What mother is telling you: The messages encoded in milk-derived extracellular vesicles: Implications for the immune system and epithelial barrier function

Abstract

Human milk assists the development of the neonatal intestinal epithelial mucosa. Human milk is composed of many bioactive macromolecular structures and it is under active investigation what the contribution of these structures is to the development of a healthy epithelial mucosa. One understudied component of human milk are the extracellular vesicles (EV). EV are lipid bilayer enclosed particles secreted by cells for the purpose of intercellular communication. EV are around 200 nm in size and contain protein, lipids, and RNA. This cargo differs depending on the type of parental cell, cellular stress and signals from the environment. This dynamic incorporation of cargo makes them excellent vehicles for multicomponent signaling. In this thesis, we describe an isolation and biobanking protocol for the reliable separation of EV from other bioactive components in human milk, while preventing contamination of the initial EV population due to cell death. Using this protocol, we were able to perform in-depth characterization of the EV proteome with increased sensitivity of detection revealing proteins which had never before been allocated to milk. The EV proteome showed that EV likely originate from immune cells and (mammary) epithelial cells. In addition, EV were enriched for the biological functions ‘signal transduction’, ‘cellular communication’, and ‘transport’. This indicates that EV could contribute to the healthy development of the epithelial mucosa. To test this hypothesis functionally we used in vitro cell systems. Human milk-derived EV enhanced the reepithelialization rate of epithelial cells lining the gastrointestinal tract through p38 MAPK. Additionally, EV were involved in the maintenance of innate immune homeostasis, as they prevented activation of TLR3 and TLR9, but not TLR2 or TLR4, by their respective ligands. This could contribute to the colonization of the intestine by the microbiota. With respect to the adaptive immune system, EV prevented activation and proliferation of CD4 T cells, without inducing regulatory T cells. Importantly, T cells were still capable of mounting immune responses in the absence of milk EV after being exposed to milk EV. Therefore, we propose that milk EV create a temporary increased threshold for T cell activation, which could provide a favorable environment for the induction of oral tolerance. Strikingly, this threshold depended on the sensitization status of the mother, as T cell activation was less inhibited during exposure to EV from milk of allergic mothers compared to those exposed to EV from non-allergic mothers. Omics-based analysis of the protein and microRNA content of milk EV from allergic and non-allergic mothers revealed that the content is altered slightly between these groups. These molecules, as well as the impact on the healthy development of the neonatal epithelial mucosa are currently under active investigation. Taken together, we here provided direct evidence that maternal EV delivered via milk are multicomponent signaling devices which can interact with and modulate cells of the epithelial mucosa, and we speculate that these milk EV create a window for regulated development of the GI tract and the immune system.