Unique miRNAs of bovine milk extracellular vesicles and their plausible role in regulating intricate pathways of milk consumers: A bioinformatics study

Milk represents a complex pool of nutrients and bioactive components that are indispensable for the growth and development of the infant. The key well-established roles played by milk's bioactive components in the infant are those at the level of the infant's intestinal and immune development. Through its bioactive components, including proteins, lipids, and oligosaccharides, milk helps the infant develop a mature intestinal identity with fully active digestive, absorptive, and barrier capacity and shapes both innate and adaptive immune responses. Recent evidence points to a new class of milk bioactive components including milk extracellular vesicles (EVs) and microRNAs, which are hypothesized to take part in contributing to infant development. The potential functions of milk microRNAs and EVs in the consumer's systems are not limited to the infant as these components can also be found in bovine milk, both raw and processed. Hence, adult consumers could also be influenced by milk EVs and microRNAs, which could affect their intestinal homeostasis particularly under pathological conditions. Nonetheless, the debate regarding the stability of milk EVs and microRNAs in the digestive tract persists, and their bioavailability and bioactivity in the consumer's tissues are still arguable. In this review, we discuss the potential functions mediated by milk microRNAs and EVs in the epithelial and immune components as well as the microbiome of the intestinal mucosa in health and disease. We also discuss the bioavailability, bioaccessibility, and bioactivity of milk EVs and microRNAs in consumer's tissues.

Boar fertility is critical for swine reproductive efficiency. Recent data demonstrates that neonatal colostrum intake is associated with enhanced sperm counts in adult boars. These data support the “Lactocrine Hypothesis,” which states that bioactive factors from maternal milk directly promote neonatal tissue development, including reproductive organs. Notably, maternal milk consumption increased the number of Sertoli cells within the testes of nursed versus formula-fed boars. However, the biological mechanisms mediating this effect are unknown. We hypothesized that milk extracellular vesicles (EVs) may contribute because EVs are abundant in milk, carry bioactive cargo (e.g., miRNAs), and protect these cargo from digestion before uptake by the intestinal mucosa and entry into the neonatal circulatory system. Our group previously demonstrated that maternal milk EVs bioaccumulate in distant neonatal tissues (e.g., brain), but testes were not evaluated. Recently, we reported that acute consumption of milk EVs alters the testicular proteome in neonatal boars, including key Sertoli cell markers (e.g., AMH, GATA-4, SOX-9). Together, these data suggest that milk EVs directly alter testis biology. The objective of this study was to determine the bioavailability of milk EVs to the neonatal testis using a transgenic swine model. Hemizygous transgenic sows ubiquitously expressing the fluorescent protein, ZsGreen1, and their male offspring, were utilized for this study. Milk was collected and pooled from 3 lactating (20–22 days post-partum) transgenic sows. Milk EVs were isolated via ultracentrifugation and filtration (220 nm). Milk EVs were validated via nano-flow cytometry (nFCM; size and concentration) and transmission electron microscopy (TEM; size and structure). The presence of ZsGreen1 (488 nm) in milk EVs was evaluated via nFCM and confocal microscopy. At necropsy, blood and testes were collected from wild type (WT) boars nursed (8–14 days) by transgenic (TSG) sows (n=4), WT boars nursed by WT sows (negative control; n=2) and a TSG boar nursed by a TSG sow (positive control; n=1). Serum was isolated for subsequent EV collection, validation, and fluorescent evaluation as described above for milk EVs. Testis samples were snap frozen and protein was extracted for HPLC/MS-MS. Results indicate that transgenic sows produce milk EVs endogenously labeled with ZsGreen1 compared with WT sow milk EVs. In addition, WT boars nursed by transgenic sows exhibited ZsGreen1-labeled milk EVs in their serum. Finally, ZsGreen1 was detectable in testes of WT boars nursed by TSG sows. The present study is the first to evaluate if maternal milk EVs are bioavailable to the neonatal testis. Using a novel swine model, these data suggest the transfer of maternal milk EVs to offspring blood and testes after a natural ingestion route (ad libitum suckling). Hence, milk EVs are bioavailable to the testis and may help mediate lactocrine programming.

