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Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages

Open ArchivePublished:February 25, 2015DOI:https://doi.org/10.3168/jds.2014-9076

      Abstract

      We reported previously that microRNA (miRNA) are present in whey fractions of human breast milk, bovine milk, and rat milk. Moreover, we also confirmed that so many mRNA species are present in rat milk whey. These RNA were resistant to acidic conditions and to RNase, but were degraded by detergent. Thus, these RNA are likely packaged in membrane vesicles such as exosomes. However, functional extracellular circulating RNA in bodily fluids, such as blood miRNA, are present in various forms. In the current study, we used bovine raw milk and total RNA purified from exosomes (prepared by ultracentrifugation) and ultracentrifuged supernatants, and analyzed them using miRNA and mRNA microarrays to clarify which miRNA and mRNA species are present in exosomes, and which species exist in other forms. Microarray analyses revealed that most mRNA in milk whey were present in exosomes, whereas miRNA in milk whey were present in supernatant as well as exosomes. The RNA in exosomes might exert functional effects because of their stability. Therefore, we also investigated whether bovine milk-derived exosomes could affect human cells using THP-1 cells. Flow cytometry and fluorescent microscopy studies revealed that bovine milk exosomes were incorporated into differentiated THP-1 cells. These results suggest that bovine milk exosomes might have effects in human cells by containing RNA.

      Key words

      Introduction

      Bovine milk and dairy products have a long tradition and are a good source of nutrition to maintain human health (
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      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
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      Purification of RNA from milk whey.
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      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
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      Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs.
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      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
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      Purification of RNA from milk whey.
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      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ). Moreover, these RNA are stable under harsh conditions, such as low pH, and in the presence of RNase (
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      • Aoki N.
      Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs.
      ;
      • Kosaka N.
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      MicroRNA as a new immune-regulatory agent in breast milk.
      ;
      • Izumi H.
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      • Sekine K.
      • Ochiya T.
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      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ). These RNA have been detected in commercial dairy products, such as powdered infant formula, that have undergone stringent industrial processes (
      • Chen X.
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      Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products.
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      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ). Based on these studies, it is possible that RNA in milk have functions in the mammalian gastrointestinal tract.
      Most body fluids, such as blood, saliva, urine, and amniotic fluid, contain miRNA (
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      • Galas D.J.
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      The microRNA spectrum in 12 body fluids.
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      Small RNA transcriptomes of two types of exosomes in human whole saliva determined by next generation sequencing.
      ). However, these circulating RNA exist in diverse forms, including packaged in exosomes (
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      Exosomes: Proteomic insights and diagnostic potential.
      ;
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      Circulating microRNA in body fluid: A new potential biomarker for cancer diagnosis and prognosis.
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      • Sjöstrand M.
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      • Lötvall J.
      • Valadi H.
      Human saliva, plasma and breast milk exosomes contain RNA: Uptake by macrophages.
      ), in high-density lipoprotein (
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      • Shamburek R.D.
      • Remaley A.T.
      MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.
      ), and as complexes with RNA-binding proteins (
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      Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.
      ). For example, our previous study (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Purification of RNA from milk whey.
      ) showed that the RNA concentration in bovine milk whey-derived exosomes was lower than was that in whey. Therefore, the aim of the current study was to clarify which miRNA and mRNA species in bovine milk whey are present in exosomes, and assess which species exist in other forms, using microarrays. No comprehensive reports have analyzed the presence of mRNA in bovine milk whey, although mRNA were found in rat milk whey (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ), human (
      • Maningat P.D.
      • Sen P.
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      • Sunehag A.L.
      • Hadsell D.L.
      • Bray M.
      • Haymond M.W.
      Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome.
      ;
      • Lemay D.G.
      • Ballard O.A.
      • Hughes M.A.
      • Morrow A.L.
      • Horseman N.D.
      • Nommsen-Rivers L.A.
      RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.
      ) and goat (
      • Brenaut P.
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      • Bevilacqua C.
      • Rebours E.
      • Cebo C.
      • Martin P.
      Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection.
      ) milk fat globules, and bovine milk somatic cells (
      • Wickramasinghe S.
      • Rincon G.
      • Islas-Trejo A.
      • Medrano J.F.
      Transcriptional profiling of bovine milk using RNA sequencing.
      ) using microarrays or next-generation sequencing. Our previous study using rat milk whey (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ) detected >10,000 mRNA. Therefore, to our knowledge, the current study is the first report to reveal which mRNA are present in bovine milk whey exosomes and in other forms. Previous studies revealed that bovine milk miRNA profiles differ among cows and between colostrum and mature milk (
      • Chen X.
      • Gao C.
      • Li H.
      • Huang L.
      • Sun Q.
      • Dong Y.
      • Tian C.
      • Gao S.
      • Dong H.
      • Guan D.
      • Hu X.
      • Shujian Z.
      • Li L.
      • Zhu L.
      • Yan Q.
      • Zhang J.
      • Zen K.
      • Zhang C.Y.
      Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products.
      ;
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ). In commercial dairy products, raw milk is collected and mixed from many cows and is usually used. Whey is an important fraction of milk, and is also used in a variety of products (
      • Marshall K.
      Therapeutic applications of whey protein.
      ). In the current study, we prepared whey fractions from raw milk, which is used for commercial dairy products. In addition, some studies showed that macrophages could take up milk whey-derived exosomes (
      • Lässer C.
      • Alikhani V.S.
      • Ekström K.
      • Eldh M.
      • Paredes P.T.
      • Bossios A.
      • Sjöstrand M.
      • Gabrielsson S.
      • Lötvall J.
      • Valadi H.
      Human saliva, plasma and breast milk exosomes contain RNA: Uptake by macrophages.
      ;
      • Sun Q.
      • Chen X.
      • Yu J.
      • Zen K.
      • Zhang C.Y.
      • Li L.
      Immune modulatory function of abundant immune-related microRNAs in microvesicles from bovine colostrum.
      ). Therefore, we also investigated whether bovine milk-derived exosomes could be taken up by human cells using human monocytic leukemia THP-1 cells that show macrophage-like functions after differentiation by phorbolmyristate acetate (PMA).

      Materials and Methods

      Milk Whey Sample Preparation

      Three bovine raw milk samples were prepared and stored at −80°C until use; these are commonly used for research and development in our laboratory. Whey was prepared as described previously (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ,
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Purification of RNA from milk whey.
      ). Briefly, the samples were centrifuged twice (1,200 × g, 4°C, 10 min) to remove fat, cells, and large debris. The defatted supernatant was then centrifuged (21,500 × g, 4°C, 30 min, 1 h) to remove residual fat and casein. The clear supernatant (whey) was then passed through 0.65-, 0.45-, and 0.22-μm filters to remove residual cell debris.

