Perspectives on Bovine Milk-Derived Extracellular Vesicles for Therapeutic Applications in Gut Health

miRNAs are noncoding RNAs of approximately 18 to 24 nucleotides that bind to a specific region of the mRNA and regulate it translation. Chen et al. (2010) found for the first time that substantial amounts of miRNAs are present in bovine milk and identified 245 miRNAs in raw milk and found that they are altered during lactation. miRNAs known to be involved in immune regulation were confirmed to be expressed both in bovine colostrum and mature milk exosomes. When miRNAs from colostrum and mature milk exosomes were compared, the ratio of the 10 most highly expressed miRNAs was different (Yun et al., 2021). Izumi et al. (2012) published a study comparing miRNAs from bovine colostrum and mature milk using microarray and qPCR and found that there are more miRNAs related to immunity or development, including miR-15b, miR-27b, miR-106b, and miR-223, in colostrum than in mature milk. The same researchers also found that miRNAs present in milk exosomes could be taken up by human macrophages (Izumi et al., 2015). Benmoussa et al. (2020) reported that miRNAs in milk EVs isolated from differential ultracentrifugation exhibited distinct compositions. In particular, the general length (around 22 nt) of miRNA sequence was abundant at milk EVs isolated by ultracentrifugation at 100,000×g, but the heterogeneous iso-miRNA was more abundant in milk EVs separated by ultracentrifugation at 12,000×g and 35,000×g. Additionally, when milk EVs were incubated with HeLa cells, miR-223, known to regulate immunity, was increased. To examine the possibility of using miRNAs as biomarkers, miRNAs from cow milk infected with Staphylococcus aureus were analyzed, and 14 or 18 miRNAs, including bta-miR-142-5p and bta-miR-223, were found to be differentially expressed in infection conditions (Cai et al., 2018; Sun et al., 2015). The composition and ratio of exosomal miRNAs from cow milk of two different breeds were also different. In Holstein milk, miRNAs related to milk synthesis and Doğu Anadolu Kirmizisi’s milk were expressed in milk fat and milk protein metabolism, respectively (Özdemir, 2020). Changes in feed and differences in breed of cattle have also been shown to alter milk miRNAs. Four miRNAs were upregulated and 5 miRNAs were downregulated when a fiber-rich diet was replaced with a nonforage fiber source diet (Quan et al., 2020). Inducing stress by relocation of the cow group have been shown to affect the composition of milk exosomal miRNAs. In particular, whether miRNAs and target genes such as miR-142a, miR-135, and miR-320a would be useful as potential biomarkers on response of mild stress has been postulated (Colitti et al., 2019). The functions and target miRNAs from milk-derived EVs are summarized in Table 1.

In milk-derived EVs, not only miRNAs but also proteins play a role in cell-to-cell communication and physiological activity (Fig. 1C). According to Reinhardt et al. (2012), a total of 2,107 proteins were identified in milk-derived EVs, of which 1,002 could be converted to David Resources. The same researchers also analyzed milk proteins from healthy cattle and from cattle infected with S. aureus. Interestingly, 2,350 proteins were identified in milk exosomes, and proteins related to host immunity and inflammation, including the heat shock protein family, protocadherin gamma family, and acute phase protein, were differentially expressed in S. aureus infection (Reinhardt et al., 2013). As a result of proteomics analysis of exosomes isolated from bovine colostrum and mature milk, a total of 9,430 proteins were detected in four samples, and 1,264, 1,404, 963, and 1,306 proteins were identified in colostrum and mature milk exosomes at 24 h, 48 h, and 72 h, respectively (Samuel et al., 2017). A total of 162 proteins were identified from exosomes and EV-containing pellets isolated through acid precipitation and ultracentrifugation, 43 of which were unique (Brown et al., 2020). When human and bovine milk exosomal proteins were analyzed and compared using the iTRAQ-coupled LC-MS/MS method, a total of 920 exosomal proteins were identified, and 575 proteins were differentially expressed. In particular, it was found that the proteins from human and bovine milk exosomes were clearly distinguishable (Yang et al., 2017). When the milk EV proteins from bovine leukemia virus-infected cattle were compared to that of uninfected cattle, a total of 1,330 proteins were detected in the milk EVs of infected cattle, of which 118 were uniquely expressed in the infected cattle. In addition, 26 proteins were expressed at significantly higher or lower levels than those of uninfected cattle. Through these studies, the possible use of proteins in EVs as a biomarker for specific diseases was revealed (Rahman et al., 2021). As a result of analyzing the lipids of EVs isolated by sequential ultracentrifugation and density gradient centrifugation through mass spectrometry-based lipid analysis, 8 major lipid classes were detected, and more than 200 fatty acid variations were identified (Grossen et al., 2021). Although it has been revealed through several previous studies that the lipids of EVs play an important role, there have been few lipidomics studies of bovine milk-derived EVs. This is because, due to the difficulty in reliably separating milk-derived EVs from milk lipids, there is still a limitation to the lipid analysis of milk EVs itself (Feng et al., 2021; Ong et al., 2021).

