The Glutamine Metabolic Switch Influences the Response of Neonatal Intestinal Macrophages to Breast Milk.

BACKGROUND & AIMS: Breast milk contains abundant glutamine and glutamate, yet their roles in neonatal gut health remain controversial. We aimed to investigate how these amino acids influence neonatal enteritis and the underlying mechanisms. METHODS: We used a neonatal rat necrotizing enterocolitis (NEC) model to test the effects of glutamine and glutamate given either before disease onset or during NEC progression. Previously published human neonatal NEC single-cell RNA psequencing (scRNA-seq) data were reanalyzed to assess immune cell composition and pathway activity. Bone marrow-derived macrophages (BMDMs) were used to examine inflammatory responses in vitro. Flow cytometry and transcriptomics were applied to analyze metabolic reprogramming of ileal macrophages. RESULTS: Glutamine and glutamate prevented NEC when administered prophylactically but worsened disease when given during NEC progression. scRNA-seq revealed enrichment of macrophages with activated inflammatory pathways in NEC ileum. In vitro, pretreatment with glutamine or glutamate reduced lipopolysaccharide-induced cytokine expression in BMDMs, whereas administration after stimulation had no benefit. In vivo, glutamine pretreatment decreased CD45(+)F4/80(+)CD11b/c(+)TNFα(+) macrophages, whereas treatment during NEC increased this subset. Integrated analyses showed NEC upregulated glutaminase (GLS) and downregulated glutamate dehydrogenase (GLUD1) in ileal macrophages. Pretreatment with glutamine or glutamate restored GLUD1 expression, favoring α-ketoglutarate (α-KG) rather than succinate metabolism. Supplementation with α-KG reversed glutamine-induced macrophage activation during NEC, whereas succinate abolished the protective effect of glutamine pretreatment. CONCLUSIONS: Glutamine and glutamate exert dual, context-dependent effects in neonatal enteritis by modulating macrophage glutamine metabolism. These findings provide mechanistic insight and suggest a basis for personalized glutamine supplementation strategies in neonatal gut disorders.