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.

The prevalence of allergies has increased worldwide, especially in affluent countries. In clinical trials, pre- and postnatal supplementation with ω-3 fatty acids or probiotics has been shown to reduce the incidence of allergies in children. However, the underlying mechanisms are not fully understood and might be mediated through changes in immune mediators in breast milk or infant saliva.

This thesis aimed to investigate the effects of pre- and postnatal supplementation with ω-3 polyunsaturated fatty acids (PUFAs) and Limosilactobacillus (L) reuteri on immune mediators, including immunoglobulin A (IgA) in breast milk and infant saliva, human milk oligosaccharides (HMOs), and milk-derived extracellular vesicle microRNAs (EV-miRNAs). In addition, we aimed to investigate the effects of the population demographic characteristics, such as maternal allergy, on these mediators.  We also aimed to investigate the effects of freezing and sodium citrate (SC), as a casein-micelle dissolving agent, on milk-derived EV characteristics and their miRNA contents.

All samples and data included in this thesis are derived from an ongoing allergy prevention, randomized clinical trial. In this trial, at gestational week 20, pregnant women were randomized into four supplementation groups receiving: ω-3 PUFA + L. reuteri (OL), ω-3 PUFA + Placebo (OP), Placebo + L. reuteri (PL), or Placebo + Placebo (PP). The ω-3 PUFA supplementation continued until 3 months postpartum, reaching the baby through breastfeeding. In contrast, the probiotic supplementation was stopped for mothers after birth and given directly to the infants for the first year of life.

In Paper I, breast milk total IgA (TIgA) levels tended to be higher in the OP group than the PP group. In the subpopulation of non-allergic mothers, higher TIgA and secretory IgA (SIgA) levels were observed in the active supplementation groups (OL, OP, and PL) than the PP group, while no effects were found among allergic mothers. In the infant saliva, the TIgA and SIgA levels were higher in the OP than in the PP group. In Paper II, supplementation with ω-3 PUFAs decreased the HMO diversity over time, from colostrum till 3 months postpartum. Furthermore, non-allergic mothers expressed significantly higher levels of several HMOs compared to allergic mothers. Additionally, breast milk IgA correlated positively with fucosylated and negatively with sialylated HMOs. In Paper III, we found no differences in EV characteristics or their miRNA cargo between fresh and frozen milk samples. In contrast, SC altered certain EV characteristics, but not their miR-148-3p levels. In Paper IV, active supplementation modulated different milk EV-miRNA expression levels among non-allergic and allergic mothers. Moreover, non-allergic mothers tended to have lower expression of miR-223-3p than allergic mothers.

In conclusion, this thesis demonstrates several immune modulatory effects of the supplements, including changes in IgA levels, HMO diversity, and EV-miRNA expression. The findings further indicate that maternal allergy modifies the supplementation effects on some of these outcomes.  In addition, freezing milk does not appear to affect EV characteristics or their miRNA content.