A Multi-Omics approach for Extracellular Vesicles characterization: Deciphering the Immunomodulatory potential of Buffalo EVs from Colostrum to Mature Milk

BACKGROUND and OBJECTIVE: Extracellular vesicles (EVs) derived from milk are promising bioactive components due to their ability to mediate intercellular communication and modulate immune responses. Naturally secreted by mammary epithelial and immune cells, they carry a complex cargo of proteins, lipids, and nucleic acids, including miRNAs, which contribute to their immunomodulatory potential. Milk EVs influence immune function, gut barrier integrity, and inflammatory processes, with potential applications in the treatment of inflammatory and autoimmune diseases. Exploring species-specific differences is crucial to identify optimal sources for clinical and therapeutic applications. This study aimed to characterize the small RNA cargo and metabolites of colostrum EVs (ColosEVs) and milk EVs (mEVs) at two lactation stages (T1 – 15 days, T2 – 60 days postparturition) in Italian Mediterranean buffalo, using two omics techniques: next-generation sequencing (NGS) and high-performance liquid chromatography–mass spectrometry (HPLC-MS). MATERIALS and METHODS: EV preparations were characterized by nanoparticle tracking analysis (NTA) and scanning electron microscopy (SEM), confirming vesicles in the expected nanometric range and typical morphology. Representative NTA profiles showed mean particle sizes of ~175.6 ± 3.2 nm in ColosEVs and ~174.0 ± 1.5 nm in mEVs, with concentrations in the order of 10^12 particles/mL (approximately 4.1 × 10^12 particles/mL in ColosEVs and 2.3 × 10^12 particles/mL in mEVs). Conventional flow cytometry was applied through both single-EV and bead-based approaches, monitoring the expression of tetraspanins including CD9, CD81, and CD63. RESULTS: Colostrum contained more EVs than mature milk, whereas mEV_T1 and mEV_T2 displayed higher molecular diversity and shared the highest number of features. We identified 51 miRNAs enriched in ColosEVs, 81 in mEV_T1, and 79 in mEV_T2. Metabolite analysis revealed 379 molecules particularly contained in ColosEVs compared to mEVs, while 619 and 764 were enriched in mEV_T1 and mEV_T2, respectively. Preliminary integrative multi-omics analyses (mixOmics, R) showed clear class-specific clustering of ColosEVs, mEV_T1, and mEV_T2 in integrated heatmaps and PCA, together with strong miRNA–metabolite associations (correlation cut-off r = 0.85), supporting stage-dependent remodeling of EV molecular cargo. Functional analysis of class-specific miRNA targets highlighted biological categories related to immune/stress response, DNA/RNA metabolism, protein modification/signaling, and transport/localization, while metabolite pathway enrichment suggested dynamic changes in amino acid, lipid, carbohydrate, and signaling pathways across lactation. CONCLUSIONS: This systems biology approach is essential to decipher the molecular messages of buffalo EVs across lactation stages and to better understand their roles in immune regulation, neonatal development, and potential clinical applications.