The Intestinal Transport of Bovine Milk Exosomes Is Mediated by Endocytosis in Human Colon Carcinoma Caco-2 Cells and Rat Small Intestinal IEC-6 Cells^,,

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 (K_m) = 55.5 ± 48.6 μg exosomal protein/200 μL of media; maximal transport rate = 0.083 ± 0.057 ng of exosomal protein · 81,750 cells⁻¹ · h⁻¹] 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 K_m, 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.

Introduction

MicroRNAs are about 22 nucleotides long (1), hybridize with complementary sequences in the 3′-untranslated regions in mRNA (1), and silence genes through destabilizing mRNA or preventing translation of mRNA (2, 3). The sequence complementarity in the “seed region” (nucleotides 2–8) in microRNAs is of particular importance for binding to target transcripts. The microRNA-dependent degradation of mRNA takes place in the RNA-induced silencing complex. If the microRNA nucleotide sequence has a high degree of complementarity to the sequence in the mRNA target, both microRNA and mRNA are degraded (4, 5). In contrast, if the sequence complementarity is imperfect, the binding of microRNA to mRNA will halt mRNA translation without causing microRNA/mRNA degradation (5, 6).

Traditionally, microRNAs have been considered endogenous regulators of genes; i.e., microRNAs synthesized by a given host regulate the expression of genes in that host. Our laboratory has refuted this paradigm. We provided strong evidence that 1) humans absorb biologically meaningful amounts of microRNAs from nutritionally relevant doses of cow milk, 2) physiologic concentrations of milk microRNAs affect human gene expression in vivo and in cell cultures, and 3) endogenous synthesis of microRNAs does not compensate for dietary microRNA deficiency in mice (7). To the best of our knowledge, our previous article is the first report providing strong evidence that microRNAs can be transferred between distinct animal species through dietary means (7). Since we published our milk paper, investigators from the NIH-supported Genboree database detected numerous dietary microRNAs in 6.8 billion sequencing reads from 528 human samples (8), and we detected bovine-specific microRNAs in human plasma and mouse livers by next-generation sequencing (SR Baier and J Zempleni, unpublished results, 2015). In contrast, previous claims by a single laboratory that microRNAs from plants affect human gene expression (9, 10) are highly controversial and have largely been dismissed by the scientific community (7, 11–14). The potential impact of our discoveries on human nutrition and health is substantial, because gene regulations by microRNAs have been implicated in numerous physiologic (15) and pathologic (16) conditions in humans including bone health, cancer, arthritis and inflammatory bowel disease, and metabolic syndrome.

Extracellular vesicles (EVs)⁴ are equally important, given that EV cargo includes various species of RNA, proteins, and lipids (17–19), and encapsulation of microRNAs in EVs confers 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 supplier’s recommendations. In select experiments, Caco-2 cells were cultured for 2 d in exosome-depleted media obtained by ultracentrifugation of FBS at 120,000 × 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 cells’ inability to form a tight monolayer.

Milk exosome isolation and fluorophore conjugation.

Skim cow milk was purchased in a local grocery store. The milk was centrifuged at 13,200 × g and 4°C for 30 min to remove somatic cells and debris. The supernatant was mixed 1:1 (by vol) with 250 mM EDTA (pH 7.0) on ice for 15 min to precipitate milk casein (30). The suspension was ultracentrifuged at 100,000 × g and 4°C for 60 min (F37L-8 × 100 rotor; Thermo Scientific) to remove precipitated protein, fat globules, and vesicles larger than the exosomes. The supernatant was ultracentrifuged at 120,000 × g for 90 min at 4°C to collect exosomes. The exosome pellet was resuspended in a small volume of PBS containing 0.01% sodium azide, filtered twice through a 0.22-μm membrane filter, and stored at 4°C and −20°C if necessary. The exosomes were labeled with the fluorophore, FM 4-64 (Molecular Probes). One microliter of a stock solution of FM 4-64 (5.9 mmol/L) was added to 1 mL of exosome suspension and incubated for 15 min at 37°C, and excess FM 4-64 was removed by ultracentrifugation at 120,000 × g at 4°C for 90 min.