Cells of all kingdoms produce extracellular vesicles (EVs); hence, they are present in most environments and body fluids. Lacticaseibacillus paracasei produces EVs that have attached biologically active proteins (P40 and P75). In this study, EV and functional proteins were found in five different commercial dairy-fermented products carrying L. paracasei. Strains present in those products were isolated, and with one exception, all produced small EVs (24-47 d.nm) carrying P40 and P75. In order to winnow bacterial EV from milk EV, products were subjected to centrifugal fractionation at 15,000 × g (15 K), 33,000 × g (33 K), and 100,000 × g (100 K). P75 was present in all supernatants and pellets, but P40 was only found in two products bound to the 15 and 33 K pellets, and 16S rDNA of L. paracasei could be amplified from all 100 K EVs, indicating the presence of L. paracasei EV. To investigate the interactions of bacterial EV and proteins with milk EV, L. paracasei BL23 EV was added to three commercial UHT milk products. Small-size vesicles (50-60 d.nm) similar to L. paracasei BL23 EV were found in samples from 100 K centrifugations, but intriguingly, P40 and P75 were bound to EV in 15 and 33 K pellets, containing bovine milk EV of larger size (200-300 d.nm). Sequencing 16S rDNA bands amplified from EV evidenced the presence of bacterial EVs of diverse origins in milk and fermented products. Furthermore, L. paracasei 16S rDNA could be amplified with species-specific primers from all samples, showing the presence of L. paracasei EV in all EV fractions (15, 33, and 100 K), suggesting that these bacterial EVs possibly aggregate and are co-isolated with EV from milk. P40 and P75 proteins would be interacting with specific populations of milk EV (15 and 33 K) because they were detected bound to them in fermented products and milk, and this possibly forced the sedimentation of part of L. paracasei EV at lower centrifugal forces. This study has solved technically complex problems and essential questions which will facilitate new research focusing on the molecular behavior of probiotics during fermentation and the mechanisms of action mediating the health benefits of fermented products.

Milk is a complex fluid that contains various types of proteins and extracellular vesicles (EVs). Some proteins can mingle with EVs, and interfere with their isolation. Among these proteins, caseins form micelles of a size comparable to milk EVs, and can thus be co-isolated with EVs. Preliminary steps that affect milk are crucial for EV isolation and impact the purity and abundance of isolated EVs. In the course of our previous works on cow's milk EVs, we found that sodium citrate (1% final), which is a biocompatible reagent capable of breaking down casein micelles into 40-nm monomers, allowed the isolation of high quantities of EVs with low coprecipitation of caseins or other contaminating proteins. Using this protocol, we successfully separated different EV subsets, characterized in depth their morphology, protein content and small RNA enrichment patterns. We were also able to describe their biological function in a mouse model of intestinal inflammation. We, hereby, detail the differential ultracentrifugation procedure that leads to high quantify, medium specificity, isolation of different milk EV subsets from the same sample. More specifically, we highlight the use of sodium citrate as a standardized approach to isolate and study milk EVs and its potential for isolation techniques other than differential ultracentrifugation.

Bone is constantly balanced between the formation of new bone by osteoblasts and the absorption of old bone by osteoclasts. To promote bone growth and improve bone health, it is necessary to promote the proliferation and differentiation of osteoblasts. Although bovine milk is known to exert a beneficial effect on bone formation, the study on the effect of bovine milk extracellular vesicles (EVs) on osteogenesis in osteoblasts is limited. In this study, we demonstrated that bovine milk EVs promoted the proliferation of human osteogenic Saos-2 cells by increasing the expression of cell cycle-related proteins. In addition, bovine milk EVs also induced the differentiation of Saos-2 cells by increasing the expression of RUNX2 and Osterix which are key transcription factors for osteoblast differentiation. Oral administration of milk EVs did not cause toxicity in Sprague-Dawley rats. Furthermore, milk EVs promoted longitudinal bone growth and increased the bone mineral density of the tibia. Our findings suggest that milk EVs could be a safe and powerful applicant for enhancing osteogenesis. PRACTICAL APPLICATIONS: Until now, calcium and vitamin D have been prescribed to promote bone formation or to prevent bone diseases such as osteoporosis. Recently, several studies to find bioactive molecules that regulate cellular functions of osteoblasts or osteoclasts are actively underway. Milk basic proteins and lactoferrin present in milk are known to promote bone formation, but they exist in small quantities and the isolation of these proteins is complicated making mass production difficult. Recently, it has been found that milk contains large quantities of EVs, and that they promote bone formation. Studies on the effect of Milk EVs on osteoblasts during osteogenesis will help in the development of biomaterials for osteogenesis.

