Commercial Dairy Cow Milk microRNAs Resist Digestion …jn.nutrition.org/content/146/11/2206.full.pdf · Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal

The Journal of Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Commercial Dairy Cow Milk microRNAs ResistDigestion under Simulated GastrointestinalTract Conditions1–3

Abderrahim Benmoussa,4 Chan Ho C Lee,4 Benoit Laffont,4 Patricia Savard,5 Jonathan Laugier,4

Eric Boilard,4 Caroline Gilbert,4 Ismail Fliss,5 and Patrick Provost4*

4University of Quebec Hospital Center Research Center/University of Laval Hospital Center, Department of Microbiology-Infectious

Disease and Immunity and Faculty of Medicine, and 5STELA Dairy Research Center, Institute of Nutrition and Functional Foods,

Universite Laval, Quebec, Canada

Abstract

Background: MicroRNAs are small, gene-regulatory noncoding RNA species present in large amounts in milk, where they

seem to be protected against degradative conditions, presumably because of their association with exosomes.

Objective: We monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined

their associations with extracellular vesicles (EVs).

Methods: We used a computer-controlled, in vitro, gastrointestinal model TNO intestinal model-1 (TIM-1) and analyzed, by

quantitative polymerase chain reaction, the concentration of 2 microRNAs within gastrointestinal tract compartments at

different points in time. EVs within TIM-1 digested and nondigested samples were studied by immunoblotting, dynamic light

scattering, quantitative polymerase chain reaction, and density measurements.

Results: A large quantity of dairy milk Bos taurusmicroRNA-223 (bta-miR-223) and bta-miR-125b (;109–1010 copies/300 mL

milk) withstood digestion under simulated gastrointestinal tract conditions, with the stomach causing the most important

decrease in microRNA amounts. A large quantity of these 2 microRNAs (;108–109 copies/300 mL milk) was detected in the

upper small intestine compartments, which supports their potential bioaccessibility. A protocol optimized for the enrichment

of dairy milk exosomes yielded a 100,0003 g pellet fraction that was positive for the exosomal markers tumor susceptibility

gene-101 (TSG101), apoptosis-linked gene 2–interacting protein X (ALIX), and heat shock protein 70 (HSP70) and containing

bta-miR-223 and bta-miR-125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of

ALIX2, HSP702/low, and TSG1012/low EVs larger than exosomes and 2–6 times more enriched in bta-miR-223 and bta-miR-

125b (P < 0.05).

Conclusions: Our findings indicate that commercial dairy cow milk contains numerous microRNAs that can resist

digestion and are associated mostly with ALIX2, HSP702/low, and TSG1012/low EVs. Our results support the existence of

interspecies transfer of microRNAs mediated by milk consumption and challenge our current view of exosomes as the

sole carriers of milk-derived microRNAs. J Nutr 2016;146:2206–15.

Keywords: digestion, exosome, extracellular vesicles, microRNA, milk, TIM-1

Introduction

MicroRNAs are small, 19- to 24-nt noncoding RNA speciesgenerated by the ribonuclease III Dicer (1) and guide Argonauteeffector complexes to regulate;60% of the genes in humans (2).

Highly conserved within the clade of mammals (3, 4) andassociated with numerous physiological and/or pathological

processes, microRNAs are expressed in plants and animals

that are part of the human diet. The provocative idea that

microRNAs may cross the barrier between species through the

diet was initially explored by Zhang et al. (5), who reported on

the uptake of exogenous plant (rice) microRNAs through food

intake. Since then, an increasing number of studies have

extended these observations on dietary microRNAs, also called

xenomiRs by Witwer (6), to various food sources, including

other plant microRNAs (7), chicken eggs (8) and cow milk (9),

although not without some controversy (10–12). Although the

1 This work was supported by Canadian Institutes of Health Research grants

319618 and 327522, through the Institute of Genetics (PP).2 Author disclosures: A Benmoussa, CHC Lee, B Laffont, P Savard, J Laugier,

E Boilard, C Gilbert, I Fliss, and P Provost, no conflicts of interest.3 Supplemental Table 1 and Supplemental Figures 1–5 are available from the

‘‘Online Supporting Material’’ link in the online posting of the article and from the

same link in the online table of contents at http://jn.nutrition.org.

*To whom correspondence should be addressed. E-mail: patrick.provost@

crchudequebec.ulaval.ca.

ã 2016 American Society for Nutrition.

2206 Manuscript received June 24, 2016. Initial review July 27, 2016. Revision accepted September 2, 2016.

First published online October 5, 2016; doi:10.3945/jn.116.237651.Downloaded from https://academic.oup.com/jn/article-abstract/146/11/2206/4584726by gueston 20 June 2018

interspecies transfer of RNA (e.g., double-stranded RNA frombacteria to nematodes) had been reported in 1998 (13), a 2014study lent tangible support to this concept by demonstrating thetransfer of microRNAs from a gastrointestinal parasitic nema-tode (Heligmosomoides polygyrus) to its mammalian (mouse)host (14).