      Exosome and Supernatant Preparation

      The whey samples (see previous section) were ultracentrifuged (100,000 × g, 4°C, 90 min) using an SRP70AT rotor (Hitachi Koki, Tokyo, Japan) according to a previous study (
      • Lässer C.
      • Alikhani V.S.
      • Ekström K.
      • Eldh M.
      • Paredes P.T.
      • Bossios A.
      • Sjöstrand M.
      • Gabrielsson S.
      • Lötvall J.
      • Valadi H.
      Human saliva, plasma and breast milk exosomes contain RNA: Uptake by macrophages.
      ). The resulting supernatant was collected carefully to avoid contaminating the pelleted exosomes. The pelleted exosomes were resuspended in PBS and then ultracentrifuged (100,000 × g, 4°C, 90 min) for washing; this wash step was performed twice. Finally, the exosomes were resuspended in PBS. The protein content of the exosomes was determined using a Micro Bradford protein assay (Bio-Rad, Hercules, CA).

      Extraction of Total RNA

      Total RNA was extracted from exosomes and supernatant samples, and was purified using an miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) as described previously (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ,
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Purification of RNA from milk whey.
      ,
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ). The quantity and integrity of the RNA were assessed using an RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA), and the miRNA-to-small RNA ratio (default settings: miRNA = 10–40 nucleotides; small RNA = 0–270 nucleotides) was examined on an Agilent 2100 Bioanalyzer (Agilent) using a Small RNA Kit (Agilent).

      miRNA Microarray

      The RNA samples were derived from an equal mixture of 3 samples. To detect the expression of miRNA in exosomes and supernatant, 40 ng of mixed total RNA was labeled with cyanine-3 and then hybridized to Bovine miRNA Microarray Rel. 17.0 arrays using an miRNA Complete Labeling Reagent and Hyb Kit (Agilent;
      • Wang H.
      • Ach R.A.
      • Curry B.
      Direct and sensitive miRNA profiling from low-input total RNA.
      ). The Bovine miRNA Microarray Rel. 17.0 analyzes the expression of 670 bovine miRNA from the Sanger miRBase Rel.17.0 (www.mirbase.org). Signals were detected with an Agilent DNA Microarray Scanner, and the scanned images were analyzed using the Feature Extraction Software (ver. 10.7.3.1; Agilent).

      mRNA Microarray

      The RNA samples were derived from an equal mixture of 3 samples. To detect the expression of mRNA in exosomes and supernatant, complementary RNA was generated from 100 ng of mixed total RNA and then labeled with cyanine-3 using a Low Input Quick Amp Labeling Kit (Agilent). Labeled cRNA was hybridized to Bovine Oligo DNA Microarray ver. 2.0 (4 × 44K) using a Gene Expression Hybridization Kit (Agilent). The Bovine Oligo DNA Microarray ver. 2.0 (4 × 44K) contains 43,713 probes (without control probes). Signals were detected with an Agilent DNA Microarray Scanner, and the scanned images were analyzed using the Feature Extraction Software (ver. 10.7.3.1).

      Microarray Data Analysis

      Raw data from Feature Extraction Software were exported to GeneSpring GX ver. 11.5.1 (Agilent). Entities were considered present if signal was judged to be present by software. For mRNA microarray data, transcripts with raw signals >20 and that were given an Entrez Gene ID were considered to be present. All microarray data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO), and are accessible through GEO Series accession number GSE61978 (www.ncbi.nlm.nih.gov/geo).

      Quantification of miRNA by qPCR

      The cDNA was generated using an miScript Reverse Transcription Kit (Qiagen). Briefly, total RNA from exosomes or supernatant (both from 3.2 mL of whey) was polyadenylated using poly(A) polymerase, and cDNA was generated using reverse transcriptase and tagged oligo-dT primers. The cDNA was then diluted in 9 volumes of nuclease-free water and subjected to quantitative (q)PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) using an miScript SYBR Green PCR Kit and miScript Primer Assay (Qiagen) according to the manufacturer’s instructions. This system was reported that show relatively high accuracy and high sensitivity among several quantification methods (
      • Mestdagh P.
      • Hartmann N.
      • Baeriswyl L.
      • Andreasen D.
      • Bernard N.
      • Chen C.
      • Cheo D.
      • D’Andrade P.
      • DeMayo M.
      • Dennis L.
      • Derveaux S.
      • Feng Y.
      • Fulmer-Smentek S.
      • Gerstmayer B.
      • Gouffon J.
      • Grimley C.
      • Lader E.
      • Lee K.Y.
      • Luo S.
      • Mouritzen P.
      • Narayanan A.
      • Patel S.
      • Peiffer S.
      • Rüberg S.
      • Schroth G.
      • Schuster D.
      • Shaffer J.M.
      • Shelton E.J.
      • Silveria S.
      • Ulmanella U.
      • Veeramachaneni V.
      • Staedtler F.
      • Peters T.
      • Guettouche T.
      • Wong L.
      • Vandesompele J.
      Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.
      ) and was used in our previous studies (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ,
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Purification of RNA from milk whey.
      ,
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ). The following real-time PCR protocol was used: initial activation of HotStarTaq DNA Polymerase (from miScript kit; 95°C, 15 min); 40 to 50 cycles of denaturation (94°C, 15 s), annealing (55°C, 30 s), and extension (70°C, 34 s); and melting curve analysis. The miScript Primer Assays (Qiagen) used for the target miRNA are listed in Supplemental Table S1 (http://dx.doi.org/10.3168/jds.2014-9076). The data were analyzed using 7500 Software ver. 2.0.4. (Applied Biosystems) with the fixed cycle threshold (Ct) setting (ΔRn = 0.02, where ΔRn is the fluorescence of the reporter dye minus the baseline) to assign baseline values and the threshold for Ct determination.

      Quantification of mRNA by qPCR

      The cDNA to quantify mRNA was generated from total RNA extracted from exosomes or supernatant (both from 3.2 mL of whey) using a High Capacity RNA-to-cDNA Kit (Applied Biosystems). The cDNA was diluted in 9 volumes of nuclease-free water, and was then subjected to qPCR on a 7500 Fast Real-Time PCR System (Applied Biosystems) using TaqMan Fast Advanced Master Mix (Applied Biosystems) and TaqMan Probes according to the manufacturer’s protocol. The following real-time PCR protocol was used: uracil-N-glycosylase incubation (50°C, 2 min), polymerase activation (95°C, 20 s), 40 to 50 cycles of denaturation (95°C, 3 s), and annealing and extension (60°C, 30 s). The TaqMan Probes used for the target mRNA are listed in Supplemental Table S2 (http://dx.doi.org/10.3168/jds.2014-9076). The data were analyzed using 7500 Software ver. 2.0.4. (Applied Biosystems) with the fixed Ct setting (ΔRn = 0.2) to assign baseline values and the threshold for Ct determination.