ABSTRACTStudies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Background/Objectives: Donkey milk (DM) has been considered a valuable alternative to human and bovine counterparts as well as to infant formulas. Milk extracellular vesicles (EVs) have been proposed to influence key biological processes. The purpose of this study is to provide a comprehensive characterization of the protein composition of extracellular vesicles (EVs) by extending quantitative proteomic comparisons to EVs derived from donkey colostrum (DC) and mature donkey milk (MDM). Methods: The EVs were isolated from DC and MDM samples, characterized, and subjected to proteomic analysis using the tandem mass tag-based quantitative approach. Results: In addition to typical milk proteins and EV markers, EVs from DC and MDM both contain components associated with the immune system, immune response, or promoting tissue repair, and assisting with communication between the infant and their environment. The EVs from DC were enriched in proteins associated with protein turnover, specific defense functions, and regenerative processes. Conclusions: Overall, the results can contribute to the broader characterization of the overall protein composition of DC and MDM and might help to predict the beneficial effects of the corresponding EVs on various mammalian cells. They may also provide valuable insights for the development of novel DM-based products for food, pharmaceutical, and biotechnological applications.

Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients' cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean±SD EV numbers (3.4× 108± 1.2× 108-2.8× 108± 0.3× 107 compared with 3.1× 108± 1.2× 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2± 3.4ng/μL in raw milk to 17.7± 5.4-23.3± 10.0mg/μL in processed milk, P<0.05). Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk.

Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.

BackgroundSubclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively.ResultsWe found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals.ConclusionsOur results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. For example, HM is the best-known preventative for the deadly neonatal intestinal inflammatory disease, necrotizing enterocolitis (NEC), for which there is no cure. How HM reduces NEC risk is relatively unknown. HM contains thousands of molecular components, including extracellular vesicles (EVs). EVs are lipid bilayer-encased particles released from cells that carry biological cargo. EVs are putative regulators of intestinal function and HM EVs offer a mechanism for the transfer of proteins from the mom to the infant’s gut. It is unknown if milk EVs or protein cargo survive digestion. This limits the ability to leverage HM EV proteins as treatments for NEC, additives to infant nutrition, or therapeutics for other diseases. The objective of this proposal is to examine if HM EV protein cargo survives in vivo human digestion and if surviving cargo confers anti-inflammatory benefits in neonatal human enteroids. Hypothesis: HM EVs transport key protein cargo to intercellular targets and protect against intestinal inflammation. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from naso- or orojejunal sampling tubes. EVs were isolated from HM and digesta by density-gradient ultracentrifugation. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blot. EV protein cargo from n=3 paired HM and digesta samples were profiled by a C18-UPLC with Orbitrap mass spectrometry. Apical-out neonatal human enteroids were validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid EV uptake was assessed with CMPTX dye-labeled EVs. Effects of digesta EVs on inflammation were assessed by qPCR in LPS-treated enteroids. Results: EVs are enriched for markers CD63, CD81, CD9, and TSG101 and de-enriched for casein, milk fat globules. Only 4.68% ± 0.02 (p=0.0012 vs HM) of EV cargo proteins from HM reach the human intestine, but nearly half 64.08% ± 0.02 (p=0.007 vs HM) of the protein diversity is preserved. Human mammary-derived protein BTN1A1 is present in digesta EVs. Apical-out enteroids gene expression is consistent with a more differentiated epithelium, e.g. elevated ChgA (7.60 ± 4.04 vs 1.37 ± 0.39, p=0.0026) and down-regulated Ki67 (0.13 ± 0.04 vs 1.00 ± 0.04, p&lt;0.0001) Enteroids take up milk EVs. Filtered (0.22μm) neonatal digesta (contains EVs) attenuates LPS-induced TNFα gene expression. Conclusion: These novel findings demonstrate that a mostly pure population of EVs can be isolated from a 1mL starting volume. A subset of HM EVs reaches the neonatal human intestine where they can be taken up by the epithelium and may act to reduce inflammation. These data demonstrate the importance of examining EV cargo that survives to the human intestine when investigating potential HM-mediated mechanisms of disease prevention. NIH K01DK129401, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Milk extracellular vesicles (EVs) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EVs with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2-quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EVs' size, concentration, or morphology. We neither detected changes in the EV cargo regarding the amount of protein and RNA nor the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low somatic cell counts. Overall, we observed fewer CD81 and CD9 markers in quarters with a high SCC. Moreover, there was no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to one week is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EVs are highly stable in the raw milk matrix.

