The Journal of Nutrition

Biochemical, Molecular, and Genetic Mechanisms

The Intestinal Transport of Bovine Milk

Exosomes Is Mediated by Endocytosis in Human
Colon Carcinoma Caco-2 Cells and Rat Small
Intestinal IEC-6 Cells1–3
Tovah Wolf, Scott R Baier, and Janos Zempleni*

Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE

Abstract
Background: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs in tissues and body
fluids is encapsulated in exosomes, thereby conferring protection against degradation and a pathway for intestinal
transport. MicroRNAs in cow milk are bioavailable in humans.
Objective: This research assessed the transport mechanism of bovine milk exosomes, and therefore microRNAs, in
human and rodent intestinal cells.
Methods: The intestinal transport of bovine milk exosomes and microRNAs was assessed using fluorophore-labeled
bovine milk exosomes in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. Transport kinetics and
mechanisms were characterized using dose-response studies, inhibitors of vesicle transport, carbohydrate competitors,
proteolysis of surface proteins on cells and exosomes, and transepithelial transport in transwell plates.
Results: Exosome transport exhibited saturation kinetics at 37°C [Michaelis constant (Km) = 55.5 6 48.6 mg exosomal
protein/200 mL of media; maximal transport rate = 0.083 6 0.057 ng of exosomal protein
81,750 cells21
h21] and
decreased by 64% when transport was measured at 4°C, consistent with carrier-mediated transport in Caco-2 cells.
Exosome uptake decreased by 61–85% under the following conditions compared with controls in Caco-2 cells: removal of
exosome and cell surface proteins by proteinase K, inhibition of endocytosis and vesicle trafficking by synthetic inhibitors,
and inhibition of glycoprotein binding by carbohydrate competitors. When milk exosomes, at a concentration of 5 times the
Km, were added to the upper chamber in transwell plates, Caco-2 cells accumulated miR-29b and miR-200c in the lower
chamber, and reverse transport was minor. Transport characteristics were similar in IEC-6 cells and Caco-2 cells, except
that substrate affinity and transporter capacity were lower and higher, respectively.
Conclusion: The uptake of bovine milk exosomes is mediated by endocytosis and depends on cell and exosome surface
glycoproteins in human and rat intestinal cells. J Nutr 2015;145:2201–6.

Keywords: endocytosis, extracellular vesicles, intestinal cells, milk exosomes, uptake