      Cell Culture

      The THP-1 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated FBS (HyClone, Logan, UT), 1% penicillin-streptomycin-glutamine (Gibco, Palo Alto, CA), 1% NEAA (Gibco), and 0.1% β-mercaptoethanol (Gibco; complete RPMI 1640). Cells were incubated at 37°C in humid atmosphere with 5% CO2. The FBS was ultracentrifuged (100,000 × g, 4°C, 90 min) before use to eliminate bovine serum exosomes.

      Exosome Staining

      The exosomes were labeled with PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions and a previous study (
      • Lässer C.
      • Alikhani V.S.
      • Ekström K.
      • Eldh M.
      • Paredes P.T.
      • Bossios A.
      • Sjöstrand M.
      • Gabrielsson S.
      • Lötvall J.
      • Valadi H.
      Human saliva, plasma and breast milk exosomes contain RNA: Uptake by macrophages.
      ), with minor modifications. Briefly, 8 μL of PKH67 dye was added to 1 mL of Diluent C (from the kit), and was then added to the exosomes and the control sample (PBS). The samples were mixed gently for 4 min, and then 2 mL of 2% FBS (exosome-depleted) Cell Wash (BD Biosciences, San Diego, CA) were added to bind the excess dye. The samples were then transferred to 100-kDa filters (Amicon, Millipore, Billerica, MA). The samples were washed 3 times with 2% FBS (exosome-depleted) Cell Wash, before being transferred to new 100-kDa filters and washed twice with complete RPMI 1640 (see previous sections).

      Uptake of Milk-Derived Exosomes by THP-1 Cells

      The THP-1 cells were cultured in 12-well plate for flow cytometry and were cultured in 8-well Lab-Tek Chamber Slide (Thermo Fisher Scientific, Waltham, MA) for fluorescence microscopy. The THP-1 cells were differentiated using 50 nM PMA (Sigma) for 60 h or were maintained without PMA, and were washed 3 times with complete RPMI. Ten micrograms of PKH67-labeled exosomes or the same volume of the PKH67-PBS control was added per 1 × 105 cells (differentiated or undifferentiated THP-1 cells), which were then incubated for 2 h at either 37°C or 4°C. The incorporation of exosomes into the macrophages was analyzed using FACSCanto (BD Biosciences) and visualized using a fluorescence microscope BX53 (Olympus, Tokyo, Japan). For flow cytometry, cells were washed twice with PBS, treated with 0.05% trypsin-0.02% EDTA-4Na solution (Gibco), washed twice with 2% FBS (exosome-depleted) Cell Wash, and suspended in 2% FBS (exosome-depleted) Cell Wash. The data were then acquired using FACSCanto. The flow cytometric data were analyzed using the FlowJo software (Tree Star Inc., Ashland, OR). For fluorescence microscopy, cells were washed twice with PBS, fixed with CellFIX (BD Biosciences) for 15 min, and washed 3 times with PBS. Five microliters of 7-AAD (BD Biosciences) in 200 μL of PBS was added for 15 min to label nuclei, and the samples were mounted using Vectashield (Vector Laboratories Inc., Burlingame, CA). The fluorescence microscopy data were analyzed using cellSens (Olympus).

      Statistical Analyses

      The values are expressed as mean ± SEM. The significance of differences was determined using t-test for comparisons between 2 groups, and the Tukey-Kramer honestly significant difference (HSD) test was used for multiple comparisons (JMP software; SAS Institute Inc., Cary, NC). A P-value <0.05 was considered significant difference.

      Results

      Bioanalyzer Analyses

      Total RNA was extracted from bovine milk whey-derived exosomes and ultracentrifuged supernatant, and was analyzed using a bioanalyzer. No or very little ribosomal RNA (18S and 28S) was seen, but small RNA (<300 nucleotides) were present (Figure 1). The RNA concentration in the exosome fraction was significantly higher than was that in the supernatant fraction (Figure 2A). The RNA content ratio of exosomes to supernatant was ~7:3 (Figure 2B). The miRNA-to-small RNA ratio (default setting: miRNA = 10–40 nucleotides; small RNA = 0–270 nucleotides) was significantly higher in the supernatant fraction compared with the exosome fraction (Figure 2C).
      Figure thumbnail gr1
      Figure 1Analysis of RNA purified from bovine raw milk whey-derived exosomes and ultracentrifuged supernatants using Bioanalyzer (Agilent Technologies, Santa Clara, CA). (A, B) Bovine raw milk whey-derived exosomes; (C, D) ultracentrifuged supernatants from bovine raw milk whey. A and C were analyzed using an RNA 6000 Pico Kit (Agilent); B and D were analyzed using a Small RNA Kit (Agilent). fu = fluorescence unit; nt = nucleotide. Color version available online.
      Figure thumbnail gr2
      Figure 2RNA concentrations, RNA concentrate ratio, and microRNA (miRNA)-to-small RNA ratio in bovine raw milk whey. (A) RNA concentrations in bovine raw milk whey-derived exosomes and ultracentrifuged supernatants; (B) ratios of the RNA concentrations in exosomes to those in supernatants from 3 raw milk (RM) whey samples; (C) miRNA-to-small RNA ratio. The values are means ± SEM (n = 3). *P < 0.05 (compared with supernatant), **P < 0.01 (compared with exosome).

      Microarray Analyses

      The miRNA microarray analyses revealed 79 miRNA in the exosomes, and 91 in the supernatant (Figure 3A). A total of 39 miRNA were common to both fractions (Figure 3A), but their signal intensities differed (Figure 3B). The top 50 highly expressed miRNA in exosomes and supernatant, and their normalized signal intensities, are listed in Table 1. The miRNA that were expressed at high levels differed between exosomes and supernatant (Table 1). The mRNA microarray analyses detected 19,320 mRNA probes in exosomes and 2,634 mRNA in supernatant (Figure 3C). A total of 2,538 mRNA probes (most of the mRNA probes that were detected in supernatant) were common to both exosomes and supernatant (Figure 3C); however, the signal intensity differed between fractions (Figure 3D). The top 50 highly expressed mRNA probes and their signal intensities in exosomes and supernatant are listed in Tables 2 and 3, respectively. Some mRNA probes were expressed highly in both exosomes and supernatant. However, most of highly expressed mRNA probes were different between exosomes and supernatant (Tables 2 and 3).
      Figure thumbnail gr3
      Figure 3MicroRNA (miRNA) and mRNA microarray results of bovine raw milk whey-derived exosomes and ultracentrifuged supernatants. (A) Venn diagram showing the numbers of miRNA expressed in exosomes and supernatants; (B) heat map of the normalized array data for miRNA detected in exosomes or supernatants; (C) Venn diagram showing the numbers of mRNA probes expressed in exosomes and the supernatants; (D) heat map of the normalized array data for mRNA detected in exosomes or supernatants.
      Table 1Top 50 most highly expressed microRNA in bovine whey exosome or supernatant
      ExosomeSupernatant
      RankNameSignal