We and others have shown that microRNAs may betransferred between mammalian cells through the release (anduptake) of the extracellular vesicles (EVs)6 known as micropar-ticles (<1 mm). EVs may form a barrier against degradativecomponents/conditions present in the extracellular milieu orvarious biological fluids, such as blood and maternal milk(15), and protect microRNAs from degradation, which wouldfacilitate their transfer between cells (16), organs (17), andindividuals (18).

Humans of all ages consume milk from various sources (e.g.,human breast, dairy cows). That milk is a complete food fornewborns and consumed worldwide, microRNAs are highlyconserved in mammals and may mediate the beneficial effects ofdairy milk consumption in rheumatoid arthritis (19) or inimmune functions (20), microRNAs contained in milk can beabsorbed by human cells and mouse model organs (9, 21), andbovine milk microRNAs exhibit relatively high stability underdegradative conditions (22) impelled us to characterize thekinetics of commercial dairy milk microRNAs during digestionunder simulated gastrointestinal tract conditions.

We had shown that platelet-derived microRNAs could betransferred to endothelial cells (23), macrophages (24), or neutro-phils (25) through EVs, which may protect microRNAs fromdegradation in the extracellular milieu (e.g., blood). The leadingtheory is that microRNAs present in milk are resilient to adverseconditions, likely because they are contained within and protectedfrom degradation by lipidic EVs—more specifically, exosomes (22,26, 27). Exosomes are small EVs (diameter: 30–100 nm ) producedby the invagination of multivesicular bodies and released outsidethe cells by fusion of the multivesicular bodies with the cellularmembrane (28). Well-established vehicles mediating the intercellu-lar transfer of microRNAs (29, 30), exosomes were the initial focusof our investigations.

Reasoning that this type of EVs may help protect food-derived, or dietary, microRNAs from the degradative environ-ment of the gastrointestinal tract, which is a prerequisite for theabsorption of microRNAs through the diet, we observed thatdairy milk microRNAs are relatively well protected from thebiophysical, biochemical, and enzymatic conditions of thegastrointestinal tract and associated with different types ofEVs, including exosomes, which is likely to contribute to theirbioavailability.

Methods

Dairy milk samples

For all of the experiments, we used commercially available filtered dairyskim milk (Lactantia PurFiltre, Parmalat Canada) bought at a grocery

store in Quebec City, Canada, on the day of the experiments.

TNO GI in vitro model

Description and settings. The dairy milk samples were digested in thecomputer-controlled Toegepast Natuurwetenschappelijk Onderzoek

(TNO, Applied Scientific Research) in vitro gastrointestinal model

(TIM-1) (Food & Nutrition), which is composed of 4 connected

compartments that simulate the stomach, duodenum, jejunum, andileum (Supplemental Figure 1), as described elsewhere (31, 32). In these

experiments, all of the settings and variables of the TIM-1 system were

continuously computer monitored and controlled to ensure highly

reproducible and reliable duplicate experiments (n = 2), as validatedand published elsewhere (33–37). The TIM-1 parameters were set to

mimic healthy adult GI conditions after milk consumption (Supplemen-

tal Table 1).

Media for TIM-1 experiments. All of the working solutions [pepsin

from porcine gastric mucosa (EC 3.4.23.1; 3200–4500 U/mg protein,

P6887, Sigma-Aldrich), trypsin from porcine pancreas (EC 3.4.21.4;$7500Na-benzoyl-L-arginine ethyl ester U/mg protein, T9201, Sigma-

Aldrich), pancreatin from porcine pancreas (4 3 USP, P1750, Sigma-

Aldrich), and lipase from Rhizopus oryzae DF 15 (150 U/mg, Amano

Enzyme USA Co.)] were freshly prepared with deionized sterile water andused. The electrolyte solutions included gastric electrolyte solution (NaCl

6.2 g/L, KCl 2.2 g/L, CaCl2 0.3 g/L, NaHCO3 1.5 g/L) and small intestinal

electrolyte solution (NaCl 5.0 g/L, KCl 0.6 g/L, and CaCl2 0.3 g/L).

Sampling in TIM-1 experiments. Samples (9 mL) from each of the 4

TIM-1 compartments were collected at baseline (0), 30, 60, and 120 min

after initiating digestion. At each point in time, the effluent fraction wascollected and weighed. Aliquots of these samples (4 3 250 mL) were

immediately flash frozen in liquid nitrogen and stored at 280�C for

subsequent qPCR analysis. The rest of the aliquots were heated at 55�C for

10 min to stop the enzymatic reactions and immediately stored at280�C.

