Colostrum and milk EVs from Water Buffalo enhance phagocytosis in macrophages and improve ATP production in human intestinal cell line

Milk and its derivatives are rich sources of bioactive components with documented health benefits. Extracellular vesicles (EVs) play a crucial role in pathophysiology and are found in various body fluids, including blood, urine, cerebrospinal fluid, tears, saliva, nasal secretions, ascites, semen, and milk. EVs transfer different cargoes that can be universal or cell-type-specific, triggering a wide range of physiological responses. EVs derived from bovine milk are considered biocompatible and are promising candidates as nanocarriers for drug delivery. This study aims to characterize and evaluate the effects on bovine macrophages and human intestinal cells of EVs from colostrum (colo), 15- and 60-day milk (15-dm and 60-dm) of Water Buffaloes (Bubalus bubalis). Briefly,15m and 60m and colo from Water Buffaloes were centrifuged to collect the supernatant, and the pellet with residual fat and debris was discarded with residual fat and debris. The samples were filtered (450 nm) and labelled with LCD, anti-CD63, anti-CD81, and anti-CD9 to characterize EVs by flow cytometry (FC). Furthermore, EVs from ultracentrifugation of selected colo, 15-dm and 60-dm samples were analyzed using Nanoparticle Tracking Analysis (NTA), FC, and Transmission Electron Microscopy (TEM). The EVs were employed to analyze the uptake after 4 and 24h in bovine macrophage cell line (BoMac) and Caco-2 human intestinal cells. 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 confocal microscopy. Of note, phagocytosis in buffalo macrophages (by the pHrodo Green Zymosan Bioparticles kit) was increased, particularly in cells pretreated with EV 60-dm. Intriguingly, CD14 mean fluorescence intensity was increased in cells treated by both colo and 60-dm EVs. Furthermore, the ATP production rates generated from mitochondrial respiration (OXPHOS) and glycolysis were measured by the Seahorse XF Real-time ATP rate assay, and an increase in glycoATP was induced in EV-treated cells. Common to bovine and human cells was the decrease of basal ROS species (by MitoSOX and DCF probes). These initial findings highlight the potential beneficial role of colostrum and milk-derived EVs in promoting human gastrointestinal health and various functions in bovine and buffalo cells. Additional experiments are currently ongoing to confirm the overall results. This research was funded by the Italian Ministry for Health (grant number RC IZSME 03/23 RC). Thanks to Professor Gaetano Donofrio for the generous donation of bovine macrophage cell line (BoMac).