      intensity
      RankNameSignal

      intensity
      1miR-247819.161miR-1777a20.02
      2miR-1777b14.112miR-1777b19.74
      3miR-1777a13.443miR-230518.93
      4let-7b12.784miR-134317.76
      5miR-122412.605miR-241217.48
      6miR-241212.546miR-288817.42
      7miR-230511.707miR-231616.97
      8let-7a11.678miR-2328*16.86
      9miR-200c11.519miR-122416.04
      10miR-14111.3210miR-247816.02
      11miR-288111.2911miR-289315.92
      12miR-2328*9.9212miR-288115.89
      13let-7c9.8913miR-237414.91
      14miR-148a9.8714miR-158414.84
      15miR-3209.8115miR-124914.47
      16miR-28889.7416miR-67114.45
      17let-7f9.7317miR-239114.22
      18miR-200b9.5518miR-234813.75
      19miR-15849.5119miR-289213.64
      20miR-26a9.3020miR-288512.76
      21miR-20a9.1821miR-242812.41
      22miR-1039.1522miR-288212.39
      23miR-29c8.8323miR-248611.57
      24miR-30d8.8024miR-245511.36
      25miR-928.6325miR-289811.31
      26miR-23048.4126miR-1225–3p11.13
      27miR-3758.4127miR-240711.12
      28miR-23918.4028miR-2436–5p10.89
      29let-7g8.3929miR-238910.85
      30miR-30a-5p8.2430miR-32010.82
      31miR-13438.1931miR-14110.58
      32miR-125b8.1732miR-247610.38
      33miR-30b-5p8.1633miR-48910.24
      34miR-26b7.9934miR-345–3p10.16
      35miR-28927.9635miR-239210.11
      36miR-24–3p7.9436miR-30d10.01
      37miR-12497.8837miR-365–3p9.99
      38miR-423–5p7.8538miR-23099.95
      39miR-6647.7539miR-23409.91
      40miR-28877.7540miR-29029.74
      41miR-2284d7.6841miR-24549.64
      42miR-23167.6342miR-2373*9.61
      43miR-23747.6143miR-18359.56
      44miR-29a7.6144miR-28999.45
      45let-7d7.5345miR-30a-5p9.42
      46miR-200a7.5146miR-425–3p9.40
      47miR-151*7.4647miR-2300a-5p9.39
      48miR-30f7.3848miR-133a9.39
      49miR-22917.3249miR-12819.38
      50miR-2284l7.2450miR-133b9.31
      Table 2Top 50 most highly expressed mRNA probes in bovine whey exosome
      RankProbe nameGene

      symbol
      Gene nameSignal

      intensity
      1A_73_P100256ITPR1Inositol 1,4,5-triphosphate receptor, type 118.27
      2A_73_113728LALBAα-LA18.26
      3A_73_P459006PAEPProgestagen-associated endometrial protein (also known as β-LG)18.14
      4A_73_P311621CSN2Casein β18.04
      5A_73_116854CSN2Casein β18.03
      6A_73_P393463CSN1S1Casein α-s117.95
      7A_73_P084401RPS28Ribosomal protein S2817.95
      8A_73_P100346ITPR1Inositol 1,4,5-triphosphate receptor, type 117.94
      9A_73_P504783ITPR1Inositol 1,4,5-triphosphate receptor, type 117.92
      10A_73_111101CSN3Casein kappa17.91
      11A_73_P287836CSN1S2Casein α-S217.90
      12A_73_P050936CSN1S1Casein α-s117.88
      13A_73_P468825CSN1S1Casein α-s117.88
      14A_73_115729PAEPProgestagen-associated endometrial protein (also known as β-LG)17.84
      15A_73_P232452CSN2Casein β17.79
      16A_73_111985RPS3Ribosomal protein S317.77
      17A_73_112949RPLP0Ribosomal protein, large, P017.77
      18A_73_P411496PAEPProgestagen-associated endometrial protein (also known as β-LG)17.76
      19A_73_P038871LALBAα-LA17.76
      20A_73_P107731CSN3Casein kappa17.76
      21A_73_P097536RPL23Ribosomal protein L2317.74
      22A_73_120857RPL21Ribosomal protein L2117.73
      23A_73_107578H1FXH1 histone family, member X17.70
      24A_73_P045606CSN2Casein β17.68
      25A_73_114056RPS2Ribosomal protein S217.63
      26A_73_P093916RPS16Ribosomal protein S1617.63
      27A_73_115443RPL18Ribosomal protein L1817.63
      28A_73_106136RPS8Ribosomal protein S817.62
      29A_73_P462036RPLP1Ribosomal protein, large, P117.59
      30A_73_111582FABP3FA binding protein 3, muscle and heart (mammary-derived growth inhibitor)17.58
      31A_73_109246RPS10Ribosomal protein S1017.58
      32A_73_120860RPL31Ribosomal protein L3117.58
      33A_73_P073166RPL23ARibosomal protein L23a17.56
      34A_73_P103236RPS28Ribosomal protein S2817.55
      35A_73_100438CSN1S1Casein α-s117.54
      36A_73_P291436RPS18Ribosomal protein S1817.54
      37A_73_P041941EEF1A1Eukaryotic translation elongation factor 1 α 117.53
      38A_73_P068826RPS3ARibosomal protein S3A17.52
      39A_73_P056536RPLP2Ribosomal protein, large, P217.51
      40A_73_P040046UBA52Ubiquitin A-52 residue ribosomal protein fusion product 117.50
      41A_73_P057311RPL31Ribosomal protein L3117.49
      42A_73_P044606PLIN2Perilipin 217.49
      43A_73_P035681RPS27ARibosomal protein S27a17.48
      44A_73_P046936RPS16Ribosomal protein S1617.46
      45A_73_115118RPL10Ribosomal protein L1017.44
      46A_73_P097551RPS8Ribosomal protein S817.43
      47A_73_114339GLYCAM1Glycosylation-dependent cell adhesion molecule 117.43
      48A_73_P041201TPT1Tumor protein, translationally-controlled 117.43
      49A_73_P097526RPL10Ribosomal protein L1017.42
      50A_73_P266861CSN1S2Casein α-S217.41
      Table 3Top 50 most highly expressed mRNA probes in bovine whey supernatant
      RankProbe nameGene symbolGene nameSignal