Dairy milk exosome isolation and enrichment

To study the association of digested and nondigested commercial cow

milk microRNAs with exosomes, we developed a protocol for dairy milkexosome isolation and enrichment that combined differential ultracen-

trifugation steps (38) with density-based separation (39). For both

samples, exosomes were isolated by following a previously described

protocol (38), with slight modifications. We replaced common sucrosewith OptiPrep (iodixanol; Sigma-Aldrich) and diluted milk samples with

sodium citrate buffer to avoid casein gel formation upon high-speed

ultracentrifugation (40). Dairy milk or TIM-1 samples were mixed

with 1 vol of 2% sodium citrate buffer in Milli-Q water (MilliporeCorporation) that had been filtered with 0.2-mm membrane microfilters

(Corning). The samples were then subjected to successive differential

ultracentrifugation steps to remove milk fat globules, cell debris, orcasein aggregates at 12,0003 g, 35,0003 g, and 70,0003 g for 1 h each

at 4�C in the Sorvall WX ultracentrifuge TL-100, equipped with a

T-1250 rotor (both Thermo Fisher). After each step, the pellets were

resuspended in 1 mL PBS containing 100 nM EDTA, pH 7.4, and storedat 280�C for subsequent analysis. The supernatant fluid (SN) was then

filtered through a 0.45-mm membrane microfilter (Corning) and

subjected to the next centrifugation step. The exosome pellet resulting

from the final ultracentrifugation step (100,0003 g, 1 h, 4�C) was gentlyresuspended in 1 mL PBS containing 100 nM EDTA, pH 7.4, and

incubated overnight at 4�C on a rocking table for further dispersion of

exosome aggregates. The suspended pellet was diluted in 3 vol of PBS

containing 0.1 mM EDTA and layered on top of a 10–40% discontin-uous iodixanol density gradient (IDG) (OptiPrep, in PBS). The loaded

IDG underwent centrifugation at 100,000 3 g for 18 h at 4�C. Afterremoving the loaded volume (4 mL SN), twelve 1-mL fractions werecollected from the top to the bottom of the IDG. The refractive index of

each fraction was measured using a refractometer (ABBE 3-L, Bausch &

Lomb), allowing density calculations of the IDG fractions by use of the

formula Density = 3.3411 3 refractive index 2 3.4584.

Particle size measurements

The hydrodynamic size of the particles was measured through use of the

Zetasizer Nano-ZS (Malvern) light-scattering measurement system. Atotal of 100 mL of each sample was loaded in a UV cuvette micro

(BRAND), and the particle size was measured at 4�C. Each data point

represents the mean of 3 measurements of 12–15 assays each.

6 Abbreviations used: ALIX, apoptosis-linked gene 2–interacting protein X;

bta-miR, Bos taurus microRNA; EV, extracellular vesicle; HSP70, heat shock

protein 70; IDG, iodixanol density gradient; SN, supernant fluid; TIM-1, TNO

intestinal model 1; TSG101, tumor susceptibility gene-101.

Dairy milk miRNAs resist digestion in the GI tract 2207

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MicroRNA detection by qPCR

In the milk digestion experiments, we monitored the relative stability of

dairymilkmicroRNAsBos taurusmicroRNA-223 (bta-miR-223) and bta-miR-125b during digestion under gastrointestinal tract conditions by

following the experimental scheme depicted in Supplemental Figure 2. We

duplicated these experiments in nondigested milk; for all of the experi-

ments, total RNAwas extracted using TRIzol-LS reagent (Invitrogen) andsubjected to DNase 1 treatment following the manufacturer�s recommen-

dations. For all of the RNA extractions, an exogenous synthetic control

microRNA (Caenorhabditis elegans let-7-as mutated, 2 fmol; Integrated

DNA Technologies, Inc.) was spiked in at the TRIzol-LS homogenizationstep, as described previously (41). Total RNAwas then reverse transcribed

with the HiFlex miScript RTII kit (Qiagen), and mature bta-miR-223 and

bta-miR-125b were detected by SYBR Green qPCR through use of themiScript Primer Assay kit (Qiagen) and oligonucleotides specific to bta-

miR-223 (MS00051065; Qiagen) and bta-miR-125b (MS00050197;

Qiagen). Standard curves of synthetic bta-miR-223 and bta-miR-125b

were constructed and used for copy number determination, as describedpreviously (42). We used the synthetic let-7-as mutated microRNA as a

qPCR internal control to calculate the efficiency of microRNA extraction,

as described elsewhere (43).

Western blot analysis

Proteins in all of the liquid samples were precipitated with 150 mL

trichloroacetic acid (100%) per milliliter sample. After 30 min on ice, theprecipitated proteinswere pelleted by centrifugation at 16,0003 g for 15min

at 4�C. The SN was discarded, and the protein pellets were rinsed with

800 mL acetone, quickly vortexed, and spun again for 5 min. After 3 washes

with 800 mL acetone, the pellets were dried and heated at 100�C for 2 minbefore being resuspended in 50 mLT8 lysis buffer [7 M urea, 2 M thiourea,

3%3-{(3-cholamidopropyl) dimethylammonio}-1-propanesulfonate), 20mM

dithiothreitol, 5 mM tris-carboxyethyl-phosphine]. The proteins were

subjected to 10% (wt:vol) SDS-PAGE, followed by immunoblottingthrough use of mouse monoclonal anti-tumor susceptibility gene-101

(TSG101; clone 4A10, ab83, Abcam), anti-heat shock protein 70

(HSP70; cat. no. 554243, BD Pharmingen), or anti-apoptosis-linked

gene 2–interacting protein X (ALIX; clone 3A9, sc-53538; Santa CruzBiotechnology, Inc.) antibodies for TSG101, HSP70, and ALIX are

highly enriched in exosomes (44). The bands were revealed by enhanced

chemiluminescence detection and densitometric analyses, as previouslyreported (23).

