Exosomes participate in cell-to-cell communication, facilitated by the transfer of RNAs, proteins and lipids from donor to recipient cells. Exosomes and their RNA cargos do not exclusively originate from endogenous synthesis but may also be obtained from dietary sources such as the inter-species transfer of exosomes and RNAs in bovine milk to humans. Here, we assessed the bioavailability and distribution of exosomes and their microRNA cargos from bovine, porcine and murine milk within and across species boundaries. Milk exosomes labeled with fluorophores or fluorescent fusion proteins accumulated in liver, spleen and brain following suckling, oral gavage and intravenous administration in mice and pigs. When synthetic, fluorophore-labeled microRNAs were transfected into bovine milk exosomes and administered to mice, distinct species of microRNAs demonstrated unique distribution profiles and accumulated in intestinal mucosa, spleen, liver, heart or brain. Administration of bovine milk exosomes failed to rescue Drosha homozygous knockout mice, presumably due to low bioavailability or lack of essential microRNAs.

Extracellular vesicles (EVs) in milk harbor a variety of compounds, including lipids, proteins, noncoding RNAs, and mRNAs. Among the various classes of EVs, exosomes are of particular interest, because cargo sorting in exosomes is a regulated, nonrandom process and exosomes play essential roles in cell-to-cell communication. Encapsulation in exosomes confers protection against enzymatic and nonenzymatic degradation of cargos and provides a pathway for cellular uptake of cargos by endocytosis of exosomes. Compelling evidence suggests that exosomes in bovine milk are transported by intestinal cells, vascular endothelial cells, and macrophages in human and rodent cell cultures, and bovine-milk exosomes are delivered to peripheral tissues in mice. Evidence also suggests that cargos in bovine-milk exosomes, in particular RNAs, are delivered to circulating immune cells in humans. Some microRNAs and mRNAs in bovine-milk exosomes may regulate the expression of human genes and be translated into protein, respectively. Some exosome cargos are quantitatively minor in the diet compared with endogenous synthesis. However, noncanonical pathways have been identified through which low concentrations of dietary microRNAs may alter gene expression, such as the accumulation of exosomes in the immune cell microenvironment and the binding of microRNAs to Toll-like receptors. Phenotypes observed in infant-feeding studies include higher Mental Developmental Index, Psychomotor Development Index, and Preschool Language Scale-3 scores in breastfed infants than in those fed various formulas. In mice, supplementation with plant-derived MIR-2911 improved the antiviral response compared with controls. Porcine-milk exosomes promote the proliferation of intestinal cells in mice. This article discusses the above-mentioned advances in research concerning milk exosomes and their cargos in human nutrition. Implications for infant nutrition are emphasized, where permitted, but data in infants are limited.

Exosomes are natural nanoparticles that play an important role in cell-to-cell communication. Communication is achieved through the transfer of cargos, such as microRNAs, from donor to recipient cells and binding of exosomes to cell surface receptors. Exosomes and their cargos are also obtained from dietary sources, such as milk. Exosome and cell glycoproteins are crucial for intestinal uptake. A large fraction of milk exosomes accumulates in the brain, whereas the tissue distribution of microRNA cargos varies among distinct species of microRNA. The fraction of milk exosomes that escapes absorption elicits changes in microbial communities in the gut. Dietary depletion of exosomes and their cargos causes a loss of circulating microRNAs and elicits phenotypes such as loss of cognitive performance, increase in purine metabolites, loss of fecundity, and changes in the immune response. Milk exosomes meet the definition of bioactive food compounds.

Background: Bovine milk exosomes are studied for their roles as bioactive food compounds and as vehicles for drug delivery. Both lines of investigation converge on immune function, e.g., immune regulation by absorption of microRNAs encapsulated in milk exosomes across species boundaries, and the possibility of exosomes and their cargos triggering an immune response if used in drug delivery. This study assessed the bioavailability of immune-related microRNAs from bovine milk and changes in plasma cytokine concentrations after milk consumption in humans, and the secretion of cytokines by human peripheral blood mononuclear cells (PBMCs) cultured with milk exosomes transfected with immune-relevant microRNAs. Results: Human plasma samples were collected before and at timed intervals after a milk meal and analyzed for concentrations of six immune-relevant microRNAs and nine cytokines. The peak plasma concentrations of miR-15b-5p, miR-21-5p, miR-106b-5p, and miR-223-3p were 60 ± 9.80% to 162 ± 31.80% higher after milk consumption (C t values 23 ± 1.2 to 26 ± 1.1 cycles) compared to baseline values (P < 0.05). Plasma concentrations of TNF-alpha were not significantly different before versus after milk consumption; eight other cytokines were below detection limit. PBMCs were collected before and six hours after milk consumption and cultured with or without concanavalin A (ConA). TNF-alpha, IL-1β, IL-6 and IL-10 were detectable in culture media, but concentrations did not depend on milk consumption prior to PBMC isolation (P > 0.05). When PBMC cultures from fasted subjects were supplemented with milk exosomes that had been transfected with immune-relevant microRNAs, the concentrations of IL-1β, IL-6, IL-10 and TNF-alpha were 29 ± 12% to 220 ± 33% higher than controls cultured with non-transfected exosomes (P < 0.05), but cytokine concentrations were not different compared with control exosomes transfected with scrambled microRNA (P > 0.05). Conclusions: MicroRNAs in bovine milk exosomes are bioavailable. Milk exosomes do not elicit an increase of plasma cytokines following oral administration.

Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

BackgroundBovine milk exosomes contain cargos such as miRNAs, mRNAs, DNA and proteins. We have demonstrated that dietary bovine exosomes deliver functional RNA species and regulate genes and metabolism in humans and other non‐bovine species. Preliminary studies suggest that RNAs obtained through milk exosomes accumulate in the brain of mice.HypothesisDepletion of dietary bovine milk exosomes affects cognitive performance in C57BL/6 mice.MethodsC57BL/6 mice were fed AIN‐93G based, bovine milk exosome‐defined diets for up to 20 weeks. Exosome‐depleted diets are denoted E−; exosome‐sufficient controls are denoted E+. Spatial learning and memory were assessed using the Barnes maze and Morris water maze. Sensorimotor gating was assessed using an acoustic startle response (ASR) system. The statistical significance of differences was assessed by unpaired, two‐tailed t‐test.ResultsThe time needed to locate the escape hole in the Barnes maze increased by up to 130% in mice fed the E− diet compared to E+ controls (Fig. 1). Likewise, the time needed to locate and reach the submerged escape platform in the Morris water maze was significantly greater in female mice fed E− diet than female controls (Fig. 2). Prepulse inhibition (PPI) of the ASR is a measure of sensorimotor gating, and was significantly lower in female mice fed the E− diet than in female controls (Fig. 3). Diet did not affect PPI in male mice (p>0.05).Conclusion & future plansDiets defined by their content of bovine milk exosomes affect sensorimotor gating and cognitive performance in mice. Future studies will investigate which regions and cells in the brain accumulate dietary milk exosome RNA cargos, and whether a distinct species of RNA is responsible for the phenotypes reported here.Support or Funding InformationSupport: NIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.

Hibiscus sabdariffa L. (sorrel) has been widely used in the development of tropical beverages and folk medicine. This study's objective was to investigate the anti-obesity potential of sorrel calyx extracts (methanol and water) on 3T3-L1 adipocyte cells. Phytochemical content, antioxidant potential as DPPH (1, 1-Diphenyl-2-picrylhydrazyl) radical scavenging activity and ferric reducing antioxidant power (FRAP) and enzyme (α-glucosidase, α-amylase, and pancreatic lipase) inhibitory activities were determined in sorrel methanol extracts (SME) and sorrel water extracts (SWE). Effect of SWE and SME on lipid accumulation, lipolysis and apoptosis were tested in 3T3-L1 adipocyte differentiation and maintenance stage of cells at selected concentrations (200-1000 µg/ml) was studied. The total phenolic (GAE mg/100g dry weight) and total flavonoid (mg catechin equi/ 100g dry weight) contents in SME and SWE were 158.31 and 317.27 and 90.77 and 100.08. DPPH% inhibition (IC-50-mg/ml) and FRAP (mmol Fe [II]/100g dry weight) were 0.82 and 0.33 and 1799.13 and 2296.38 for SWE and SME, respectively. SME and SWE inhibited α-glucosidase, α-amylase, and pancreatic lipase activities by more than 40% at 4mg/ml. Significant (p < 0.05) reduction in lipid accumulation and increased glycerol release in 3T3-L1 cells was observed at concentrations ranged from 600 mg/ml of both extracts. Treating cells with SME-1000 µg/ml at differentiation resulted inhibition (p < 0.05) of lipid accumulation by 45% compared to untreated cells. Highest (p < 0.05) (35%) decrease in triglyceride content as well as higher glycerol release was seen in cells exposed to SME at the differentiation stage. Sorrel extracts induced apoptosis in adipocytes at higher concentrations with prominent effect of treating cells at differentiation stage. The results of this study showed effect of sorrel extracts in reduction of lipid accumulation and increase in lipolysis of 3T3-L1 cells.

Background Milk exosomes and their microRNA (miR) cargos are bioavailable. The content of exosomes and miRs is negligible in infant formulas compared to human milk, and dietary depletion of exosomes led to changes in bacterial communities and impaired gut health in juvenile mice. Adverse effects of formula feeding may be compounded by using soy formulas due to exosome binding by abundant lectins in that matrix. The purpose of this study was to assess the bioavailability of milk exosomes and their miR cargos added to soy formula in adults, as well as the potential role of soy lectins in exosome bioavailability. Methods Eleven healthy adults (6 men, 5 women) enrolled in this randomized crossover study. Participants consumed 1.0 l of soy formula without (SF) or with (SFE) bovine milk exosomes added. Concentration-time curves of six plasma miRs were analyzed using reverse transcription quantitative PCR. Lectin affinity chromatography was used to assess the binding of exosomes by soy lectins. Data were analyzed by using paired t test. P < 0.05 was considered statistically significant. Results Consumption of SF and SFE did not elicit postprandial increases in plasma miRs. Approximately 39% of bovine milk exosome particles were retained by lectin columns. Conclusions We conclude that fortification of soy formulas with milk exosomes, in the absence of removing lectins, is not a viable strategy for delivering bioavailable exosomes and their miR cargos. Lectins in soy formulas bind glycoprotein on the surfaces of milk exosomes, thereby preventing exosome absorption. Trial registration ISRCTN registry ID: 16329971. Retrospectively registered on February 7th, 2019.