      intensity
      1A_73_P504783ITPR1Inositol 1,4,5-triphosphate receptor, type 117.40
      2A_73_P153516POLR2KPolymerase (RNA) II (DNA directed) polypeptide K, 7.0kDa17.36
      3A_73_P455626TSPYL4TSPY-like 416.52
      4A_73_P361931FBXO16F-box protein 1616.06
      5A_73_P491198TMEM173Transmembrane protein 17315.65
      6A_73_P215177TLR2Toll-like receptor 215.46
      7A_73_P093286C3H1orf87Uncharacterized protein C1orf87-like15.36
      8A_73_117796IL1RL1Interleukin 1 receptor-like 115.30
      9A_73_P420956PFDN5Prefoldin subunit 515.29
      10A_73_P190192AGTR1Angiotensin II receptor, type 115.28
      11A_73_P213062ANKRD39Ankyrin repeat domain 3914.90
      12A_73_P180692ACOT11Acyl-CoA thioesterase 1114.83
      13A_73_P486468TNS4Tensin 414.80
      14A_73_P224702ANKRD39Ankyrin repeat domain 3914.70
      15A_73_121245LOC511936Cytochrome P450, family 2, subfamily J14.70
      16A_73_P051356MGC152278Similar to Myeloid-associated differentiation marker14.66
      17A_73_P048836ZNF674Zinc finger protein 67414.64
      18A_73_P405601AGTR1Angiotensin II receptor, type 114.61
      19A_73_P240340HRASLSHRAS-like suppressor14.59
      20A_73_P459006PAEPProgestagen-associated endometrial protein (also known as β-LG)14.58
      21A_73_P191827AGTR1Angiotensin II receptor, type 114.54
      22A_73_103111GTF2H5General transcription factor IIH, polypeptide 514.48
      23A_73_107578H1FXH1 histone family, member X14.46
      24A_73_P192282CHRNA1Cholinergic receptor, nicotinic, α 1 (muscle)14.46
      25A_73_P476773SLC5A12Solute carrier family 5 (sodium/glucose cotransporter), member 1214.41
      26A_73_P033441H2BHistone H2B14.40
      27A_73_P242615CHMP1BChromatin modifying protein 1B14.40
      28A_73_P461676HERC1Hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 114.38
      29A_73_P246691C3H1orf109Hypothetical LOC50802114.34
      30A_73_P374271SECTM1Secreted and transmembrane 114.31
      31A_73_P195057NIPSNAP1Nipsnap homolog 1 (C. elegans)14.28
      32A_73_P134616ACPPAcid phosphatase, prostate14.26
      33A_73_P132081ACPPAcid phosphatase, prostate14.18
      34A_73_P100256ITPR1Inositol 1,4,5-triphosphate receptor, type 114.06
      35A_73_P187832ACPPAcid phosphatase, prostate14.05
      36A_73_P200172POLHPolymerase (DNA directed), eta14.04
      37A_73_P218867IFT80Intraflagellar transport 80 homolog (Chlamydomonas)14.02
      38A_73_P171747LOC100139208Hypothetical protein LOC10013920814.02
      39A_73_P149206FGF2Fibroblast growth factor 2 (basic)13.96
      40A_73_P288576CWC25CWC25 spliceosome-associated protein homolog (S. cerevisiae)13.91
      41A_73_P211172MBL2Mannose-binding lectin (protein C) 2, soluble13.85
      42A_73_P504858NEPNNephrocan13.83
      43A_73_P211497C12H13orf18Uncharacterized protein C13orf18 homolog13.83
      44A_73_P465708ACPPAcid phosphatase, prostate13.75
      45A_73_P134026ACOT11Acyl-CoA thioesterase 1113.74
      46A_73_P465498ACPPAcid phosphatase, prostate13.72
      47A_73_113343LOC517043Leucine-rich repeat-containing G protein-coupled receptor 6-like13.71
      48A_73_P187927ACOT11Acyl-CoA thioesterase 1113.69
      49A_73_P465423PIGMPhosphatidylinositol glycan anchor biosynthesis, class M13.63
      50A_73_100232LOC508118Zinc finger and BTB domain containing 313.63

      qPCR Analyses of miRNA

      Twenty miRNA were selected from the top 50 list (Table 3) and were analyzed using qPCR to assess their expression levels in exosomes and supernatants that were isolated from the same volume of whey (Figure 4). Almost all of the selected miRNA (except for miR-133b and miR-1281) were expressed at significantly higher levels in exosomes compared with supernatants.
      Figure thumbnail gr4
      Figure 4Quantitative PCR analysis [cycle threshold (Ct) values] of select microRNA (miRNA) in exosomes and supernatants isolated from equal volumes of bovine raw milk whey. Total RNA from exosomes and supernatants isolated from 3.2 mL of raw milk whey was used. The values are means ± SEM (n = 3); *P < 0.05 (compared with supernatant), **P < 0.01 (compared with supernatant).

      qPCR Analyses of mRNA

      Twenty-three mRNA were selected from those shown in Tables 2 and 3, and were analyzed using qPCR to quantify their expression in exosomes and supernatants that were isolated from the same volume of whey (Figure 5). The selected mRNA were milk- and milk-derived exosome-related mRNA (Figure 5A, B) or mRNA that were reported in other studies to be milk RNA (milk cells and milk fat globule; Figure 5C;
      • Maningat P.D.
      • Sen P.
      • Rijnkels M.
      • Sunehag A.L.
      • Hadsell D.L.
      • Bray M.
      • Haymond M.W.
      Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome.
      ;
      • Brenaut P.
      • Bangera R.
      • Bevilacqua C.
      • Rebours E.
      • Cebo C.
      • Martin P.
      Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection.
      ;
      • Wickramasinghe S.
      • Rincon G.
      • Islas-Trejo A.
      • Medrano J.F.
      Transcriptional profiling of bovine milk using RNA sequencing.
      ;
      • Lemay D.G.
      • Ballard O.A.
      • Hughes M.A.
      • Morrow A.L.
      • Horseman N.D.
      • Nommsen-Rivers L.A.
      RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.
      ). All the selected mRNA were expressed at significantly higher levels in exosomes compared with supernatants (Figure 5).
      Figure thumbnail gr5
      Figure 5Quantitative PCR analysis [cycle threshold (Ct) values] of select mRNA in exosomes and supernatants isolated from equal volumes of bovine raw milk whey. Total RNA from exosomes and supernatants from 3.2 mL of raw milk whey was used. (A) Selected transcripts from the 50 species expressed at the highest level in exosomes; (B) milk-derived exosome-related transcripts; (C) selected transcripts identified in previous studies of milk somatic cells and the milk fat layer. The values are the mean ± SEM (n = 3); *P < 0.05 (compared with supernatant), **P < 0.01 (compared with supernatant). Actb = β-actin; EEF1α1 = eukaryotic translation elongation factor 1 α 1; FAS = fatty acid synthase; FTH1 = ferritin, heavy polypeptide 1; GLYCAM1 = glycosylation-dependent cell adhesion molecule 1; LF = lactoferrin; LPO = lactoperoxidase; MFG-E8 = milk fat globule-EGF factor 8; MUC1 = mucin 1; pIgR = poly Ig receptor; SPP1 = secreted phosphoprotein 1; TPT1 = tumor protein, translationally controlled 1; XDH = xanthine dehydrogenase; CD36 = cluster of differentiation 36, CD63 = cluster of differentiation 63; ApoE = apolipoprotein E, CD74 = cluster of differentiation 74.