Statistical analyses

All of the statistical analyses were performed using Prism 6 (GraphPadSoftware). TIM-1 experiments were conducted as described elsewhere

(35, 45–47) in duplicate (n = 2), and the results were shown as individual

data points with the mean. Experiments on nondigested milk were

performed to validate TIM-1 results in biological triplicates (n = 3); analyzedwith 1-way 1-tailed Student�s t tests, comparing each pellet with the

100,0003 g exosome-enriched pellet; and P# 0.05 considered significant.

Results

Dairy milk microRNAs withstand digestion under

simulated GI tract conditions. We aimed to determine whetherdairy milk microRNAs are able to withstand in vitro digestionwithin the TIM-1 system. At baseline, the commercial dairy milkpreparation (300 mL), poured into the stomach compartment ofthe TIM-1 digestion system, contained >8.3 3 109 copies of bta-miR-223 (Figure 1A, Input) and 3.8 3 1010 copies of bta-miR-125b (Figure 1B, Input), indicating that commercial dairy milk ishighly enriched in these 2 microRNAs.

After 30 min of digestion, several microRNAs could bedetected within the digested sample (Supplemental Figure 3). Wechose to focus our investigation on bta-miR-223 and bta-miR-125b. At the 30-min-of-digestion time point, 5.33 109 copies ofbta-miR-223 (64% of input) seemed to have been degraded,compared with 8.0 3 109 copies of bta-miR-125b (23% of

FIGURE 1 Absolute quantification and distribution of dairy milk

microRNAs bta-miR-223 and bta-miR-125b in the gastrointestinal tract

during digestion under simulated conditions. RT-qPCR detection and

absolute quantification of bta-miR-223 (A) and bta-miR-125b (B) within

the TIM-1 in vitro digestion system are shown. Results are shown as

individual data points with the mean (n = 2 independent experiments).

Absolute quantification of the 2 microRNAs are expressed in copy

numbers [bar graphs (top)]. Distribution of each microRNA (percent-

age of remaining microRNAs) is shown, at each point in time, within

the TIM-1 system [donut charts (bottom)]. bta-miR, Bos taurus

microRNA.

2208 Benmoussa et al.


input), pointing to differential digestion kinetics for these 2microRNAs. Despite this loss, >3.03 109 copies of bta-miR-223(Figure 1A, 30 min) and 3.0 3 1010 copies of bta-miR-125b(Figure 1B, 30 min) were still available for quantification at thispoint in time and, presumably, bioaccessible. A major propor-tion of these microRNAs (67.1% of bta-miR-223 and 73.0% ofbta-miR-125b) that had resisted the highly degradative condi-tions of the stomach reached the small intestine compartments(Figure 1A, B, donut charts, 30 min). Moreover, study of themicroRNA distribution within the TIM-1 system implies that alarge proportion of the preserved bta-miR-223 (22.8%) and bta-miR-125b (15.2%) had already flowed through the entire uppergastrointestinal tract and into the effluent fraction (E) in <30min(Figure 1A, B, donut charts, 30 min), suggesting that thesemicroRNAs withstood the entire digestion process.

Similarly, after 60 min of digestion, the total microRNA copynumber within the whole system decreased to 1.3 3 109 copiesfor bta-miR-223 (Figure 1A, 60 min) and to 1.0 3 1010 for bta-miR-125b (Figure 1B, 60 min). At this point in time, most of thedetected microRNAs were contained within the small intestinecompartments or had flowed out of the system Figure 1A, B,donut charts, 60 min).

Finally, after 120 min of digestion, the total microRNA copynumber remained unchanged compared with the previous (60min)time point (Figure 1A, B, 120 min), and most bta-miR-223 andbta-miR-125b microRNAs were detected within the jejunumand ileum compartments (Figure 1A compared with 1B, donutcharts, 60 min).

Taken together, these results indicate that a high quantity(;109–1010 copies/300 mLmilk) of dairy milk bta-miR-223 andbta-miR-125b withstood digestion under simulated gastrointes-tinal tract conditions. These data also imply that although someof these 2 microRNAs seem to have been degraded in thestomach compartment during the first hour of digestion, a largenumber of microRNAs can reach the small intestine, where mostof the nutrients are absorbed.

