The bioactivity of colostrum and milk exosomes of high, average, and low immune responder cows on human intestinal epithelial cells

Bovine milk contains bioactive components that are nutritionally and immunologically important to calves and humans. Dairy cows classified as high (H) immune responders using the patented high immune response technology have higher concentrations of immunoglobulin and specific antibodies in sera and milk compared with average (A) and low (L) responders. MicroRNA post-transcriptionally regulate expression of milk bioactive components and are enriched in extracellular vesicles known as exosomes, which protect them from degradation. The bioactivity of colostrum and milk exosomes at the human intestinal epithelial barrier remains to be explored, particularly in the context of the high immune response technology. Therefore, the purpose of this study was to evaluate the functional role of bovine milk exosomes compared with colostrum exosomes from H, A, and L responders at the intestinal interface using human colorectal adenocarcinoma epithelial (Caco-2) cells. Exosomes were isolated by successive ultracentrifugation and confirmed by western blot analysis for the presence of common exosomal proteins (CD9, CD63, and heat shock protein 70). Fluorescent labeling of exosomes using PKH67 dye confirmed their uptake by Caco-2 cells, demonstrating their potential bioavailability. The MTT assays showed that colostrum and milk exosomes maintain Caco-2 metabolic activity and are not cytotoxic to these cells. Specifically, metabolic activity after co-incubation with colostrum and milk exosomes from H responder cows was significantly greater than after co-incubation with exosomes from L responders. Caspase 3 activity, an indicator of apoptosis, was significantly lower after co-incubation of Caco-2 cells with milk exosomes compared with colostrum exosomes, suggesting that unlike colostrum exosomes, particularly those from L responders, milk exosomes do not activate the caspase 3 pathway in Caco-2 cells. This study helps us better understand the functional importance of colostrum and milk exosomes from dairy cows and emphasizes differences in functionality among exosomes from H, A, and L immune responders.