Introduction
(1), and silence genes through destabilizing mRNA or preventing
MicroRNAs are about 22 nucleotides long (1), hybridize with translation of mRNA (2, 3). The sequence complementarity in the
complementary sequences in the 3#-untranslated regions in mRNA ‘‘seed region’’ (nucleotides 2–8) in microRNAs is of particular
importance for binding to target transcripts. The microRNA-
1
Supported by the National Institute of Food and Agriculture, USDA, under award
dependent degradation of mRNA takes place in the RNA-induced
number 2015-67017-23181; Public Health Service grant NIH 1P20GM104320; the silencing complex. If the microRNA nucleotide sequence has a
Gerber Foundation; the Egg Nutrition Center; the University of Nebraska Agricultural high degree of complementarity to the sequence in the mRNA
Research Division (Hatch Act); and USDA multistate group W3002 (all to JZ).
2 target, both microRNA and mRNA are degraded (4, 5). In contrast,
Author disclosures: T Wolf, SR Baier, and J Zempleni, no conflicts of interest.
The granting agencies had no influence on the study design; the collection, if the sequence complementarity is imperfect, the binding of
analysis, and interpretation of the data; the writing of the manuscript; or the microRNA to mRNA will halt mRNA translation without causing
decision to submit the manuscript for publication. microRNA/mRNA degradation (5, 6).
3
Supplemental Table 1 is available from the ‘‘Online Supporting Material’’ link in
Traditionally, microRNAs have been considered endogenous
the online posting of the article and from the same link in the online table of
contents at http://jn.nutrition.org. regulators of genes; i.e., microRNAs synthesized by a given host
* To whom correspondence should be addressed. E-mail: jzempleni2@unl.edu. regulate the expression of genes in that host. Our laboratory has
ã 2015 American Society for Nutrition.
Manuscript received June 8, 2015. Initial review completed July 2, 2015. Revision accepted July 15, 2015. 2201
First published online August 12, 2015; doi:10.3945/jn.115.218586.
refuted this paradigm. We provided strong evidence that 1) Thermo Scientific) to remove precipitated protein, fat globules, and
humans absorb biologically meaningful amounts of microRNAs vesicles larger than the exosomes. The supernatant was ultracentrifuged at
from nutritionally relevant doses of cow milk, 2) physiologic 120,000 3 g for 90 min at 4°C to collect exosomes. The exosome pellet
concentrations of milk microRNAs affect human gene expression was resuspended in a small volume of PBS containing 0.01% sodium
azide, filtered twice through a 0.22-mm membrane filter, and stored at 4°C
in vivo and in cell cultures, and 3) endogenous synthesis of
and 220°C if necessary. The exosomes were labeled with the fluorophore,
microRNAs does not compensate for dietary microRNA defi- FM 4-64 (Molecular Probes). One microliter of a stock solution of FM
ciency in mice (7). To the best of our knowledge, our previous 4-64 (5.9 mmol/L) was added to 1 mL of exosome suspension and incubated
article is the first report providing strong evidence that micro- for 15 min at 37°C, and excess FM 4-64 was removed by ultracentrifu-
RNAs can be transferred between distinct animal species through gation at 120,000 3 g at 4°C for 90 min.
dietary means (7). Since we published our milk paper, investigators Absence of aggregation and exosome purity were assessed as
from the NIH-supported Genboree database detected numerous recommended by the International Society for Extracellular Vesicles
dietary microRNAs in 6.8 billion sequencing reads from 528 (28). Briefly, absence of exosome aggregation was confirmed using
human samples (8), and we detected bovine-specific microRNAs transmission electron microscopy (Hitachi H7500; Hitachi) in the
in human plasma and mouse livers by next-generation sequencing Microscopy Core Facility at the University of Nebraska-Lincoln (Figure
1A). ImageJ (http://imagej.nih.gov/ij/index.html) was used to analyze the
(SR Baier and J Zempleni, unpublished results, 2015). In contrast,
exosome size distribution, which averaged 69 6 20 nm in diameter, as
previous claims by a single laboratory that microRNAs from expected for exosomes that are smaller than microvesicles, apoptotic
plants affect human gene expression (9, 10) are highly controver- bodies, and fat globules (28, 31). Exosome purity and identity were
sial and have largely been dismissed by the scientific community confirmed using whole protein extracts from exosomes as described
(7, 11–14). The potential impact of our discoveries on human previously (32), using mouse anti-bovine CD63 (AbD Serotec) as a marker
nutrition and health is substantial, because gene regulations by for exosomes, rabbit antiserum to bovine a-s1-casein as a marker for the
microRNAs have been implicated in numerous physiologic (15) animal species of exosome origin, and goat anti-bovine histone H3 (Santa
and pathologic (16) conditions in humans including bone health, Cruz Biotechnology) as a negative control (all at 1000-fold dilutions).
cancer, arthritis and inflammatory bowel disease, and metabolic Bands were visualized using an Odyssey infrared imaging system (Licor,
syndrome. Inc.) and IRDye 800CW-labeled secondary antibodies at a 50,000-fold
dilution (Figure 1B).
Extracellular vesicles (EVs)4 are equally important, given that
EV cargo includes various species of RNA, proteins, and lipids Transport studies. Caco-2 cells and IEC-6 cells were seeded at a density
(17–19), and encapsulation of microRNAs in EVs confers of 20,000 and 7000 cells/well, respectively, in 96-well plates, and allowed
protection against degradation (20–24) and a pathway for cellular
uptake by endocytosis (25, 26). EVs are secreted by donor cells for
delivery to recipient cells, and EV cargo has emerged as an
important mediator in intercellular communication (27). There
are 3 major classes of EVs, i.e., exosomes, microvesicles, and
apoptotic bodies (28). Exosomes are of particular interest because
they are loaded with microRNAs in a targeted, nonstochastic
process that involves sorting mechanisms (29). Here, we tested
the hypothesis that the intestinal transport of bovine milk exosomes
is mediated by endocytosis in human and rat mucosal cells.
We further determined whether mucosal cells transport milk
microRNAs across the basolateral membrane for subsequent
delivery to peripheral tissues.

