Flow cytometry-based analysis of Water Buffalo colostrum and milk EVs: phenotypes, uptake and immunomodulatory effects on macrophages

Extracellular vesicles (EVs) have garnered attention for their ability to transport bioactive molecules and mediate intercellular communication (1). However, their qualitative and quantitative analysis in colostrum (colo) and milk is challenged by the presence of fat globules and casein micelles, which contribute to the complexity and heterogeneity of these biological fluids (2). This study aims to evaluate phenotypes and subcellular effects of colo and milk EVs from Water Buffaloes (Bubalus bubalis), on both no ultracentrifuged whey samples and EV pellets obtained through ultracentrifugation, by conventional Flow Cytometry (FC). Indeed, subcellular immunomodulation effects in water buffalo macrophages, were investigated. No ultracentrifuged whey samples were analyzed post-collection, adhering to MISEV2023 guidelines. EDTA treatment were employed to reduce the presence of casein micelles, which are a significant obstacle in EV isolation. Phenotypic EVs characterization is based on stain with anti-CD9, and -CD63 mAbs. Ultracentrifuged EVs from colo, 15- and 60-day milk (dm) were counted by NTA, labelled with PkH67 and then added to BoMac cells for uptake studies at 4 and 24 hours. Indeed, Water Buffaloes PBMCs were employed to evaluate phagocytosis modulation. Preliminary experiments highlight that both colo and milk samples contain EVs with diameters ranging from ~70 nm to ~350 nm (FACSCanto acquisition) (Fig. 1A) and with diameters ranging from ~85 nm to ~300 nm (Cytoflex acquisition) (Fig. 1B). Colo samples are enriched in Small EVs, measuring <200 nm. Large EVs (200–350 nm) appear more abundant in 15 dm. Furthermore, colo samples exhibit a higher EVs content, with a greater proportion of CD63⁺ and CD9⁺ vesicles than those from 15 dm and 60 dm. Uptake experiments revealed increased internalisation of colo EVs at both 4 and 24 hours in BoMac cells (Fig. 2A), promoting cell re-adhesion (Fig. 2B) and demonstrating a major persistence of EVs (Fig. 2C). An increase in the phagocytic activity was observed in ex vivo buffalo macrophages after pre-treatment with 60-dm EVs, assessed by pHrodo Green Zymosan uptake (Fig. 3A-3B). Furthermore, CD14 surface density was higher in macrophages treated with either colo or 60-dm Evs (Fig. 3C-3D). These results confirm that Conventional FC is suitable for rapid and affordable EV detection, as well as determining their size and phenotype in colo and milk samples. Tetraspanins can be employed to highlight sample heterogeneity. Finally, our preliminary data reveal 60 dm EVs as the most effective in boosting phagocytosis.