      Cell-Based Experiments

      Fluorescence microscopy revealed that bovine raw milk-derived exosomes were taken up by differentiated THP-1 cells (Figure 6A–C). The extent of exosome incorporation into THP-1 cells was analyzed using flow cytometry. Undifferentiated THP-1 cells did not take up exosomes. In contrast, differentiated THP-1 cells (macrophage-like) could uptake exosomes at both 4 and 37°C; however, exosome incorporation was significantly higher at 37 than at 4°C (Figure 6D).
      Figure thumbnail gr6
      Figure 6Uptake of bovine raw milk whey-derived exosomes by macrophages. (A–C) Fluorescence microscopy images; 7-AAD (BD Biosciences, San Diego, CA) was used to label the nuclei of THP-1 cells (red), and PKH67 (Sigma-Aldrich, St. Louis, MO) was used to label the exosomes (green). (A) PBS-PKH67 was added to differentiated THP-1 cells, followed by incubation at 37°C. (B) PKH67-labeled exosomes were added to differentiated THP-1 cells, followed by incubation at 4°C. (C) PKH67-labeled exosomes were added to differentiated THP-1 cells, followed by incubation at 37°C. (D) The uptake of fluorescence-labeled exosomes into THP-1 cells at various conditions was evaluated using a flow cytometer. PBS 37°C: PBS-PKH67 was added to differentiated THP-1 cells and cells were incubated at 37°C. UD Exo 37°C: PKH67-labeled exosomes were added to undifferentiated THP-1 cells, followed by incubation at 37°C. Exo 37°C: PKH67-labeled exosomes were added to differentiated THP-1 cells, followed by incubation at 37°C. Exo 4°C: PKH67-labeled exosomes were added to differentiated THP-1 cells, followed by incubation at 4°C. The values are the mean ± SEM (n = 3). Means without common letter (a–c) differ significantly (P < 0.05). Exo = exosome; MFI = mean fluorescence intensity; UD = undifferentiated.

