Inhalation of Mesenchymal Stromal Cell-Derived Extracellular Vesicles Activates Macrophage Polarization through the miR-22-3p/NLRP3/IL-1β Pathway, Ameliorating Lung Ischemia-Reperfusion Injury

Abstract

Background: Lung ischemia–reperfusion injury (IRI) is a major contributor to primary graft dysfunction (PGD) after lung transplantation. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as promising therapeutic agents in inflammatory diseases by ameliorating tissue damage and promoting repair. However, the anti-inflammatory efficacy of these approaches and the underlying mechanisms in lung ischemia–reperfusion injury remain incompletely understood. Methods: The protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) against lung ischemia–reperfusion injury were evaluated using two delivery approaches, inhalation and intravenous injection. Both in vivo and in vitro models were employed to assess the biological activity of MSC-EVs and to elucidate the underlying molecular mechanisms. In addition, a rat orthotopic lung transplantation (OLT) model was established to further examine the translational relevance of MSC-EVs. Results: MSC–EVs treatment significantly ameliorated lung IRI, with inhalation showing superior efficacy over intravenous delivery. Mechanistically, miR-22-3p within MSC-EVs targeted macrophage NLRP3, suppressing activation of the NLRP3/Caspase-1/IL-1β pathway and promoting M2 polarization. The protective efficacy was confirmed in a clinically relevant rat OLT model, underscoring their translational potential. Conclusions: Our findings indicate that inhaled MSC-derived extracellular vesicles attenuate lung ischemia–reperfusion injury by promoting macrophage polarization via the miR-22-3p/NLRP3/IL-1β pathway, supporting their potential as a cell-free therapeutic approach to mitigate primary graft dysfunction after lung transplantation.

1. Introduction

Lung ischemia-reperfusion injury (IRI) is a pivotal pathological process underlying the development of primary graft dysfunction (PGD), which remains the leading cause of early morbidity and mortality following lung transplantation [1, 2]. PGD typically manifests within 72 h post-transplant as diffuse alveolar damage, severe hypoxemia, and pulmonary edema, and remains a major obstacle to improved post-transplant outcomes [3]. Mechanistically, lung IRI is initiated during the ischemic phase by metabolic derangements, which are further exacerbated upon reperfusion by a burst of oxidative stress, inflammatory mediator release, and immune cell infiltration [4]. Despite decades of investigation, no effective pharmacological agents are currently available to prevent or treat lung IRI in the transplant setting.

Mesenchymal stem cells (MSCs)-based therapies have emerged as promising candidates for mitigating IRI, owing to their potent immunomodulatory properties, capacity to promote tissue repair, and favorable safety profile observed in early-phase clinical trials [5–7]. Increasing attention has focused on MSC-derived extracellular vesicles, which are nanosized lipid bilayer vesicles enriched in proteins, lipids, and nucleic acids, and which mediate much of the paracrine activity of MSCs [8, 9]. Compared with direct MSCs administration, MSC-EVs offer several advantages: they retain MSCs bioactivity without the risk of uncontrolled differentiation, display superior tissue penetration due to their small size, and exhibit low immunogenicity, rendering them particularly suitable for allogeneic applications [10–12]. Recent studies have demonstrated that MSC-EVs exert tissue-protective and reparative effects by transferring bioactive cargoes such as microRNAs (miRNAs) or mitochondria and modulating immune cell function, thereby alleviating injury and promoting regeneration [10, 13–15]. Beyond identifying the therapeutic cargoes of EVs, it is essential to elucidate the mechanisms by which they exert therapeutic benefits, including the regulation of immune responses, mitigation of oxidative stress, and promotion of tissue regeneration. However, the role and mechanisms of MSC-EVs in lung IRI remain poorly defined [16]. Clarifying these effects is crucial for the potential application of MSC-EVs in alleviating PGD after lung transplantation.