Methods
Cell cultures. Human colon carcinoma Caco-2 cells were purchased
from American Type Culture Collection and were used from passages
52 to 72 for experiments cultured following the supplierÕs recommen-
dations. In select experiments, Caco-2 cells were cultured for 2 d in
exosome-depleted media obtained by ultracentrifugation of FBS at
120,000 3 g for 6 h. Cell media were replaced with fresh media every
2–3 d. Transport studies were conducted at 75% confluence in 96-well
plates and at 100% confluence in transwell plates. IEC-6 primary rat small
intestinal cells were obtained at passage 14 and cultured as described for
Caco-2 cells except that culture media contained 0.1 U/mL bovine insulin,
and no transwell studies were conducted because of the IEC-6 cellsÕ inability
to form a tight monolayer.

Milk exosome isolation and fluorophore conjugation. Skim cow FIGURE 1 Milk exosome preparations from cow milk. (A) Trans-
milk was purchased in a local grocery store. The milk was centrifuged at mission electron microscope images of exosome preparations. The
13,200 3 g and 4°C for 30 min to remove somatic cells and debris. The large field image was obtained at 15,0003 magnification; the insert
supernatant was mixed 1:1 (by vol) with 250 mM EDTA (pH 7.0) on ice for depicts a single particle selected from an image that was obtained at
15 min to precipitate milk casein (30). The suspension was ultra- 60,0003 magnification and electronically enlarged when assembling
centrifuged at 100,000 3 g and 4°C for 60 min (F37L-8 3 100 rotor; this figure. (B) Exosome extracts were probed using anti-CD63, anti–
a-s1-casein, and anti–histone H3. Protein extracts were run on the
4
Abbreviations used: BFA, brefeldin A; Cyt D, cytochalasin D; EV, extracellular same gel, but membranes were cut for probing with the 3 antibodies
vesicle; Km, Michaelis constant; LY, Lucifer yellow. and reassembled after probing.

