Single-cell transcriptomics reveals that human milk feeding shapes neonatal immune cell interleukin signaling pathways in a nonrandomized clinical trial

Authors: SALINAS, MICHAEL - Texas A&M University; MULAKALA, BHARATH - Texas A&M University; DAVIDSON, LAURIE - Texas A&M University; CAI, JAMES - Texas A&M University; DONOVAN, SHARON - University Of Illinois; CHAPKIN, ROBERT - Texas A&M University; Yeruva, Venkat

Submitted to: The American Journal of Clinical Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2025
Publication Date: 4/26/2025
Citation: Salinas, M.L., Mulakala, B.K., Davidson, L.A., Cai, J.J., Donovan, S.M., Chapkin, R.S., Yeruva, V. 2025. Single-cell transcriptomics reveals that human milk feeding shapes neonatal immune cell interleukin signaling pathways in a nonrandomized clinical trial. The American Journal of Clinical Nutrition. https://doi.org/10.1016/j.ajcnut.2025.04.024.
DOI: https://doi.org/10.1016/j.ajcnut.2025.04.024

Interpretive Summary: Clinical and epidemiological studies support health benefits of human milk feeding (HMF) compared to formula feeding (FF), including reduced upper respiratory tract and gastrointestinal tract infections during infancy, lower rates of necrotizing enterocolitis, reduced risk of allergy, and decreased incidence of obesity in childhood. Therefore, the World Health Organization recommends exclusive human milk feeding for the first 6 months of life and continued breastfeeding for two years and beyond. Although previous studies have documented the benefits of exclusive human milk feeding, the mechanisms by which human milk contributes to the development of the infant’s innate and adaptive immune function are still unknown. Thus, in this study, we report the effects of human milk vs formula on the ontogeny of the systemic immune system in healthy newborn infants. We demonstrated that human milk feeding shapes cell signaling pathways in peripheral immune cell subsets that likely link to lower allergic inflammation and infection.

Technical Abstract: Background: Several studies have indicated the benefits of human milk feeding to infants however, mechanisms behind positive health outcomes have not been investigated. Objectives: The study aimed to characterize circulating immune cell subpopulation gene expression in human milk-fed (HMF) compared with cow milk formula-fed (FF) infants using single-cell transcriptomics. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy HMF (n ¼ 6), and FF (n ¼ 3) infants who were 3–3.5 mo old and enrolled in a nonrandomized clinical trial. Single-cell RNA sequencing was used to generate a PBMC atlas and evaluate gene expression in immune cell subsets. Differential expression analysis was performed on each cell type independently after clustering the cells by similar marker gene expression using the scGEAToolbox. Differentially expressed genes were subjected to pathway analyses using an online functional enrichment analysis program. Results: The relative abundance (%) of T and B lymphocytes, natural killer (NK) cells, and plasmacytoid dendritic cells were similar, whereas monocytes were higher in FF infants than in HMF infants (22.6 _ 10.7 compared with 8.3 _ 5.6; P ¼ 0.0314). In addition, innate and adaptive immune cells from FF infants exhibited a higher activation state compared with HMF infants. We identified 16 distinct cell subsets from the major immune cell types: 3 monocyte subsets, 4 NK subsets, 2 B cell subsets, and 7 T cell subsets. Transcriptional profiles of each peripheral innate and adaptive immune cell subtype varied between HMF and FF infants. Pathway enrichment analysis of cell-specific transcriptional changes within subsets of major cell types revealed that the interleukin (IL)-4/IL-13 signaling pathways were upregulated in FF infants relative to HMF infants. Conclusions: These findings suggest that human milk downregulates peripheral immune cell cytokine transcriptional signatures linked to allergic inflammation and infection relative to formula feeding.