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Flow cytometric profiling of water buffalo milk EVs: how lactation stage drives phenotypic evolution and macrophage
Barbara Canonico1, Mariele Montanari1, Ludovica Di Fabrizio1
1Department of Biomolecular Sciences, DISB, University of Urbino Carlo Bo, Urbino, Italy.
Introduction:
Milk is more than a traditional nutrient source; it serves as a crucial carrier for milk-derived extracellular vesicles (mEVs), food-derived nanoparticles with diverse effects connecting animal health and human nutrition to ecological stability. The primary goal of this study was to characterize the phenotype and functional impact of mEVs from water buffalo (Bubalus bubalis) across the lactation curve. The researchers aimed to determine how the transition from colostrum (Colo EVs) to early milk (EM-EVs) and mature milk (MM-EVs) alters the vesicles' physical properties and their ability to modulate the immune response in macrophages.
Material And Methods:
The study utilized Flow Cytometry (FC) as a core tool, first validating specific antibody clones for use in the buffalo species. The functional impact was tested on both a bovine cell line (BoMac) and primary water buffalo macrophages, focusing on cellular uptake, mitochondrial oxidative stress (ROS) and phagocytic activity.
Results And Conclusion:
MM-EVs play the most important role in boosting phagocytosis in bubaline macrophages, accompanied by the highest increase in CD14 surface density, whereas Colo-EVs demonstrated the highest degree of internalization and the strongest antioxidant activity. In conclusion, the study demonstrates that mEVs provide a biological bridge during the calf's development. While Colo-EVs focus on antioxidant protection and systemic signaling, MM-EVs actively enhance the innate immune defense. This provides a mechanistic explanation for why continued consumption of maternal milk beyond the colostral phase is vital for the immunological resilience and survival of buffalo calves. Furthermore, BoMac cells and bubaline macrophages exhibit distinct cytokine profiles, as IL-1α displays an increasing trend that did not reach statistical significance in MM-EV-treated BoMac cells, whereas a non-significant increasing trend is observed in macrophages treated by both Colo EVs and MM-EVs. Ultimately, the conserved nature of tetraspanins and miRNA profiles underscores the broad cross-species translational potential of buffalo mEVs, extending to human applications.