2202 Wolf et al.

to adhere for 48 h, when cells were ;75% confluent. Transport studies
were conducted using FM4-64–labeled exosomes using 3–110 mg of
exosomal protein/well (Caco-2 cells) or 27–652 mg of exosomal protein/
well (IEC-6 cells) and incubating cells for periods of time described in the
Results section to assess saturation kinetics; blanks were created using
solvent. Assays were calibrated by quantifying the fluorescence of a known
mass of exosomes labeled with FM 4-64. When indicated, cells or
exosomes were treated with 100 mg/mL proteinase K (Caco-2 cells) to
remove surface proteins, 10 mg/mL endocytosis inhibitor cytochalasin D
(Cyt D), 20 mg/mL brefeldin A (BFA) to inhibit vesicle trafficking, and 150
mmol/L carbohydrate competitors D-glucose or D-galactose 30 min before
initiation of and continuing for the duration of transport studies. IEC-6
cells did not survive proteinase K treatment. Therefore, surface proteins
were removed by treating IEC-6 cells and exosomes using 0.105 mmol/L
trypsin for 5 and 30 min, respectively, at room temperature. Exosome
uptake was analyzed by measuring the cell fluorescence at 515 (excitation)
and 640 nm (emission) using a Biotek FLx800 plate reader (BioTek Instru-
ments). Fluorescence readings were corrected for cell autofluorescence by
subtracting signals measured in cells incubated with exosome-depleted
media. Transport kinetics was modeled using the Michaelis-Mentenequation
and nonlinear regression; modeling was conducted using GraphPad Prism
6.0 (GraphPad Software).
In transwell studies Caco-2 cells were seeded at a density of 9000 cells/
well with 75 mL of media in 96-well polycarbonate plates with a pore
size of 0.4 mm (EMD Millipore). The cells were allowed to grow a
differentiated monolayer for 21–24 d (33). Caco-2 cell monolayer
integrity was formally confirmed using the Lucifer yellow (LY) rejection
assay according to the manufacturerÕs instructions (33). LY fluorescence FIGURE 2 Time courses of bovine exosome uptake in Caco-2 cells
was measured in the transwell apical and basolateral chambers after 1 h of and IEC-6 cells. (A) Exosome uptake into human colon carcinoma
incubation at 37°C. In parallel experiments, Caco-2 cells were cultured in Caco-2 cells as a function of time at a concentration of 110 mg
exosome-depleted media to which milk exosomes were added back to exosome protein/200 mL media and a temperature of 37°C (n = 6). (B)
produce a concentration of 275 mg/100 mL exosomal protein in either the Exosome uptake into rat primary intestinal IEC-6 cells as a function of
upper, apical chamber or the lower, basolateral chamber. Controls were time at a concentration of 55 mg exosome protein/200 mL media and a
cultured in exosome-depleted media. Aliquots of media were collected temperature of 37°C (n = 3). Values are means 6 SDs.
from the upper chamber and bottom chamber after 2 h of incubation for
analysis of microRNAs. Twenty-five attomoles of internal standard
(miSPIKE Synthetic RNA; IDT Technologies) was added to samples before mechanisms. Transport kinetics was modeled using the Michaelis-
microRNA extraction and subsequent analysis of miR-29b and miR-200c
Menten equation. In Caco-2 cells Michaelis constant (Km) and
in transwell chambers by quantitative real-time PCR and microRNA-
specific primers (Supplemental Table 1); miSpike was also used for PCR
maximal transport rate were 55.5 6 48.6 mg exosomal protein/
calibration (7). Values were corrected for the internal standard to 200 mL medium and 0.08 6 0.06 ng of exosomal protein
81,750
normalize for extraction efficiency. cells21
h21, respectively (r2 = 0.75; Figure 3A). In IEC-6 cells Km
and maximal transport rate were 152 6 39.5 mg/200 mL and
Statistics. Homogeneity of variances was assessed using the Brown- 0.14 6 0.01 ng of exosomal protein
36,375 cells21
30 min21,
Forsythe test (34, 35). The data variation for IEC-6 cells was heteroge- respectively (r2 = 0.56; Figure 3B). When the incubation temper-
nous; i.e., those data were log transformed before statistical analysis. ature was decreased from 37°C to 4°C, the transport rate
Statistical significance of differences among treatment groups was decreased from 100% 6 56% to 54% 6 13% using a substrate
assessed using 1-factor ANOVA and DunnettÕs t test for post hoc concentration of 55.5 mg exosomal protein/200 mL in Caco-2 cells
comparisons between treatment groups and control. Time course exper-
(P < 0.05; n = 3). Likewise, when the incubation temperature was
iments were analyzed using linear regression analysis. Analyses were
performed using GraphPad Prism. Differences were considered signifi-
decreased from 37°C to 4°C, the transport rate decreased from
cant at P < 0.05. Results were presented as means 6 SDs and represent 100% 6 11% to 44% 6 25% using a substrate concentration of
independent biological replicates. 153 mg exosomal protein/200 mL in IEC-6 cells (P < 0.05; n = 3).
Subsequent transport studies were conducted using substrate
concentrations of 55 mg/200 mL and 153 mg/200 mL in Caco-2 cells
and IEC-6 cells, respectively, except for the transwell studies.
Results
Time course of bovine milk exosome uptake. In Caco-2 cells Roles of surface glycoproteins and endocytosis. The uptake
exosome uptake was linear for up to 120 min if transport was of bovine milk exosomes into human and rat intestinal cells
measured using nonsaturating substrate concentrations (Figure depended on surface proteins in both exosomes and cells. When
2A): y = 0.0012x + 0.014 (r2 = 0.97; P < 0.05). In IEC-6 cells surface proteins were removed from exosomes or Caco-2 cells
exosome uptake was linear for only up to 60 min if transport was were treated with proteinase K, exosome uptake decreased to
measured using nonsaturating substrate concentrations (Figure 32% 6 25% and 18% 6 16% of controls (P < 0.05; n = 3). When
