yu WS, Son J. Donor selection, management, and procurement for lung transplantation. J Chest Surg. 2022; 55(4): 277-282.

[23] Kurihara C, Lecuona E, Wu Q, et al. Crosstalk between nonclassical monocytes and alveolar macrophages mediates transplant ischemia-reperfusion injury through classical monocyte recruitment. JCI Insight. 2021; 6(6): e147282.

[24] Chen-Yoshikawa TF. Ischemia-reperfusion injury in lung transplantation. Cells. 2021; 10(6): 1333.

[25] Wang J, Huang R, Xu Q, et al. Mesenchymal stem cell-derived extracellular vesicles alleviate acute lung injury via transfer of miR-27a-3p. Crit Care Med. 2020; 48(7): e599-e610.

[26] Potter DR, Miyazawa BY, Gibb SL, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate pulmonary vascular permeability and lung injury induced by hemorrhagic shock and trauma. J Trauma Acute Care Surg. 2018; 84(2): 245-256.

[27] Zamani P, Oskuee RK, Atkin SL, Navashenaq JG, Sahebkar A. MicroRNAs as important regulators of the NLRP3 inflammasome. Prog Biophys Mol Biol. 2020; 150: 50-61.

[28] Mann M, Mehta A, Zhao JL, et al. An NF-κB-microRNA regulatory network tunes macrophage inflammatory responses. Nat Commun. 2017; 8(1): 851.

[29] Wu X, Yang D, Zhao F, et al. Quantification of dynamic protein interactions and phosphorylation in LPS signaling pathway by SWATH-MS. Mol Cell Proteomics. 2019; 18(6): 1054-1069.

[30] He Y, Hara H, Núñez G. Mechanism and regulation of NLRP3 inflammasome activation. Trends Biochem Sci. 2016; 41(12): 1012-1021.

[31] Liu G, Wu J, Chen G, Shang A. The potential therapeutic value and application prospect of engineered exosomes in human diseases. Front Cell Dev Biol. 2022; 10: 1051380.

[32] Ma X, Liu B, Fan L, et al. Native and engineered exosomes for inflammatory disease. Nano Res. 2022.

[33] Wang S, Yang Y, Suen A, et al. Role of extracellular microRNA-146a-5p in host innate immunity and bacterial sepsis. iScience. 2021; 24(12): 103441.

[34] Zhao R, Wang L, Wang T, Xian P, Wang H, Long Q. Inhalation of MSC-EVs is a noninvasive strategy for ameliorating acute lung injury. J Controlled Release. 2022; 345: 214-230.

[35] Popowski KD, López de Juan Abad B, George A, et al. Inhalable exosomes outperform liposomes as mRNA and protein drug carriers to the lung. Extracell Vesicle. 2022; 1: 100002.

[36] Wong A, Zamel R, Yeung J, et al. Potential therapeutic targets for lung repair during human ex vivo lung perfusion. Eur Respir J. 2020; 55(4): 1902222.

[37] Nakajima D, Watanabe Y, Ohsumi A, et al. Mesenchymal stromal cell therapy during ex vivo lung perfusion ameliorates ischemia-reperfusion injury in lung transplantation. J Heart Lung Transplant. 2019; 38(11): 1214-1223.

[38] Nykänen AI, Mariscal A, Duong A, et al. Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment. Mol Ther Methods Clin Dev. 2021; 23: 184-197.

[39] Yang D, Zhang W, Zhang H, et al. Progress, opportunity, and perspective on exosome isolation—efforts for efficient exosome-based theranostics. Theranostics. 2020; 10(8): 3684-3707.

[40] Brossa A, Tapparo M, Fonsato V, et al. Coincubation as miR-loading strategy to improve the anti-tumor effect of stem cell-derived EVs. Pharmaceutics. 2021; 13(1): 76.

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2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.