Dairy milk microRNAs bta-miR-223 and bta-miR-125b that

resist digestion are associated with exosomes. MilkmicroRNAs often are associated with exosomes; therefore, wedecided to monitor this association within digested milksamples. We chose to focus our analyses on the effluent samplecollected after 30 min of digestion because it had passed throughthe entire gastrointestinal tract and contained high amounts ofmicroRNAs bta-miR-223 and bta-miR-125b that withstooddigestion (Figure 1, donut chart, 30 min). We found that the100,000 3 g pellet contained large amounts of bta-miR-223(2.4 3 107 copies) and bta-miR-125b (4.5 3 108 copies; ABenmoussa and P Provost, unpublished data, 2015) that arelikely to be associated with exosomes, because a centrifugationspeed of 100,0003 g is used commonly to pellet this type of EV(48). To confirm this hypothesis we subjected this 100,000 3 gpellet to IDG fractionation, which attempted to isolate digesteddairy milk exosomes. The buoyant density of exosomes iniodixanol ranges between 1.1 and 1.2 g/mL (49), suggesting thatIDG fractions F5–F8 likely contain most of the digested dairy

FIGURE 2 Dairy milk microRNAs bta-

miR-223 and bta-miR-125b that resist

digestion are associated with exosomes.

The effluent sample obtained after 30 min

of digestion was subjected to the exo-

some purification and enrichment proto-

col (A and B). The buoyant density of

each IDG fraction was calculated (A),

and the hydrodynamic size (diameter in

nanometers) of the particles present in

each IDG fraction was measured (B).

Proteins from fractions F3–F8 were ana-

lyzed by Western blot for the presence of

exosome-enriched proteins TSG101, ALIX,

and HSP70 (C). qPCR detection and abso-

lute quantification of bta-miR-223 (D) and

bta-miR-125b (E) are shown as in Figure 1.

Results are shown as individual data

points with the mean (n = 2 independent

experiments). ALIX, apoptosis-linked gene

2–interacting protein X; bta-miR, Bos taurus

microRNA; F, fraction; HSP70, heat shock

protein 70; IDG, iodixanol density gradient;

TIM-1, TNO intestinal model 1; TSG101,

tumor susceptibility gene-101.



milk exosomes (Figure 2A). Fractions F5 and F6 also contain thesmallest particles (diameter: 130 nm), close to the size expectedfor exosomes (;100 nm) (50) (Figure 2B). Western blot analysesconfirmed that fractions F5 and F6 are enriched in TSG101 andALIX protein and that fraction F6 is positive for the exosome-enriched protein HSP70, thereby supporting the presence ofexosomes in the 100,000 3 g pellet (Figure 2C).

Absolute qPCR quantification of bta-miR-223 and bta-miR-125b revealed that fraction F5 was the most enriched inmicroRNAs, with 6.1 3 106 copies of bta-miR-223 (Figure2D) and 9.1 3 107 copies of bta-miR-125b (Figure 2E).Moreover, we observed that large amounts of microRNAs bta-miR-223 (1.1 3 107 copies) and bta-miR-125b (2.0 3 108

copies) were associated with the exosomal fractions F5–F8combined, further supporting the idea that dairy milk microRNAsare protected from digestion by exosomes.

Majority of digested dairy milk microRNAs bta-miR-223

and bta-miR-125b is associated with particles sedimenting

at a centrifugation speed lower than that for exosomes.

Wealso observed that the combined nonexosomal fractions (F1–F3 +F8–F12) were associated with more bta-miR-223 (1.4 3 107

copies) and bta-miR-125b (2.0 3 108 copies) than the sum ofexosomal fractions F4–F7. Moreover, the total number of bta-miR-223 and bta-miR-125b copies detected in the IDG fractionscorresponded to only 3.5% and 9.1%, respectively, of the totalbta-miR-223 (6.9 3 108 copies) and bta-miR-125b (4.3 3 109

copies) copy numbers detected in the entire effluent sample at 30minpostdigestion. Together, these results suggest that most of thedairy milk bta-miR-223 and bta-miR-125b that resisted diges-tion are neither contained in the IDG analysis of the 100,000 gpellet nor associated with canonical exosomes. This promptedus to hypothesize that most of these 2 microRNAs are associatedwith particles sedimenting at a centrifugation speed lower than100,000 3 g.

Quantification of bta-miR-223 and bta-miR-125b copynumbers revealed that most of the microRNAs present in theeffluent sample [73.6% of all bta-miR-223 (Figure 3A, C) and75.6% of all bta-miR-125b (Figure 3B, C) sedimented at 12,000or 35,000 3 g. The remaining microRNAs (22.2% of all bta-miR-223 and 23.7% of all bta-miR-125b) pelleted at highercentrifugation speeds of 70,000 and 100,000 3 g. Most of theTSG101-positive EVs (exosomes) contained in the digestedeffluent sample sedimented at 100,000 3 g (Figure 3D, lane 4),thereby excluding the possibility that exosomes or exosomeaggregates were pelleted during the 12,000 and 35,000 3 gultracentrifugation steps. A positive TSG101 signal in the100,0003 g SN fraction (Figure 3D, lane 5) suggests that 1 h maynot be long enough to pellet all of the exosomes at 100,000 3 g.The absence of a TSG101 signal in the 12,000, 35,000, or70,000 3 g pellets (Figure 3D, lanes 1–3) is consistent with theabsence of exosomes, which are expected to sediment atcentrifugation speeds $100,000 3 g (51). Indeed, particle sizemeasurements showed that most of the particles that sedimentedat a speed #100,000 3 g are larger than exosomes (diameter:200–300 nm) (Figure 3E). Collectively, these results suggest thatmost (;90%) dairy milk microRNAs that resisted digestionunder simulated gastrointestinal tract conditions are not likelyassociated with exosomes.

