ROLE OF MIRNAS SHUTTLED BY LPS-PRIMED MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES IN MODULATING MACROPHAGE ACTIVATION

Mesenchymal stem cells (MSCs) have regenerative and immunomodulatory properties largely mediated by extracellular vesicles (EVs). Priming MSCs with lipopolysaccharide (LPS) enhances the therapeutic efficacy of their EVs, particularly in acute lung injury. This study investigates the role of differentially expressed miRNAs between EVs from naïve (C-EVs) and LPS-primed MSCs (LPS-EVs) and their immunomodulatory effects. miRNA profiling of C-EVs and LPS-EVs was performed using GeneChip miRNA 4.0 Array (n=2). Differentially expressed miRNAs (fold-change ≥ 1.5) were identified, and pathway enrichment was analyzed using GSEA with KEGG, GO, Biocarta, Broad Hallmarks, and PID databases. Selected overexpressed miRNAs were validated by RT-qPCR and tested for immunomodulatory effects in Pseudomonas aeruginosa-infected THP-1 cells, individually and in combination (MIMIX). Twenty-one miRNAs were differentially expressed between C-EVs and LPS-EVs (16 upregulated, 5 downregulated in LPS-EVs). Enriched pathways included inflammation, epithelial regeneration, and endothelial barrier integrity. RT-qPCR confirmed overexpression of miR-297, let-7b-5p, and miR-93-5p in LPS-EVs, which reduced IL1B, IL6, and IL8 expression in infected THP-1 cells. Notably, only the combined administration of these three miRNAs induced M2 polarization, increasing CD206, CD163, and TGFβ expression. LPS priming enhances the immunomodulatory potential of MSC-derived EVs, partly through overexpression of miR-297, miR-93-5p, and let-7b-5p, which synergistically promote M2 macrophage polarization.