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

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 = 6), and FF (n = 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 = 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.