Term-dependent Changes in Human Milk Extracellular Vesicle Composition and Immunomodulatory Effects in Vitro

Abstract

Human milk (HM) provides essential nutrition and immune protection for infants, with a varying composition based on gestational age (GA). The timing of birth – term or preterm, influences infants’ vulnerability to inflammatory diseases. Premature infants have an increased risk of developing necrotizing enterocolitis (NEC), a devastating intestinal disease. The contributing factors to the NEC inflammatory cascade include formula feeding and intestinal dysbiosis. HM contains membrane bound extracellular vesicles (EVs), which could prevent the development of NEC via its cargo that includes mRNA, microRNAs (miRs), and proteins. Since mothers deliver infants at varying GA with unique needs, I hypothesized that the contents and biological functions of HM EVs are distinct. I characterized the GA-specific HM EVs using proteomics and RNA sequencing, and investigated the ability of HM EVs to mediate inflammation in cell culture models. The results revealed significant differences in the cargo of EVs isolated from term and preterm HM. While the total number of proteins and mRNAs in EVs was similar between the two types of milk, functional analyses revealed enrichment of immunoregulatory contents in preterm EVs, whereas term EVs were enriched in metabolism-related processes. Similarly, miR profiling identified 12 significantly different miRs between term and preterm HM EVs, with both groups exhibiting functional enrichments in metabolism and immune-related pathways. GA-specific HM EVs had a further differential effect on human gut epithelial cells, primary macrophages, and in a leukemia monocytic cell line – THP-1 cells. Both term and preterm EVs upregulated secretion of epidermal growth factor, while distinct effects were measured for pro-inflammatory cytokines in human intestinal cells and macrophages. Only term EVs inhibited secretion of IL-6, expression of IL-1β following inflammatory stimuli, and apoptosis, while EVs from both groups inhibited IL-1β secretion in macrophages and inflammasome-induced cell death in THP-1 cells. These findings suggest that HM EVs, particularly those from term mothers, may regulate IL-1β, the inflammasome, and cell death, thereby promoting a tolerogenic response. The results highlight GA-specific differences in HM EV cargo and their specific functional effects. Targeting the EV-mediated mechanisms could support the development of specialized therapeutic interventions to mitigate the risk of NEC and optimize neonatal health.