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

Overview

This prospective observational study aims to identify and characterize immunoregulatory microRNAs (miRNAs) associated with regulatory T cells (Tregs) in human breast milk and to evaluate their temporal expression patterns. Breast milk samples will be collected longitudinally, including colostrum, transitional milk (days 10-15), and mature milk at 1, 4, 6, and/or 9 months postpartum. The study will investigate miRNAs that may influence the development and function of Treg cells and assess changes in their expression over time. In addition, a single blood sample will be obtained from infants during routine clinical visits (6-9 months postpartum) to analyze Treg-related miRNAs and target genes and to evaluate their relationship with breast milk components. The study also aims to explore the association between breast milk miRNAs, Treg-related immune mechanisms, and the development of 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.