Breast milk-derived extracellular vesicles influence the development of the epithelial and immunological gastrointestinal barrier

Introduction: The gastrointestinal barrier is both a physical barrier and an immunological site, which needs to mature in neonates. Breast milk reduces intestinal permeability and supports neonatal immunity and oral tolerance. However, the specific contribution of the different milk components in these processes is not fully understood. Here, we investigated the potential role of breast milk-derived extracellular vesicles (EV) on components of the innate and adaptive immune system, as well as on the epithelial barrier. Methods: We developed a density gradient-based (optiprep) protocol for isolation of naturally occurring EV from breast milk. The function of breast milk EV was assessed in vitro by determining their effects on activation and cytokine production of adult and neonatal CD4+ T cells, on activation of Tolllike receptor (TLR) reporter cell lines and downstream signalling and on migration/proliferation of epithelial cells in a wound-healing assay. Results: Breast milk EV strongly inhibited aCD3/aCD28-induced T cell activation and proliferation and resulted in an overall reduced cytokine production. In this assay, high levels of CD45RA were maintained on T cells, indicating a more naïve and unresponsive state while no increase of FoxP3 regulatory T cells was observed. EV also inhibited triggering of intracellular TLR while activation of extracellular TLR2 and TLR4 was not inhibited. Finally, epithelial cells showed a substantially increased wound-healing capacity in the presence of breast milk EV. Summary/conclusion: Breast milk EV reduces immune activation by inhibiting both T-cell responses and endosomal TLR activation, while stimulating epithelial cell proliferation and migration. Taken together, our data suggest a role for breast milk EV in both the development of the intestinal barrier and the immune system.