How probiotics and omega-3s reshape breast milk to prevent allergies

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.