Absence of aggregation and exosome purity were assessed as recommended by the International Society for Extracellular Vesicles (28). Briefly, absence of exosome aggregation was confirmed using transmission electron microscopy (Hitachi H7500; Hitachi) in the Microscopy Core Facility at the University of Nebraska-Lincoln (Figure 1A). ImageJ (http://imagej.nih.gov/ij/index.html) was used to analyze the exosome size distribution, which averaged 69 ± 20 nm in diameter, as expected for exosomes that are smaller than microvesicles, apoptotic bodies, and fat globules (28, 31). Exosome purity and identity were confirmed using whole protein extracts from exosomes as described previously (32), using mouse anti-bovine CD63 (AbD Serotec) as a marker for exosomes, rabbit antiserum to bovine α-s1-casein as a marker for the animal species of exosome origin, and goat anti-bovine histone H3 (Santa Cruz Biotechnology) as a negative control (all at 1000-fold dilutions). Bands were visualized using an Odyssey infrared imaging system (Licor, Inc.) and IRDye 800CW-labeled secondary antibodies at a 50,000-fold dilution (Figure 1B).

FIGURE 1.

Milk exosome preparations from cow milk. (A) Transmission electron microscope images of exosome preparations. The large field image was obtained at 15,000× magnification; the insert depicts a single particle selected from an image that was obtained at 60,000× magnification and electronically enlarged when assembling this figure. (B) Exosome extracts were probed using anti-CD63, anti–α-s1-casein, and anti–histone H3. Protein extracts were run on the same gel, but membranes were cut for probing with the 3 antibodies and reassembled after probing.

Transport studies.

Caco-2 cells and IEC-6 cells were seeded at a density of 20,000 and 7000 cells/well, respectively, in 96-well plates, and allowed to adhere for 48 h, when cells were ∼75% confluent. Transport studies were conducted using FM4-64–labeled exosomes using 3–110 μg of exosomal protein/well (Caco-2 cells) or 27–652 μg 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 μg/mL proteinase K (Caco-2 cells) to remove surface proteins, 10 μg/mL endocytosis inhibitor cytochalasin D (Cyt D), 20 μg/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 Instruments). 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-Menten equation 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 μL of media in 96-well polycarbonate plates with a pore size of 0.4 μm (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 manufacturer’s instructions (33). LY fluorescence was measured in the transwell apical and basolateral chambers after 1 h of incubation at 37°C. In parallel experiments, Caco-2 cells were cultured in exosome-depleted media to which milk exosomes were added back to produce a concentration of 275 μg/100 μL exosomal protein in either the upper, apical chamber or the lower, basolateral chamber. Controls were cultured in exosome-depleted media. Aliquots of media were collected 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 microRNA extraction and subsequent analysis of miR-29b and miR-200c in transwell chambers by quantitative real-time PCR and microRNA-specific primers (Supplemental Table 1); miSpike was also used for PCR calibration (7). Values were corrected for the internal standard to normalize for extraction efficiency.

Statistics.

Homogeneity of variances was assessed using the Brown-Forsythe test (34, 35). The data variation for IEC-6 cells was heterogenous; i.e., those data were log transformed before statistical analysis. Statistical significance of differences among treatment groups was assessed using 1-factor ANOVA and Dunnett’s t test for post hoc comparisons between treatment groups and control. Time course experiments were analyzed using linear regression analysis. Analyses were performed using GraphPad Prism. Differences were considered significant at P < 0.05. Results were presented as means ± SDs and represent independent biological replicates.

Results

Time course of bovine milk exosome uptake.

In Caco-2 cells exosome uptake was linear for up to 120 min if transport was measured using nonsaturating substrate concentrations (Figure 2A): y = 0.0012x + 0.014 (r² = 0.97; P < 0.05). In IEC-6 cells exosome uptake was linear for only up to 60 min if transport was measured using nonsaturating substrate concentrations (Figure 2B): y = 0.0033x + 0.033 (r² = 0.75; P < 0.05). Subsequent transport studies were conducted using incubation times of 60 and 30 min for Caco-2 cells and IEC-6 cells, respectively.

FIGURE 2.

Time courses of bovine exosome uptake in Caco-2 cells and IEC-6 cells. (A) Exosome uptake into human colon carcinoma Caco-2 cells as a function of time at a concentration of 110 μg exosome protein/200 μL media and a temperature of 37°C (n = 6). (B) Exosome uptake into rat primary intestinal IEC-6 cells as a function of time at a concentration of 55 μg exosome protein/200 μL media and a temperature of 37°C (n = 3). Values are means ± SDs.

Transport kinetics.

