Allergic disease often begins in early life, and the increased prevalence has been linked to reduced microbial exposure and dietary changes associated with modern lifestyle. These changes in environmental stimuli are thought to negatively influence immune maturation and thereby increase the risk of allergic sensitization. Supplementation with probiotics and ω-3 polyunsaturated fatty acids (PUFAs), have been proposed as strategies to mitigate the lack of stimuli, promoting immune tolerance and preventing subsequent allergic disease in the infant. Human breast milk contains several immune relevant components, including extracellular vesicles (EVs) and microRNAs (miRNAs), which may mediate vertical immune signaling from the mother to baby, contributing to infant immune maturation.

This thesis is based on a randomized placebo-controlled study, where pre- and postnatal supplementation with the probiotic bacteria Limosilactobacillus (L) reuteri and/or ω-3 PUFAs was given from gestational week 20 until delivery (L. reuteri) and ω-3 three months postpartum. After birth, the child continued with L. reuteri for a year and was exposed to ω- 3 through breastfeeding. The overall aim was to study human milk-derived EVs and miRNAs in relation to infant immune maturation, and to investigate how the supplementations modulate these breast milk components.

In Paper I, we show that several breast milk miRNAs moderately correlate with the proportions of resting and activated regulatory T cells in infants at 6 and 24 months of age, suggesting a potential role for milk-derived miRNAs in tolerance development. However, maternal supplementation did not alter the relative expression of 24 immune-related miRNAs in colostrum or in mature milk.

Following this, we evaluated the impact of milk sample handling on EVs and their miRNA cargo. In Paper II, Frozen milk samples had comparable EV characteristics and miRNA expression as fresh samples, supporting their use in large-scale studies with biobank materials. However, casein removal with sodium citrate seem to affect some EV characteristics between fresh and frozen samples, highlighting the importance of methodological considerations in EV research.

In Paper III, we demonstrate that there are effects of maternal supplementation on expression of EV-associated miRNAs in breast milk, but these effects seem to depend on the maternal allergy status. Also, miR-223-3p was higher in milk-EVs from allergic compared to non-allergic mothers, without any supplemental interactions. Thus, both maternal allergy and dietary interventions might shape aspects of the miRNA composition in milk-derived EVs, potentially influencing infant immune maturation and allergy risk.

Finally, in Paper IV, we provided an in-depth proteomic characterization of milk-derived EVs, revealing that surface-associated proteins were enriched in immune-related functions, whereas luminal cargo proteins were primarily associated with intracellular processes and exosome biogenesis. The results suggest that milk-derived EVs originate from multiple cellular sources, both the mammary gland and immune cells, and that EV surface proteins may play key roles in vertical immune communication.

Together, these findings support the concept where human milk-derived EVs and their miRNA cargo are influenced both by maternal allergy status and dietary interventions. Furthermore, milk EVs and their cargo may contribute to early-life immune programming, potentially affecting tolerance development and allergy risk in the infant.