Single-cell transcriptomics reveals that human milk feeding shapes neonatal immune cell interleukin signaling pathways in a non-randomized clinical trial
Objective: The study aimed to characterize circulating immune cell subpopulation gene expression in human milk-fed (HMF) compared to cow's 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 to 3.5 months old and enrolled in a non-randomized clinical trial. Single-cell RNA sequencing (scRNA-seq) 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 (DEGs) 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, while monocytes were higher in FF infants than in HMF infants (22.6 ± 10.7 vs 8.3 ± 5.6; P = 0.0314). In addition, innate and adaptive immune cells from FF infants exhibited a higher activation state compared to HMF infants. We identified 16 distinct cell subsets from the major immune cell types: three monocyte subsets, four NK subsets, two B cell subsets, and seven 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.  Conclusion: These findings suggest that human milk downregulates peripheral immune cell cytokine transcriptional signatures linked to allergic inflammation and infection relative to formula feeding. Overall design: Peripheral blood mononuclear cells isolated from human milk-fed or formula-fed infants.