ABSTRACTHuman milk is rich in extracellular vesicles (EV) that may contribute to shaping neonatal immunity. Here, we evaluated whether freezing, and the addition of sodium citrate (SC), affect the characteristics of human milk EVs and their miRNAs. Freezing may compromise the milk EV population and their miRNA profile by creating artificial vesicles due to cell lysis. Furthermore, SC can be added to clear the EV fraction of micelles, that is, protein aggregates that co‐isolate with milk EVs, and may affect certain downstream analyses. To investigate potential differences between milk EV and their miRNA cargo when isolated from fresh and frozen samples, mature milk samples were collected from 10 women and subjected to four different treatments: fresh and frozen; freshSC and frozenSC. Ultracentrifugation was used for EV isolation, and subsequently characterized by Nanoparticle tracking analysis, flow cytometry, Western blot and electron microscopy. While freezing without SC has no impact on the evaluated EV parameters, freezing with SC significantly altered particle mean size as measured by NTA and protein levels as studied by MACSPlex flow cytometry. Importantly, neither freezing nor SC had an impact on the EV miRNA cargo, measured by qPCR. These findings also suggest that EV isolates from frozen samples, in comparison to freshly isolated ones, can produce valid results concerning morphology, size, surface markers and the EV miRNA profile.

Extracellular vesicles facilitate intercellular communication by transferring bioactive molecules with the potential to elicit host responses. Raw bovine milk contains functional extracellular vesicles that are taken up by humans. By comparing with extracellular vesicles from unprocessed bovine milk, the impact of pasteurisation, hydrodynamic cavitation treatment, or homogenisation on purity of milk extracellular vesicles isolated by a series of centrifugation steps followed by size-exclusion chromatography was assessed. Extracellular vesicles could be isolated from all four source materials, though to varying extents as evident from Western blots. In particular, homogenisation resulted in co-isolation of non-extracellular vesicle material such as β-casein, β-lactoglobulin, and triacylglycerol. RNA concentrations were highest in extracellular vesicles derived from the three processed source materials, thus indicating co-isolation of other RNA-containing detergent-labile particles in addition to native milk extracellular vesicles. Overall, this study shows that the history of milk processing impacts the purity of milk extracellular vesicles.

Accumulating evidence of the critical role of Ferrostatin-1 (Fer-1, ferroptosis inhibitor) in cerebral ischemia has intrigued us to explore the molecular mechanistic actions of Fer-1 delivery by bone marrow mesenchymal stem cells-derived extracellular vesicles (MSCs-EVs) in cerebral ischemia-reperfusion (I/R) injury. In vivo middle cerebral artery occlusion (MCAO) in mice and in vitro oxygen-glucose deprivation/reperfusion (OGD/R) in hippocampal neurons were developed to simulate cerebral I/R injury. After Fer-1 was confirmed to be successfully delivered by MSCs-EVs to neurons, we found that MSCs-EVs loaded with Fer-1 (MSCs-EVs/Fer-1) reduced neuron apoptosis and enhanced viability, along with curtailed inflammation and ferroptosis. The regulation of Fer-1 on GPX4/COX2 axis was predicted by bioinformatics study and validated by functional experiments. The in vivo experiments further confirmed that MSCs-EVs/Fer-1 ameliorated cerebral I/R injury in mice. Furthermore, poor expression of GPX4 and high expression of COX-2 were witnessed in cerebral I/R injury models. MSCs-EVs/Fer-1 exerted its protective effects against cerebral I/R injury by upregulating GPX4 expression and inhibiting COX-2 expression. Taken together, our study indicates that MSCs-EVs/Fer-1 may be an attractive therapeutic target for the treatment of cerebral I/R injury due to its anti-ferroptotic properties.