Large amounts of nondigested commercial dairy milk

microRNAs are associated with exosomes. The sameexperimental approach applied to nondigested commercial dairymilk yielded similar results; buoyant density analysis revealed

that nondigested dairy milk IDG fractions F5–F8 are likely tocontain most of the dairy milk exosomes (Figure 4A). Study ofthe particle hydrodynamic size showed that the particles present

FIGURE 3 The majority of digested dairy milk microRNAs bta-miR-223

and bta-miR-125b is associated with 200-nm particles sedimenting at a

centrifugation speed lower than that for exosomes. The effluent sample

obtained after 30 min of digestion was subjected to the successive

ultracentrifugation protocol. qPCR detection and absolute quantification of

bta-miR-223 (A) and bta-miR-125b (B) are shown as in Figure 1. Results

are shown as individual data points with the mean (C). Proteins from each

pellet and from the 100,000 3 g SN were analyzed by immunoblotting to

detect the exosome-enriched protein TSG101. The hydrodynamic size

(diameter in nanometers) of the particles present in each sample was

measured (D). Results are shown as individual data points with the mean

(n = 2 independent experiments). bta-miR, Bos taurus microRNA; SN,

supernatant fluid; TSG101, tumor susceptibility gene-101.



in these fractions range between 118 and 160 nm in diameter,similar to the results obtained with the digested TIM-1 effluentsample (Figure 4B). Fraction F6 (density: 1.12 g/mL) seems tocontain the smallest particles (diameter: 118 nm) and is thuslikely to be enriched in exosomes (Figure 4B). Western blotmonitoring 3 of the most commonly used exosome-enrichedmarkers (TSG101, HSP70, and ALIX proteins) revealed that

most of the ALIX protein was associated with fraction F5,whereas most of the TSG101 protein was detected in fraction F7[Figure 4C, lanes 2 and 4 (52)]. These results suggest thatcommercial dairy milk likely contains $2 subpopulations ofexosomes, with different densities and relative enrichment inTSG101 and ALIX proteins, as reported previously (53).

Between fractions F5 and F7 is fraction F6, which may beenriched equally in TSG101 and ALIX (Figure 4C, lane 3) andis positive for HSP70. Analysis of fraction F6 by transmissionelectron microscopy revealed that most of the particlescontained within this fraction exhibit the shape and featuresof exosomes (Figure 4D) (54). Moreover, preliminary qPCRanalysis of bta-miR-223 and bta-miR-125b concentrationssuggests that this fraction also is the most enriched in these2 microRNAs (Supplemental Figure 4). Most nondigesteddairy milk microRNAs within the IDG, however, seem to beassociated with exosomal fractions F5–F8 rather than withthe lower-density fractions F1–F4, as previously observedfor the digested sample, suggesting that digestion causes adecrease in exosome density or a differential effect on dif-ferent types of exosomes that are present in commercial dairymilk.

Most commercial dairy milk microRNAs bta-miR-223 and

bta-miR-125b are associated with 200-nm particles

sedimenting at a centrifugation speed lower than that

for exosomes. Absolute microRNA quantification displayed acopy number profile in undigested milk that was similar to theone obtained for the digested effluent fraction, with most bta-miR-223 (79.7%) and bta-miR-125b (85.6%) detected in the12,000 and 35,000 3 g pellets (Figure 5A–C). Again, a smallproportion of these 2 microRNAs could be pelleted at higherultracentrifugation speeds (70,000 and 100,000 3 g). Interest-ingly, the bta-miR-223 and bta-miR-125b profiles were slightlydifferent, because only 5.2% of bta-miR-223 is contained withinthe 70,000 and 100,000 3 g pellets, compared with 20.2% forbta-miR-125b. Moreover, more bta-miR-223 was detected inthe SN than in the 100,000 3 g pellet, whereas this profile isinverted for bta-miR-125b (Figure 5A, B, last 2 bars). We alsofound that these results could be extended to 2 other microRNAs(Supplemental Figure 5). These findings support the idea thatthese 2 microRNAs may be differentially enriched in differenttypes of EVs.

As observed with the digested dairy milk, the profiles ofTSG101, ALIX, and HSP70 proteins in the undigested dairymilk imply that the 12,000 and 35,000 3 g pellets contain noexosomes to extremely few exosomes (Figure 5D, lanes 1–6).Most of the exosomes was confined within the 100,000 3 gpellet (Figure 5D, lanes 10–12). Some of the TSG101 and HSP70proteins could be detected within the 70,000 3 g pellet (Figure5D, lanes 7–9), supporting the existence of denser exosomes innondigested dairy milk.