Milk and milk-derived EVs are important for the development of the immune system of calves, and EVs express a large number of immune-related miRNAs and proteins and exert immunomodulatory functions. Matic et al. (2020) reported that bovine milk exosomes promoted the proliferation of RAW 264.7 cells, decreased cisplatin-induced cytotoxicity, and regulated the expression of proteins related to the cell cycle and proliferation. Additionally, a previous study reported that the pretreatment with milk EVs in LPS-treated RAW 264.7 cells alleviated the production of inflammatory cytokines and decreased NK-κB activity (Ascanius et al., 2021). According to Pieters et al. (2015), commercial cow milk contains exosomes that are stable under low pH and boiling-freezing conditions and can carry bioactive immunoregulatory TGF-β. In addition, cow milk exosomes alone could not regulate immune cells but enhanced the production of interferon-γ cells by NK cells and γδT cells in inflammatory conditions (Komine-Aizawa et al., 2020). A previous study examined whether agricultural dust-derived inflammatory responses were modulated by bovine milk-derived EVs in a mouse model. When mice were fed a diet containing milk EVs and then exposed to organic dust, the inflammatory response was improved, and M1 polarization of macrophages was promoted, suggesting that milk-derived exosomes promote immune responses in inflammatory conditions (Nordgren et al., 2019).

In addition to regulating the immune system, milk-derived EVs are absorbed in the intestine and exert beneficial effects on intestinal health. In particular, they not only affect intestinal epithelial cells and the microbiota but may also have a protective effect in diseases that occur in the intestine. When milk EVs were fed to mice for 8 weeks, expression levels of genes important for the integrity of the mucus layer, including Muc2, RegIIIγ, and Myd88, were increased (Tong et al., 2020). Additionally, when oxidative stress was induced in intestinal crypt epithelial cells pretreated with bovine exosomes, superoxide dismutase, glutathione peroxidase activity and heme-oxygenase 1 (HO-1) protein levels was increased. In addition, ROS levels and the activity of adenosine deaminase and xanthine oxidase were decreased, suggesting that milk EVs could enhance the energy state of damaged cells to exert a protective effect against oxidative stress (Wang et al., 2021a; Wang et al., 2021b). Maghraby et al. (2021) reported that malnutrition-associated intestinal dysfunction was significantly alleviated by milk EV-induced intestinal stem cell proliferation and villus architecture restoration. Necrotizing enterocolitis (NEC), a type of colitis that can occur in premature infants, is characterized by significant intestinal damage and decreased mucin production. Treatment of milk exosomes with experimental NEC pups can alleviate the symptoms of NEC by increasing the expression of goblet cells and regulating genes involved in protein synthesis (Li et al., 2019). In addition, previous studies reported that treatment using cow milk-derived EVs alleviated colitis in both a DSS-induced colitis model and a genetic mouse model of ulcerative colitis (Benmoussa et al., 2019; Reif et al., 2020; Stremmel et al., 2020; Tong et al., 2021).

Milk-derived EVs have been reported to exert a beneficial effect on intestinal health, which may be due to milk EVs bringing positive changes to the gut microbiota. Therefore, studies are being conducted to determine how milk EVs affect the intestinal microbiota. Yu et al. (2019) proposed that when exosomes isolated from milk were cocultured with bacteria, they promoted the growth of Escherichiacoli K-12 MG1655, a commensal bacterium, and Lactobacillus plantarum WCFS1, a probiotic, and influenced gene expression. Feeding C57BL/6 mice a diet containing milk exosomes and RNA was shown to affect the cecum microbiota composition in mice, a non-bovine species (Zhou et al., 2019). Similarly, when C57BL/6 mice were fed milk EVs containing 1.0×10¹⁰ particles per gram body weight, they had an effect on the gut microbiota and serum metabolites. In particular, among the intestinal microbiota, Akkermansia and Muribaculum, which are known to be beneficial, increased, and Desulfovibrio, which is known to be harmful, decreased. In addition, serum metabolite analysis revealed that milk EVs affected lipid and amino acid metabolism, especially anti-inflammatory factors such as lysophosphatidylcholines, deoxycholic acid, eicosapentaenoic acid, related to immune and metabolic diseases (Du et al., 2021). In addition to previous studies showing that milk EVs affect the intestinal microbiota and bacteria, studies have also investigated how these changes may affect intestinal immunity and colitis. When milk EVs were administered to mice, as in previous results, they had an effect on intestinal microbes and their metabolites, short-chain fatty acids, and the expression of genes related to intestinal immunity was increased (Tong et al., 2020). The same researchers reported that milk EVs alleviated ulcerative colitis by altering the gut microbiome and modulating gut immunity. Feeding milk EVs to an experimental mouse model of ulcerative colitis restricted the TLR4 signaling pathway and NLRP3 activation and restored DSS-induced intestinal dysbiosis (Tong et al., 2021).