      Discussion

      Extracellular circulating RNA can be present in forms other than exosomes and microvesicles (
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      Exosomes: Proteomic insights and diagnostic potential.
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      • Kosaka N.
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      • Ochiya T.
      Circulating microRNA in body fluid: A new potential biomarker for cancer diagnosis and prognosis.
      ;
      • Arroyo J.D.
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      • Tait J.F.
      • Tewari M.
      Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.
      ;
      • Lässer C.
      • Alikhani V.S.
      • Ekström K.
      • Eldh M.
      • Paredes P.T.
      • Bossios A.
      • Sjöstrand M.
      • Gabrielsson S.
      • Lötvall J.
      • Valadi H.
      Human saliva, plasma and breast milk exosomes contain RNA: Uptake by macrophages.
      ;
      • Vickers K.C.
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      • Remaley A.T.
      MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.
      ). In the current study, data revealed that RNA were also present in ultracentrifuged supernatants of whey fraction (Figure 1), although the RNA concentration was significantly lower in supernatants compared with exosomes (Figure 2A). In addition, the RNA content ratio of exosome to supernatant was ~7:3 (Figure 2B); therefore, most RNA was present in exosomes. Ninety-one miRNA were detected in the supernatant; however, 52 miRNA of these were present in only the supernatant. Therefore, the miRNA expression pattern differed between the supernatant and exosomes (Figure 3A, B).
      Most (2,538 mRNA transcripts) mRNA detected in the supernatant were also present in exosomes, and the detected number of mRNA only in the supernatant was very few (96 mRNA transcripts), although 2,634 mRNA transcripts were detected in supernatant (Figure 3C). In contrast, large numbers of mRNA (19,320 mRNA transcripts) were present in exosomes. Therefore, it might be thought that most mRNA detected in milk whey were concentrated in exosomes.
      The 50 miRNA expressed at the highest level in exosomes and supernatant according to microarrays are presented in Table 1. Fourteen miRNA (miR-2478, miR-2412, miR-2305, miR-2881, miR-2328*, miR-2888, miR-2304, miR-2391, miR-2892, miR-2887, miR-2316, miR-2374, miR-2291, miR-2284l) in exosomes and 28 miRNA (miR-2305, miR-2412, miR-2888, miR-2316, miR-2328*, miR-2478, miR-2893, miR-2881, miR-2374, miR-2391, miR-2348, miR2892, miR-2885, miR-2428, miR-2882, miR-2486, miR-2455, miR-2898, miR-2407, miR-2436–5p, miR-2389, miR-2309, miR-2340, miR-2902, miR-2454, miR-2373*, miR-2899, miR-2300a-5p) in supernatant are bovine-specific. Generally, species-specific miRNA are likely to be evolutionally younger and expressed at lower levels than highly conserved miRNA. The biological roles of these miRNA are unknown; however, some (miR-2478, miR-2412, miR-2305, miR-2881, miR-2328*, miR-2888, miR-2391, miR-2374, miR-2882, miR-2455, miR-2898) are predicted to regulate carbohydrate or lipid metabolism (
      • Romao J.M.
      • Jin W.
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      MicroRNAs in bovine adipogenesis: Genomic context, expression and function.
      ).
      • Zhou Q.
      • Li M.
      • Wang X.
      • Li Q.
      • Wang T.
      • Zhu Q.
      • Zhou X.
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      • Gao X.
      • Li X.
      Immune-related microRNAs are abundant in breast milk exosomes.
      ) and
      • Gu Y.
      • Li M.
      • Wang T.
      • Liang Y.
      • Zhong Z.
      • Wang X.
      • Zhou Q.
      • Chen L.
      • Lang Q.
      • He Z.
      • Chen X.
      • Gong J.
      • Gao X.
      • Li X.
      • Lv X.
      Lactation-related microRNA expression profiles of porcine breast milk exosomes.
      ) reported the top 10 miRNA expressed in human and in pig milk exosomes, respectively. Seven (miR-148a, miR-30b-5p, let-7f, miR-29a, let-7a, miR-141, and miR-200a) of the top 10 miRNA in human milk exosomes and 5 miRNA (miR-148a, miR-30a-5p, miR-30d, miR-200c, and let-7a) from pig milk exosomes were also present in the 50 miRNA expressed at the highest levels in bovine milk exosomes. Nevertheless, 14 of the top 50 miRNA were bovine-specific. These results suggest that milk exosome miRNA have some common roles among species, whereas other functions are species-specific. The biological functions of most miRNA remain unknown. In the current study, we selected 20 miRNA from the top 50 whose function was relatively well characterized (Table 1) and quantified them using qPCR in exosomes and supernatants that were isolated from the same volume of whey (Figure 4). Data revealed almost all of the miRNA (except for miR-133b and miR-1281) were expressed at significantly higher levels in exosomes than in supernatants. Both miR-133b and miR-1281 were among the 50 species expressed at the highest levels in supernatants, but not in exosomes. Therefore, these data do not contradict the microarray results because we used same amount of RNA from exosomes and supernatants in the microarrays. Two miRNA (miR-148a and let-7a) are common in human and pig milk exosomes (
      • Gu Y.
      • Li M.
      • Wang T.
      • Liang Y.
      • Zhong Z.
      • Wang X.
      • Zhou Q.
      • Chen L.
      • Lang Q.
      • He Z.
      • Chen X.
      • Gong J.
      • Gao X.
      • Li X.
      • Lv X.
      Lactation-related microRNA expression profiles of porcine breast milk exosomes.
      ;
      • Zhou Q.
      • Li M.
      • Wang X.
      • Li Q.
      • Wang T.
      • Zhu Q.
      • Zhou X.
      • Wang X.
      • Gao X.
      • Li X.
      Immune-related microRNAs are abundant in breast milk exosomes.
      ). These 2 miRNA were also expressed highly in bovine raw milk exosomes (Table 1, Figure 4). A previous study reported that miR-148a is a negative regulator of the innate immune response and antigen-presenting function of dendritic cells (
      • Liu X.
      • Zhan Z.
      • Xu L.
      • Ma F.
      • Li D.
      • Guo Z.
      • Li N.
      • Cao X.
      MicroRNA-148/152 impair innate response and antigen presentation of TLR-triggered dendritic cells by targeting CaMKIIα.
      ). It is also upregulated during intestinal cell differentiation (
      • Hino K.
      • Tsuchiya K.
      • Fukao T.
      • Kiga K.
      • Okamoto R.
      • Kanai T.
      • Watanabe M.
      Inducible expression of microRNA-194 is regulated by HNF-1alpha during intestinal cell differentiation.
      ), and targets DNA methyltransferase 3b (
      • Sato F.
      • Tsuchiya S.
      • Meltzer S.J.
      • Shimizu K.
      MicroRNAs and epigenetics.
      ). Let-7a inhibits Th17 differentiation by downregulating IL-6 secretion (
      • Zhang Y.
      • Wang X.
      • Zhong M.
      • Zhang M.
      • Suo Q.
      • Lv K.
      MicroRNA let-7a ameliorates con A-induced hepatitis by inhibiting IL-6-dependent Th17 cell differentiation.
      ). In addition, let-7a is upregulated during intestinal cell differentiation (
      • Papetti M.
      • Auqenlicht L.H.
      Mybl2, downregulated during colon epithelial cell maturation, is suppressed by miR-365.
      ). It is very interesting that the miRNA that are expressed highly in milk exosomes from several species exhibit common functions, such as regulating immune function and intestinal maturation.
      The 50 mRNA that were expressed at the highest level in exosomes and supernatants according to microarrays are shown in Tables 2 and 3, respectively. Most of mRNA expressed in exosomes are milk protein- and ribosomal protein-related mRNA. We observed similar results with rat milk whey-derived mRNA previously (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ). Consistent with this, previous microarray analysis of RNA extracted and purified from rat milk whey revealed that milk protein-derived and ribosomal mRNA transcripts were expressed highly (
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ). Few studies of milk mRNA have been conducted. However,
      • Maningat P.D.
      • Sen P.
      • Rijnkels M.
      • Sunehag A.L.
      • Hadsell D.L.
      • Bray M.
      • Haymond M.W.
      Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome.
      ) and
      • Lemay D.G.
      • Ballard O.A.
      • Hughes M.A.
      • Morrow A.L.
      • Horseman N.D.
      • Nommsen-Rivers L.A.
      RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.
      ) studied human milk fat-derived mRNA. Both of these studies showed that milk protein- and ribosomal protein-related mRNA transcripts were expressed highly in the milk fat layer (
      • Maningat P.D.
      • Sen P.
      • Rijnkels M.
      • Sunehag A.L.
      • Hadsell D.L.
      • Bray M.
      • Haymond M.W.
      Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome.
      ;
      • Lemay D.G.
      • Ballard O.A.
      • Hughes M.A.
      • Morrow A.L.
      • Horseman N.D.
      • Nommsen-Rivers L.A.
      RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.
      ). However,
      • Wickramasinghe S.
      • Rincon G.
      • Islas-Trejo A.
      • Medrano J.F.
      Transcriptional profiling of bovine milk using RNA sequencing.
