Background: The immunomodulatory capacity of breast milk may partially be mediated by microRNAs (miRNA), small RNA molecules that regulate gene expression on a post‐transcriptional level and are hypothesized to be involved in modulation of immunological pathways. Here, we evaluate the expression of immune‐related miRNAs in breast milk after pre‐ and postnatal supplementation with Limosilactobacillus reuteri and omega‐3 (ω‐3) polyunsaturated fatty acids (PUFAs), and the association to infant regulatory T cell (Treg) frequencies.Methods: One‐hundred and twenty women included in a double‐blind, randomized, placebo‐controlled allergy intervention trial received L. reuteri and/or ω‐3 PUFAs daily from gestational week 20. Using Taqman qPCR, 24 miRNAs were analyzed from breast milk obtained at birth (colostrum) and after 3 months (mature milk) of lactation. The proportion of activated and resting Treg cells were analyzed in infant blood using flow cytometry at 6, 12, and 24 months.Results: Relative expression changed significantly over the lactation period for most of the miRNAs; however, the expression was not significantly influenced by any of the supplements. Colostrum miR‐181a‐3p correlated with resting Treg cell frequencies at 6 months. Colostrum miR‐148a‐3p and let‐7d‐3p correlated with the frequencies of activated Treg cells at 24 months, as did mature milk miR‐181a‐3p and miR‐181c‐3p.Conclusion: Maternal supplementation with L. reuteri and ω‐3 PUFAs did not significantly affect the relative miRNA expression in breast milk. Interestingly, some of the miRNAs correlate with Treg subpopulations in the breastfed children, supporting the hypothesis that breast milk miRNAs could be important in infant immune regulation.Trial registration: ClinicalTrials.gov‐ID: NCT01542970.

Objectives: Extremely preterm (EPT; gestational week < 28 + 0, < 1000 g) neonates are vulnerable to infections and necrotising enterocolitis, important contributors to mortality and morbidity. However, knowledge regarding their immune maturation remains limited. We here investigated the longitudinal development of functional T‐cell capacity in EPT infants.Methods: Peripheral blood mononuclear cells were isolated at 14th and 28th day (D) and at gestational week 36 + 0 (Gw36) from EPT infants, participated in a randomised, double‐blind, placebo‐controlled study of Lactobacillus reuteri DSM 17938 probiotic supplementation. Blood collected from 25 full‐term (FT) infants at D14 was used as control. The secretion of immune mediators was determined through comprehensive Luminex panels after stimulation with human T‐cell activator CD3/CD28 beads.Results: The levels of many mediators were low in EPT infants at D14, whereas the secretion of several chemokines was higher in EPT than in FT infants. Furthermore, Th2:Th1 cytokine ratios were higher in EPT than in FT infants. Progressively elevated secretion of, for example, IFN‐γ, TNF and IL‐17A in EPT infants was observed from D14 to D28 and then at Gw36. Elevated levels were observed for many proinflammatory mediators at D28. Probiotic supplementation or perinatal factors (e.g. clinical chorioamnionitis, preeclampsia and delivery mode) did not influence the cytokine and chemokine responses.Conclusions: Immune mediators induced by T‐cell activation in EPT infants were mainly reduced at D14 and Th2 skewed compared to those in FT infants, but mostly recovered at Gw36, indicating immune maturation. Increased proinflammatory responses at D28 may be related to the heightened risk of severe immune‐associated complications seen in EPT infants.