Tregs and miRNAs in Breast Milk: Effects on Infant Food Tolerance

Breast milk contains microRNAs (miRNAs) that contribute to the maturation of the infant immune system by influencing immune cell development, inflammatory mediator secretion, and maintenance of immunological homeostasis. Certain miRNAs, such as miR-92 and miR-17, play a role in B- and T-cell maturation, while miRNA-10a is associated with regulatory T cells (Tregs), which can modulate food allergic responses. This study aims to identify immunoregulatory Treg-associated miRNAs in breast milk, particularly those involved in the development and function of Treg cells; to analyze their expression in colostrum, transitional milk, and mature breast milk; to investigate their effects on Treg cells; and to examine the relationship between food tolerance and miRNA-Treg interactions.

Laboratory analyses will be conducted on maternal breast milk and infant blood samples. Breast milk samples will be manually expressed between 09:00 and 14:00 into sterile collection containers, with 7-10 mL collected per session. Five milliliters of each sample will be transferred into Eppendorf tubes and transported on ice within 30 minutes for molecular analysis. Each sample will be aliquoted into sterile 2 mL cryotubes, flash-frozen, and stored at -80°C until RNA isolation. The remaining 2-5 mL of each breast milk sample will be transported on ice within 30 minutes for immunologic and cytometric analyses. Infant blood samples obtained during routine follow-up visits will be analyzed for Treg-associated miRNAs and their target gene expression to evaluate correlations with maternal breast milk miRNA profiles and infant immune tolerance.

All laboratory analyses will be conducted using standard equipment and procedures in certified Genetics and Immunology laboratories. Through this approach, the study will examine longitudinal changes in Treg-associated miRNAs in breast milk, their correlation with infant immune parameters, and their potential role in promoting immune tolerance and preventing food allergy development. The results are expected to provide insights into early-life immune programming and may inform novel therapeutic interventions and preventive strategies.

Inclusion criteria

• Lactating mothers and their infants attending the Child Health and Diseases Clinic at Istanbul University Faculty of Medicine, or the Child Health and Diseases Clinic at Esencan Hospital.
• Infants who are exclusively breastfed for the first 6 months.
• Mothers willing to provide breast milk samples at multiple time points (colostrum, transitional milk, and mature milk at 1, 4, 6, and/or 9 months postpartum).
• Infants aged 0-9 months, born at term (≥37 weeks gestation) with no major congenital anomalies or chronic illnesses.
• Mothers and infants who provide informed consent for participation in the study.

Exclusion criteria

• Infants who are not exclusively breastfed for the first 6 months.
• Infants with chronic illnesses or diagnosed immunodeficiency, metabolic, or gastrointestinal disorders.
• Mothers who are unable or unwilling to provide informed consent.
• Mothers with chronic illnesses or immunological disorders that may affect breast milk composition.