In both Caco-2 and IEC-6 cells, the uptake of bovine milk exosomes was mediated by saturable transport mechanisms. Transport kinetics was modeled using the Michaelis-Menten equation. In Caco-2 cells Michaelis constant (K_m) and maximal transport rate were 55.5 ± 48.6 μg exosomal protein/200 μL medium and 0.08 ± 0.06 ng of exosomal protein · 81,750 cells⁻¹ · h⁻¹, respectively (r² = 0.75; Figure 3A). In IEC-6 cells K_m and maximal transport rate were 152 ± 39.5 μg/200 μL and 0.14 ± 0.01 ng of exosomal protein · 36,375 cells⁻¹ · 30 min⁻¹, respectively (r² = 0.56; Figure 3B). When the incubation temperature was decreased from 37°C to 4°C, the transport rate decreased from 100% ± 56% to 54% ± 13% using a substrate concentration of 55.5 μg exosomal protein/200 μL in Caco-2 cells (P < 0.05; n = 3). Likewise, when the incubation temperature was decreased from 37°C to 4°C, the transport rate decreased from 100% ± 11% to 44% ± 25% using a substrate concentration of 153 μg exosomal protein/200 μL in IEC-6 cells (P < 0.05; n = 3). Subsequent transport studies were conducted using substrate concentrations of 55 μg/200 μL and 153 μg/200 μL in Caco-2 cells and IEC-6 cells, respectively, except for the transwell studies.

FIGURE 3.

Saturation kinetics of bovine exosome transport in intestinal cells. (A) Exosome uptake into human colon carcinoma Caco-2 cells as a function of substrate concentration at 37°C (n = 5). (B) Exosome uptake into rat primary small intestinal IEC-6 cells as a function substrate concentration at 37°C (n = 3). Values are means ± SDs.

Roles of surface glycoproteins and endocytosis.

The uptake of bovine milk exosomes into human and rat intestinal cells depended on surface proteins in both exosomes and cells. When surface proteins were removed from exosomes or Caco-2 cells were treated with proteinase K, exosome uptake decreased to 32% ± 25% and 18% ± 16% of controls (P < 0.05; n = 3). When IEC-6 cells were treated with trypsin, exosome uptake decreased to 82% ± 8% of controls (P < 0.05; n = 3). When Caco-2 cells were treated with inhibitors of endocytosis (Cyt D), intracellular vesicle trafficking (BFA), or carbohydrate competitors, the uptake of exosomes decreased to <50% of controls (Figure 4A). The effects of these inhibitors and competitors were similar in IEC-6 cells, although the effects of treatments were not statistically significant (P = 0.11; n = 6; Figure 4B).

FIGURE 4.

Effects of inhibitors of endocytosis and vesicle trafficking, and carbohydrate competitors on the uptake of bovine milk exosomes in human and rat intestinal cells. (A) Exosome transport in Caco-2 cells (expressed as ng exosomal protein · 81,750 cells⁻¹ · h⁻¹) pretreated for 30 min with 10 μg/mL Cyt D or 20 μg/mL BFA or in the presence of 150 mmol/L carbohydrate competitors, using an exosome concentration of 55 μg/200 μL (n = 5). (B) Exosome transport in IEC-6 cells (expressed as ng exosomal protein · 36,375 cells⁻¹ · 30 min⁻¹) pretreated for 30 min with 10 μg/mL Cyt D or 20 μg/mL BFA, or in the presence of 150 mmol/L carbohydrate competitors, using an exosome concentration of 153 μg/200 μL (n = 6). *Different from control, P < 0.05. Values are means ± SDs. BFA, brefeldin A; Cyt D, cytochalasin D;.

microRNA transport across intestinal monolayers.

When Caco-2 cells were provided with exosome-supplemented media (275 μg/100 μL) in the apical chamber and incubated for 2 h, the concentration of miR-29b in the basolateral chamber increased from 0.018 ± 0.03 fmol/L to 0.094 ± 0.16 fmol/L (P < 0.05). Likewise, the concentration of miR-200c increased from 53.5 ± 91.4 fmol/L to 1864 ± 2982 fmol/L in the basolateral chamber (P < 0.05). When exosomes were provided with exosome-supplemented media (275 μg/100 μL) in the basolateral chamber and incubated for 2 h, no concentration gradient was detected in the 2 chambers, suggesting minimal reverse transport under the experimental conditions: 0.067 ± 0.068 fmol/L miR-29b in the basolateral chamber compared with 0.066 ± 0.11 fmol/L miR-29b in the apical chamber. Likewise, only 3.3% ± 4.3% of miR-200c added to the basolateral chamber was secreted into the apical chamber. Caco-2 cells formed a tight monolayer, judged by an LY rejection percentage of 99.7% ± 0.19%.

