Milk-derived EVs play a differential role in boosting innate immune functions and developmental processes on the basis of the lactation curve

BACKGROUND and OBJECTIVE: Water buffaloes, more resistant to disease and parasites, have a higher capacity to adapt and survive in different environments with distinct topography, climate and vegetation. For this reason, they are a key source of livestock in southern Italy, where they are mainly bred for milk production, as their milk has a significantly higher nutrient concentration than cow's milk. It contains bioactive compounds, including antioxidants and anti-inflammatories, and is rich in fat, lipids, vitamins and extracellular vesicles (EVs). Milk-derived EVs (mEVs) offer diverse advantages: high stability in harsh environments, making them efficient vehicles for intercellular communication and promising candidates for therapeutic delivery, and carrying a variety of bioactive molecules that contribute to immunity and development. This study aims to characterize mEVs throughout the water buffalo lactation curve and to evaluate their effects on bovine macrophages and buffalo peripheral blood mononuclear cells (PBMC) when derived from colostrum (Colo), 15-day-old milk (15-dom), and 60-day-old milk (60-dom). MATERIALS and METHODS: The samples were isolated and characterized using various techniques, including Transmission Electron Microscopy, Confocal Microscopy, Nanoparticle Tracking Analysis and Flow Cytometry, enabling a multiparametric analysis of individual EVs to obtain their concentration, size, and phenotype. EVs characterization is based on staining with anti-CD9 and -CD63 monoclonal antibodies. Finally, the lipid cationic dye (LCD) was used to trace intact EVs. Our FC experiments highlight CD9 and CD63 expression on Colo and milk samples, highlighting a higher EV concentration in Colo. Uptake experiments were conducted using PKH67-labelled EVs and revealed a greater internalization of Colo EVs at both 4h and 24h in BoMac cells, demonstrating major EV persistence over time. Phagocytosis in bovine macrophages was investigated by FITC-labelled Escherichia Coli (E. Coli), revealing no increase in phagocytosis but a minor cell adhesion of E. Coli due to pre-treatment with milk EVS, highlighted by CM. Of note, phagocytosis in buffalo macrophages was increased, particularly in cells pretreated with EV 60-dom. RESULTS: Furthermore, the cargo composition was assessed and out of the total number of microRNAs (miRNAs) found in Colo (145) and milk-EVs (179), a significant number were found to be expressed in one specific sample type (10 Colo-EV-specific and 44 milk-EV-specific) and were completely absent (or present in negligible quantities) in the other one. These miRNAs contribute to immune tolerance, intestinal cell and neonatal intestinal barrier maturation. CONCLUSIONS: These findings suggest that mEVs play a role in boosting innate immune functions and are beneficial for developmental processes.