Our results indicate that dairy milk microRNAs may not beassociated mainly with exosomes, suggesting that their resilienceto digestion may be linked to other kinds of EVs, proteincomplexes, or lipids. Hydrodynamic size measurements by light-scattering technology performed on digested dairy milk ultra-centrifugation pellets revealed that the 12,000 and 35,000 3 gpellets contained particles ;200 nm in diameter (Figure 5E).These pellets also are the most enriched in microRNAs bta-miR-223 and bta-miR-125b, suggesting that dairy milk microRNAare associated mainly with 200-nm, ALIX2, HSP702/low, andTSG1012/low particles that pellet at ultracentrifugation speeds<100,000 3 g.

FIGURE 4 Large amounts of nondigested commercial dairy milk

microRNAs are associated with exosomes. Fresh commercial dairy

milk (60 mL, n = 3) was subjected to the same exosome enrichment

protocol described previously for the digested sample. The buoyant

density of each IDG fraction was calculated (A), and the hydrodynamic

size (diameter in nanometers) of the particles present in each fraction

was measured (B); results are expressed as means 6 SEMs (n = 3).

Proteins from fractions F5–F8 containing exosomes and those from F4

and F9 used as controls were analyzed by immunoblotting to detect

the exosome-enriched proteins TSG101 and ALIX (C) (52). Fraction

F6–associated particles were studied by transmission electron mi-

croscopy (D). ALIX, apoptosis-linked gene 2–interacting protein X; F,

fraction; HSP70, heat shock protein 70; IDG, iodixanol density

gradient; TSG101, tumor susceptibility gene-101.



Discussion

We observed that microRNAs contained in a commercial dairymilk preparation resist the digestion process and that most ofthem are associated with particles that are negative or low forthe exosome-enriched proteins ALIX, HSP70, and TSG101(ALIX2, HSP702/low, and TSG1012/low). These conclusionswere reached through use of the in vitro TIM-1 digestion model,which most closely simulates the biophysical, biochemical, andenzymatic conditions that prevail during the digestion of dairymilk in the human gastrointestinal tract (55). This model alsohas the advantage of allowing tight control over digestionparameters and providing sampling sites in the various gastro-intestinal compartments at various points in time. At the sametime it prevents contamination with endogenous, conserved,gastrointestinal cell–associated microRNAs (10), because thesystem is acellular and loaded only with the dairy milk sample.

The TIM-1 digestive system contains dialysis membranes thataim to mimic intestinal absorption of small molecules. We wereunable, however, to detect any microRNA in the dialysates.Considering that the molecular weight of a single microRNAstrand (;7 kDa) is slightly lower than that of the dialysismembrane cutoff (11.8 kDa), we must assume that dairy milkmicroRNAs may be part of a larger macromolecular complex orparticle.

Although the TIM-1 digestion model does not allow monitor-ing of the cellular internalization of milk-derived microRNAs,it does provide key insights into their bioaccessibility; one wouldexpect milk-derived microRNAs to be internalized by the cells ofthe gastrointestinal tract during digestion (56). The leadingtheory for explaining how dairy milk microRNAs are able towithstand the relatively harsh conditions that prevail in thedigestive system involves their association with and protectionby EVs—more specifically, exosomes (22, 26, 27). Using aprotocol that we optimized for the study of exosomal microRNAsin commercial dairy milk preparations, we were able to obtainexosome-enriched fractions containing large amounts ofmicroRNAs,which is consistent with previous studies (49, 50) and supports aprotective role for exosomes during digestion.

We observed a slight shift in the profile of dairy milk bta-miR-223 and bta-miR-125b toward lower-density fractions upondigestion, which may be the result of partial digestion of milk EVproteins in the TIM-1 system and a corresponding decreasein the density of exosomes. Another possibility is that milkcontains subpopulations of exosomes that exhibit differentialresistance to digestion, as suggested by our TSG101 and ALIXWestern blot data and the literature (53, 57). Finally, it ispossible that the IDG centrifugation time may not have beensufficient or the speed not high enough to allow dairy milkexosomes to reach density equilibrium, as pointed out in theliterature (58, 59).

Our results show that <10% of the total bta-miR-223 andbta-miR-125b copy numbers are associated with the 100,0003 gpellets, supposedly the most enriched in exosomes. Wheninterpreting our dairy milk microRNA data, we assumed thatbta-miR-223 and bta-miR-125b microRNAs were representa-tive of all microRNAs; however, microRNA-specific patterns,changes, or processes cannot be excluded. Our results imply that

FIGURE 5 Most commercial dairy milk microRNAs bta-miR-223 and

bta-miR-125b are associated with 200-nm particles sedimenting at a

centrifugation speed lower than that for exosomes. Commercial dairy

milk was subjected to successive ultracentrifugation protocol. qPCR

detection and absolute quantification of bta-miR-223 (A) and bta-miR-

125b (B) are shown. Results are expressed as means6 SEMs (n = 3).