      ) reported that no ribosomal protein-related mRNA were among the 7 mRNA species expressed at the highest level in bovine milk somatic cells (cells from 15, 90, and 250 d of lactation), although milk protein-related mRNA were included in the list. These results suggest that some roles of mRNA might be common to milk whey, milk whey-derived exosomes, the milk fat layer, and milk cells, whereas others might differ or the roles of milk mRNA might differ among species.
      In the current study, a large number of mRNA transcripts (19,320 mRNA transcripts) were detected in exosomes. However, selecting specific mRNA transcripts is challenging. Therefore, we selected milk protein-, milk-derived exosome-, and milk fat layer-related mRNA that were expressed at high levels in milk whey, the milk fat layer, or in milk somatic cells in previous studies (
      • Maningat P.D.
      • Sen P.
      • Rijnkels M.
      • Sunehag A.L.
      • Hadsell D.L.
      • Bray M.
      • Haymond M.W.
      Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome.
      ;
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ,
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.
      ;
      • Wickramasinghe S.
      • Rincon G.
      • Islas-Trejo A.
      • Medrano J.F.
      Transcriptional profiling of bovine milk using RNA sequencing.
      ;
      • Lemay D.G.
      • Ballard O.A.
      • Hughes M.A.
      • Morrow A.L.
      • Horseman N.D.
      • Nommsen-Rivers L.A.
      RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.
      ). These mRNA transcripts were then analyzed in exosomes and supernatants isolated from same volume of whey using qPCR (Figure 5). Data revealed that all mRNA were expressed at significantly higher levels in exosomes than in supernatants. Among the selected mRNA, some transcripts were expressed highly in bovine milk somatic cells and the human milk fat layer in previous reports (
      • Maningat P.D.
      • Sen P.
      • Rijnkels M.
      • Sunehag A.L.
      • Hadsell D.L.
      • Bray M.
      • Haymond M.W.
      Gene expression in the human mammary epithelium during lactation: The milk fat globule transcriptome.
      ;
      • Wickramasinghe S.
      • Rincon G.
      • Islas-Trejo A.
      • Medrano J.F.
      Transcriptional profiling of bovine milk using RNA sequencing.
      ;
      • Lemay D.G.
      • Ballard O.A.
      • Hughes M.A.
      • Morrow A.L.
      • Horseman N.D.
      • Nommsen-Rivers L.A.
      RNA sequencing of the human milk fat layer transcriptome reveals distinct gene expression profiles at three stages of lactation.
      ), but were not listed among the 50 highly expressed transcripts in exosomes in the current study. These results support the hypothesis that mRNA might be concentrated in exosomes. Unfortunately, in the current study, we did not investigate the presence of forms of RNA in supernatant in detail or the size range of exosomes. We prepared exosomes by ultracentrifugation (most commonly used method); however, the size of exosomes ranges widely. Thus, it is possible that extensively small or light exosomes remained in supernatant. It might be the reason that small numbers of mRNA detected in supernatant.
      Exosomes isolated from bovine milk could be taken up by human macrophages (Figure 6). Consistent with this,
      • Lässer C.
      • Alikhani V.S.
      • Ekström K.
      • Eldh M.
      • Paredes P.T.
      • Bossios A.
      • Sjöstrand M.
      • Gabrielsson S.
      • Lötvall J.
      • Valadi H.
      Human saliva, plasma and breast milk exosomes contain RNA: Uptake by macrophages.
      ) reported that exosomes from human breast milk could be taken up by human macrophages. Moreover,
      • Sun Q.
      • Chen X.
      • Yu J.
      • Zen K.
      • Zhang C.Y.
      • Li L.
      Immune modulatory function of abundant immune-related microRNAs in microvesicles from bovine colostrum.
      ) reported that bovine milk exosomes could be taken up by murine macrophages, and that the functions of macrophages that incorporated bovine milk exosomes were altered. This suggests that human cells can uptake exosomes from other species, which is consistent with the current results.
      It remains unknown whether milk-derived exosomes exert functions in vivo. However, previous studies, including those from our laboratory, showed that milk whey-derived RNA are resistant to acidic conditions and RNase (
      • Hata T.
      • Murakami K.
      • Nakatani H.
      • Yamamoto Y.
      • Matsuda T.
      • Aoki N.
      Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs.
      ;
      • Kosaka N.
      • Izumi H.
      • Sekine K.
      • Ochiya T.
      MicroRNA as a new immune-regulatory agent in breast milk.
      ;
      • Izumi H.
      • Kosaka N.
      • Shimizu T.
      • Sekine K.
      • Ochiya T.
      • Takase M.
      Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.
      ). Therefore, it is possible that bovine milk-derived exosomes could exert functions in human body. In recent years, studies demonstrated that food-derived miRNA could be incorporated and exert function in vivo. For example,
      • Zhang L.
      • Hou D.
      • Chen X.
      • Li D.
      • Zhu L.
      • Zhang Y.
      • Li J.
      • Bian Z.
      • Liang X.
      • Cai X.
      • Yin Y.
      • Wang C.
      • Zhang T.
      • Zhu D.
      • Zhang D.
      • Xu J.
      • Chen Q.
      • Ba Y.
      • Liu J.
      • Wang Q.
      • Chen J.
      • Wang J.
      • Wang M.
      • Zhang Q.
      • Zhang J.
      • Zen K.
      • Zhang C.Y.
      Exogenous plant MIR168a specifically targets mammalian LDLAP1: Evidence of cross-kingdom regulation by microRNA.
      ) reported that osa-MIR-168a (from rice) could be detected in human and mouse sera, and osa-MIR-168a decreased low-density lipoprotein receptor adapter protein 1 mRNA (the predicted target mRNA of osa-MIR-168a) in the mouse liver. In addition,
      • Ju S.
      • Mu J.
      • Dokland T.
      • Zhuang X.
      • Wang Q.
      • Jiang H.
      • Xiang X.
      • Deng Z.B.
      • Wang B.
      • Zhang L.
      • Roth M.
      • Welti R.
      • Mobley J.
      • Jun Y.
      • Miller D.
      • Zhang H.G.
      Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis.
      ) reported that grape exosome-like nanoparticles mediate intestinal tissue remodeling to induce intestinal stem cells and protect intestinal tissue from dextran sulfate sodium-induced colitis. Moreover,
      • Lukasik A.
      • Zielenkiewicz P.
      In silico identification of plant miRNAs in mammalian breast milk exosomes—A small step forward?.
      ) reported that some miRNAs from Arabidopsis thaliana, Picea abies, Populus trichocarpa, Brachypodium distachyon, and Zea mays were present in human and porcine breast milk exosomes by analyzing publically available data obtained from high-throughput sequencing using bioinformatics. In contrast, studies regarding the bioavailability of plant-borne miRNA in humans are controversial (
      • Dickinson B.
      • Zhang Y.
      • Petrick J.S.
      • Heck G.
      • Ivashuta S.
      • Marshall W.S.
      Lack of detectable oral bioavailability of plant microRNAs after feeding in mice.
      ;
      • Snow J.W.
      • Hale A.E.
      • Isaacs S.K.
      • Baggish A.L.
      • Chan S.Y.
      Ineffective delivery of diet-derived microRNAs to recipient animal organisms.
      ). Recently,
      • Baier S.R.
      • Nguyen C.
      • Xie F.
      • Wood J.R.
      • Zempleni J.
      MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers.
      ) reported that meaningful amounts of miRNA were detected in plasma isolated from healthy human subjects after the ingestion of nutritionally relevant doses of cow milk. They also revealed that Brassica-specific miRNA could not be detected in a broccoli sprout-feeding study. Therefore, it is important to consider why only milk-borne miRNA were absorbed. In the current study, we showed that human macrophages could take up bovine raw milk-derived exosomes. However, it is unknown whether bovine raw milk-derived exosomes are taken up by only macrophages.
      In conclusion, the current study demonstrated that most mRNA in bovine raw milk whey were present in exosomes, whereas miRNA were present both in exosomes and in other forms. Most miRNA and mRNA that are present in bovine raw milk whey were expressed at significantly higher levels in exosomes than in other forms. In addition, human macrophages could uptake bovine raw milk whey-derived exosomes. Moreover, a recent study (
      • Baier S.R.
      • Nguyen C.
      • Xie F.
      • Wood J.R.
      • Zempleni J.
      MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers.
      ) suggested that functional RNA in milk exert functions in the human body. However, it is unclear whether bovine raw milk whey-derived exosomes affect the function of macrophages. Moreover, the different size exosomes contain different types of RNA and other exosome-like vesicles (e.g., ectosomes) might be contained in milk-derived exosomes. As such, several aspects of milk-derived RNA remain unclear. Bovine raw milk is used widely in many dairy products and is an important food source. Therefore, additional studies regarding the physiological and biological roles of these functional RNA are needed. The current data describing RNA in bovine raw milk whey-derived exosomes might facilitate future studies.

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