ABSTRACT: Acute lung injury (ALI) is characterized by excessive inflammation and alveolar damage, arising from pathogens or systemic insults such as sepsis, and can progress to severe acute respiratory distress syndrome (ARDS). Current treatments, including mechanical ventilation, remain largely supportive, emphasizing the urgent need for the potent, cell-free therapeutic modalities. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as promising candidates for lung repair, but insufficient immunosuppressive capacity often limits their efficacy. Human adipose-derived mesenchymal stem cells (hADMSCs) were primed with IFN-γ and TNF-α to enhance the immunomodulatory properties of their secreted EVs. We characterized primed MSC-EVs (P-MEVs) and unprimed control MSC-EVs (C-MEVs) by transmission electron microscopy, nanoparticle tracking analysis, and western blotting for EV markers. Functional assays in THP-1 and A549 cells examined anti-inflammatory potency and barrier regeneration against lipopolysaccharide (LPS)-induced damage. A preclinical mouse model of LPS-induced ALI was used to evaluate inflammatory cytokine expression, immune cell infiltration, pulmonary edema, and vascular leakage. Finally, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Vero E6 cells were tested to explore the antiviral and anti-inflammatory potential of P-MEVs. Primed hADMSCs exhibited elevated expression of immunosuppressive molecules (e.g., COX-2, IDO, TSG-6), without changing EV morphology or yield. P-MEVs mitigated LPS-induced inflammation more effectively than C-MEVs in THP-1 and A549 cells. In vivo , P-MEVs more robustly attenuated inflammatory cytokines, immune cell recruitment, and lung injury markers in mice challenged with LPS. In SARS-CoV-2-infected Vero E6 cells, P-MEVs suppressed cytopathic effects and inflammatory responses more potently than C-MEVs. Mechanistic analyses revealed that these enhancements were associated with elevated miRNA levels, involved in inhibiting inflammatory pathways.