Proteins from each pellet and from the 100,0003 g SN were analyzed

by immunoblotting to detect the exosome-enriched proteins TSG101,

ALIX, and HSP70 (n = 3) (C). The hydrodynamic size (diameter in

nanometers) of the particles present in each sample was measured

(D). *,**,****Statistically different from the 100,000 3 g pellet: *P ,

0.05, **P , 0.01, ****P , 0.0001. ALIX, apoptosis-linked gene

2–interacting protein X; bta-miR, Bos taurus microRNA; HSP70, heat

shock protein 70; SN, supernatant fluid; TSG101, tumor susceptibility

gene-101.



exosomes are not confined to the 100,000 3 g pellet, at least indairy milk and based on the exosome-enrichment markersTSG101, ALIX, and HSP70; it may not be prudent to believethat all exosomes are pelleted by centrifugation at 100,000 3 gfor 1 h.

Nonetheless, our study suggests that the survival of mostdairy milk microRNAs during digestion is not related toexosomes. Considering that dairy milk microRNAs would notresist digestion or withstand the relatively high ribonucleaseactivity found in milk (22, 60) without protection or carriage(a synthetic microRNA added to milk is completely degradedin <10 min in milk and we could not detect a naked syntheticmicroRNA loaded within the TIM-1 system; A Benmoussa, BLaffont, and P Provost, unpublished data, 2014), we posit thatdairy milk microRNAs that resist digestion are associatedmainly with particles that are negative (or low) in TSG101and sediment at a centrifugation speed that is lower than that forexosomes. This possibility is supported by our findings that themajority of dairy milk microRNAs sediment at 12,000 and35,000 3 g and are associated with ALIX2, HSP702/low, andTSG1012/low particles of ;200 nm in diameter. The exactnature of these new dairy milk EVs, which are larger thanexosomes and may protect microRNAs from degradation in thegastrointestinal tract, and whether they can be internalized byhuman cells or transit through the gut wall, as previouslydescribed for dairy milk exosomes (21, 56), warrant furtherinvestigation. Our results suggest that the new kind of micro-particles described here are more susceptible than exosomes tothe harsh conditions of the digestive tract (Figures 3C and 5C)but are associated with more microRNAs. Therefore, these newkinds of microparticles and exosomes may offer quantity- andquality-based strategies for microRNA transfer with milk as aunique microRNA vehicle.

The potential existence of these TSG101-negative EVsenriched in microRNAs (ALIX2, HSP702/low, and TSG1012/low)reveals amajor limitation in the study ofmilk-derivedmicroRNAsthat comes with the use of exosome-isolation protocols [e.g.,gradual ultracentrifugation, density or velocity gradients,ExoQuick (Systems Biosciences, Inc.)] involving a centrifugation-clearing step at;12,000 3 g, which is likely to discard 30–40%of milk microRNAs. One may be well advised to consider milkas a complex fluid containing different kinds of microRNA-associated EVs, not to consider exosomes as the sole carriers ofmicroRNAs in milk, and not to discard potentially importantmicroRNA-enriched EVs that sediment at centrifugation speedslower than those for exosomes in any investigation of milkmicroRNAs.

The conservation of microRNA sequences between Homosapiens and Bos taurus and that 1000 copies of a specificmicroRNA per cell may be sufficient to exert a measurablebiological activity (61) suggest that the relatively high amounts(109–1010 copies) of dairy milk bta-miR-223 and bta-miR-125bmay contribute to the regulation of gene expression by endog-enous microRNAs, provided that they are indeed absorbed bythe GI tract. The number of bioaccessible microRNAs may beunderestimated by the absence of cells within the system, whichin vivo may bind to and protect microparticles from digestion.Conversely, the limitation of a cell-free digestive system is that itcannot take into account the contribution of the cells to thedigestion process, which may affect the chemical reactantstoichiometry. Nevertheless, miR-223 is specific to the hemato-poietic cell lineage and is involved in immunity, immune celllineage differentiation, and granulopoiesis (62). It also hasbeen involved in cancer progression, HIV-1 infection, and

IL-17–induced inflammation. Therefore, high intake of dairymilk miR-223 may modulate immunity and health and worsenthe symptoms of diseases such as rheumatoid arthritis (63).Regarding miR-125b, it is ighly important in regulating celldifferentiation, proliferation, and apoptosis, and it is consideredan oncogenic microRNA in cancer development and dissemina-tion (64). In some cases, it also acts as a proinflammatorymicroRNA, possibly is involved in autoimmune diseases, andmodulates immune system development and host defense.

Dairy milk microRNA survival in the upper small intestinecompartments of the gastrointestinal tract may thus lead totheir absorption and influence the health status of dairy milkconsumers. Whether the consumption of dairy milk microRNAshas beneficial or deleterious effects on health remains to bedetermined.

Acknowledgments

We thank Richard Janvier for electron microscopy analyses andCaroline Gilbert�s team for assistance with and sharing of theiranalytical tools. PP conceived and coordinated the project andrevised and finalized the manuscript; AB led the project;designed, planned, and performed the experiments; analyzedthe data, and wrote the first draft of the manuscript; CHCL, BL,and JL performed some experiments and analyzed the data; PSperformed the TIM-1 experiments and analyzed the data; andEB, CG, and IF provided expertise, guidance, advice, andanalytical tools. All of the authors commented on and edited themanuscript and read and approved the final manuscript.

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