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 (Km) = 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 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.

Keywords: endocytosis; extracellular vesicles; intestinal cells; milk exosomes; uptake.

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.

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.

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.

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;.