2B): y = 0.0033x + 0.033 (r2 = 0.75; P < 0.05). Subsequent IEC-6 cells were treated with trypsin, exosome uptake decreased
transport studies were conducted using incubation times of 60 and to 82% 6 8% of controls (P < 0.05; n = 3). When Caco-2 cells were
30 min for Caco-2 cells and IEC-6 cells, respectively. treated with inhibitors of endocytosis (Cyt D), intracellular vesicle
trafficking (BFA), or carbohydrate competitors, the uptake of
Transport kinetics. In both Caco-2 and IEC-6 cells, the uptake exosomes decreased to <50% of controls (Figure 4A). The effects
of bovine milk exosomes was mediated by saturable transport of these inhibitors and competitors were similar in IEC-6 cells,
Transport of milk exosomes in intestinal cells 2203
FIGURE 3 Saturation kinetics of bovine exosome transport in intestinal
cells. (A) Exosome uptake into human colon carcinoma Caco-2 cells as FIGURE 4 Effects of inhibitors of endocytosis and vesicle traffick-
a function of substrate concentration at 37°C (n = 5). (B) Exosome ing, and carbohydrate competitors on the uptake of bovine milk
uptake into rat primary small intestinal IEC-6 cells as a function substrate exosomes in human and rat intestinal cells. (A) Exosome transport in
concentration at 37°C (n = 3). Values are means 6 SDs. Caco-2 cells (expressed as ng exosomal protein
81,750 cells21
h21)
pretreated for 30 min with 10 mg/mL Cyt D or 20 mg/mL BFA or in the
presence of 150 mmol/L carbohydrate competitors, using an exosome
although the effects of treatments were not statistically significant concentration of 55 mg/200 mL (n = 5). (B) Exosome transport in IEC-6
(P = 0.11; n = 6; Figure 4B). cells (expressed as ng exosomal protein
36,375 cells21
30 min21)
pretreated for 30 min with 10 mg/mL Cyt D or 20 mg/mL BFA, or in
microRNA transport across intestinal monolayers. When the presence of 150 mmol/L carbohydrate competitors, using an
Caco-2 cells were provided with exosome-supplemented media exosome concentration of 153 mg/200 mL (n = 6). *Different from
control, P , 0.05. Values are means 6 SDs. BFA, brefeldin A; Cyt D,
(275 mg/100 mL) in the apical chamber and incubated for 2 h, the
cytochalasin D;.
concentration of miR-29b in the basolateral chamber increased
from 0.018 6 0.03 fmol/L to 0.094 6 0.16 fmol/L (P < 0.05).
Likewise, the concentration of miR-200c increased from 53.5 6
human and rat intestinal cells. A previous report suggested that
91.4 fmol/L to 1864 6 2982 fmol/L in the basolateral chamber
human macrophages have the ability to absorb bovine milk
(P < 0.05). When exosomes were provided with exosome-
exosomes, but the transport mechanism is unknown (38).
supplemented media (275 mg/100 mL) in the basolateral chamber
This study provides compelling evidence that the intestinal
and incubated for 2 h, no concentration gradient was detected in
uptake of microRNAs encapsulated in exosomes is an active,
the 2 chambers, suggesting minimal reverse transport under the
saturable process and is most likely mediated by endocytosis in
experimental conditions: 0.067 6 0.068 fmol/L miR-29b in the
humans and rats. It is evident from the studies using proteases to
basolateral chamber compared with 0.066 6 0.11 fmol/L miR-
remove surface proteins that protein/protein recognition plays a
29b in the apical chamber. Likewise, only 3.3% 6 4.3% of miR-
crucial role in the intestinal uptake of exosomes. Carbohydrate
200c added to the basolateral chamber was secreted into the
competitors caused a decrease in exosome uptake, suggesting
apical chamber. Caco-2 cells formed a tight monolayer, judged by
that glycosylated proteins are important to the endocytosis of
an LY rejection percentage of 99.7% 6 0.19%.
food-borne exosomes and their cargo. We speculate that the
low bioavailability of plant-borne microRNAs compared with
animal-borne microRNAs (7, 11–14) might be because of poor
Discussion
compatibility of the glycoproteins present on plant vesicles with
Our previous observation that milk microRNAs have biological receptors on the apical surface of mammalian intestinal cells. This
activity in humans continues to gain traction (7) and is now study also provides evidence that the intestinal transport of
supported by 2 peer-reviewed meeting reports from independent microRNAs in bovine milk exosomes is primarily a unidirectional
laboratories, which have identified dietary microRNAs in human process in the apical-to-basolateral direction.
plasma and milk exosomes in mammalian circulation and tissues The capacity for transporting bovine milk exosomes was
(8, 36). In addition, we recently reported that microRNAs from higher in the primary IEC-6 cells from the upper intestine than in
another food of animal origin, chicken eggs, have biological the Caco-2 colon cell line, particularly when adjusting our data
activity in humans (37). This paper adds to the body of evidence for the much smaller number of IEC-6 cells in wells compared
that microRNAs are transferred among animal species by dietary with Caco-2 cells. This interpretation is consistent with our time
means. To the best of our knowledge, this is the first report course studies of postprandial milk microRNAs in human plasma,
assessing the transport mechanism of bovine milk exosomes in suggesting that absorption occurs in the upper intestine (7). We
2204 Wolf et al.
recognize that species effects (rat vs. human) might contribute to the primary responsibility for the final content; TW, SRB, and
transporter activity and that these effects were not formally JZ contributed to the development of this work. All authors
excluded in this study. read and approved the final manuscript.
The discovery that microRNAs from foods of animal origin are
bioavailable in humans and alter the expression of human genes
has great importance for human nutrition and health (7). Based on
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