Human breast milk-derived exosomes attenuate cell death in intestinal epithelial cells

Abstract

Human breast milk has been shown to reduce the incidence of necrotizing enterocolitis (NEC). Breast milk has many components (immunoglobulins, proteins, fat, and, of recent interest, exosomes), but the specific component that affords protection against NEC is not known. Exosomes are small-nanometer vesicles that are rich in protein, lipid, and microRNA. Here, we hypothesized that human breast milk-derived exosomes can protect intestinal epithelial cells (IECs) from cell death. Human breast milk was collected, separated using ultracentrifugation, and quantified using NanoSight tracking analysis. Purified exosomes were added to IECs that had been treated with varying concentrations of H₂O₂. Cells were then incubated overnight with the human breast milk-derived exosomes and assessed for cell viability. Western blot analysis showed that both clathrin and CD81 were present in the purified sample. Oxidative stress using H₂O₂ caused a 50% decrease in cell viability and human breast milk-derived exosomes had a protective effect in IECs. In the presence of H₂O₂, exosomes had a statistically significant protective effect. The protection seen by human breast milk-derived exosomes was not attenuated by cycloheximide. Thus, human breast milk-derived exosomes allow IECs to be protected from oxidative stress, but the mechanism is still not clear. Exosomes derived from human breast milk are an attractive treatment concept for children with intestinal injury.

Keywords: Necrotizing enterocolitis; breast milk; exosomes; intestinal epithelial cells.

Figure 1.

Characteristics of human breast milk. (a) Average size of exosome particles plotted as the number of particles per ml. (b) Western blot representation of CD81 and clathrin represented at approximately 26 and 180 kDA, respectively. (c) A 10⁹ sample of exosomes was prepared for EM. Samples for analysis were processed at the UAB Cryo-EM facility. The core EM facility uses an FEI Tecnai F20 200 kV field-emission gun transmission electron microscope. Images were collected using a 4 k × 4 k-pixel Gatan charge-coupled device camera.

Figure 2.

Viability of IEC-6 cells treated with increasing doses of human breast milk-derived exosomes. *P < 0.05 using post hoc Dunnett's test. Bright-field images of IEC-6 cells were taken at 10× magnification. (a) Control, (b) exosome concentration of 0.1 µg, (c) exosome concentration of 1 µg, (d) exosome concentration of 10 µg.

Figure 3.

Viability of IEC-6 cells treated with 100 μM H₂O₂ with increasing concentrations of human breast milk-derived exosomes.

Figure 4.

Viability of IEC-6 cells treated with 200 μM H₂O₂ with increasing concentrations of human breast milk-derived exosomes. *P < 0.05, **P < 0.01 using post hoc Dunnett's test.

Figure 5.

Viability of IEC-6 cells treated with 100 or 200 μM H₂O₂ with increasing concentrations of human breast milk-derived exosomes and cycloheximide. **P < 0.01 using post hoc Dunnett's test.