The potential role of pooled bovine milk-derived EVs in regulating epithelial cells through human primary macrophages

Extracellular vesicles (EVs) play an important role in cell-to-cell communication and modulation of numerous physiological and pathological processes. EVs are abundantly present in milk, serving to encapsulate several immune-modulating bioactive molecules, ensuring stability for these components and their subsequent regulation. Due to the availability, scalability, and low immunogenicity, bovine milk-derived extracellular vesicles (MEVs) are increasingly considered as promising carriers of nanomedicines for future therapies. Here, we investigated the effect of two EV groups, enriched from raw milk of healthy or mastitis-affected cows, isolated by differential ultracentrifugation. To study the potential effect on the cellular response, samples of the same EV group were pooled and applied to human primary macrophages. We collected the supernatants and treated the Caco-2 cell line with them. TaqMan Arrays were utilized to measure multiple gene expression levels. Among them, the HRH2 and HRH3 expression patterns in different groups are of high interest. We employed polarized enterocyte-like monolayers by culturing Caco-2 cells beyond confluence in Transwell inserts for 21 days and treated them with macrophage supernatants for transmission electron microscopy analysis. The images indicated that in the mastitis group, desmosomes tended to loosen up. Therefore, the structural integrity and adhesion between epithelial cells could be severely compromised, potentially contributing to intestinal inflammation and associated diseases. This study explores the potential molecular mechanisms behind the impact of MEVs on human cells. By examining MEVs from various bovine health conditions, including mastitis, this study provides a comprehensive view and novel insight into their future applications.