Discussion

Our previous observation that milk microRNAs have biological activity in humans continues to gain traction (7) and is now supported by 2 peer-reviewed meeting reports from independent laboratories, which have identified dietary microRNAs in human plasma and milk exosomes in mammalian circulation and tissues (8, 36). In addition, we recently reported that microRNAs from another food of animal origin, chicken eggs, have biological activity in humans (37). This paper adds to the body of evidence that microRNAs are transferred among animal species by dietary means. To the best of our knowledge, this is the first report assessing the transport mechanism of bovine milk exosomes in human and rat intestinal cells. A previous report suggested that human macrophages have the ability to absorb bovine milk exosomes, but the transport mechanism is unknown (38).

This study provides compelling evidence that the intestinal uptake of microRNAs encapsulated in exosomes is an active, saturable process and is most likely mediated by endocytosis in humans and rats. It is evident from the studies using proteases to remove surface proteins that protein/protein recognition plays a crucial role in the intestinal uptake of exosomes. Carbohydrate competitors caused a decrease in exosome uptake, suggesting that glycosylated proteins are important to the endocytosis of 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 compatibility of the glycoproteins present on plant vesicles with receptors on the apical surface of mammalian intestinal cells. This study also provides evidence that the intestinal transport of microRNAs in bovine milk exosomes is primarily a unidirectional process in the apical-to-basolateral direction.

The capacity for transporting bovine milk exosomes was higher in the primary IEC-6 cells from the upper intestine than in the Caco-2 colon cell line, particularly when adjusting our data for the much smaller number of IEC-6 cells in wells compared with Caco-2 cells. This interpretation is consistent with our time course studies of postprandial milk microRNAs in human plasma, suggesting that absorption occurs in the upper intestine (7). We recognize that species effects (rat vs. human) might contribute to transporter activity and that these effects were not formally excluded in this study.

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 these discoveries, milk microRNAs meet the National Cancer Institute’s definition of bioactive compounds as “a type of chemical found in small amounts in plants and certain foods …. Bioactive compounds have actions in the body that may promote good health. They are being studied in the prevention of … diseases” (39). Using the TargetScan algorithm (40), we identified 11,119 unique human gene targets for 175 of the 245 microRNAs in cow milk (20, 41). Although the majority of these targets have not been experimentally validated, it is reasonable to propose that milk microRNAs are important regulators in a variety of metabolic pathways. Gene regulation by microRNAs has been implicated in virtually all aspects of human health and disease including bone health (42, 43), female and male reproduction (44, 45), arthritis and inflammatory bowel disease (20, 46, 47), metabolic syndrome (48–51), and cancer (52). Note that pasteurization, fat adjustment, and cold storage of milk does not alter its microRNA content, but that a 50% loss of microRNAs was observed when milk was homogenized, presumably because of the destruction of exosomes by shear forces (53).

Note that Cyt D, BFA, and carbohydrate competitors consistently failed to decrease exosome uptake into IEC-6 cells, whereas exosome uptake was strongly inhibited in Caco-2 cells. Previous studies suggest that the expression of surface protein and proteins involved in vesicle trafficking depends on cell density in IEC-6 cell cultures (54). We speculate that under the experimental conditions of this study, the cell density was not optimal to capture effects of inhibitors of endocytosis and carbohydrate inhibitors. However, we did not formally test this theory.

Some uncertainties remain; e.g., the field of dietary microRNAs would benefit from the publication of a full-length peer-reviewed paper that unambiguously confirms the presence of dietary microRNAs in biological fluids in humans. It is safe to assume that such publications will be available soon, when considering the recent report of the identification of numerous dietary microRNAs in 6.8 billion sequencing reads from human samples (8) and our own sequencing studies identifying 4 bovine microRNAs with nucleotide sequences different than their human orthologs and 1 bovine microRNA that humans cannot synthesize in postprandial plasma samples (SR Baier and J Zempleni, unpublished results, 2015). Also, the identities of the glycoproteins involved in the endocytosis of dietary microRNAs are unknown. The identification of these proteins is an area of active investigation in our laboratory. Moreover, we know little about the biological functions of exosome cargo other than microRNAs including various species of RNA, proteins, and lipids (24, 55). For examples, as of today 1160 exosomal proteins have been identified in human urine in the Urinary Exosome Protein Database (56), >40,000 proteins have been identified in human EVs (57), and 2107 exosomal proteins have been identified in bovine milk exosomes (58). We consider these uncertainties priority areas for future research.