An Integrated Single-Cell Atlas of the Mouse Ileum Links Nutrient Metabolism with Epithelial and Immune Crosstalk

The ileum integrates nutrient absorption with mucosal immunity, yet its cell-type-specific functions remain poorly defined. Disruption of epithelial or immune pathways contributes to nutrient deficiency, Crohn's disease, and impaired barrier integrity. Single-cell RNA sequencing (scRNA-seq) provides the resolution needed to uncover epithelial differentiation and immune crosstalk that bulk approaches cannot resolve.

This study aimed to map ileal cellular heterogeneity and define epithelial differentiation, nutrient metabolism programs, and epithelial-immune interactions relevant to health and disease.

scRNA-seq was performed on ileal cells from 8-wk-old male C57BL/6J mice. Gene expression and clustering were analyzed using Seurat, with pseudotime trajectory, cell-cell communication, and pathway enrichment analyses applied to characterize intestinal dynamics.

A total of 32,076 ileal cells were identified, including 6 epithelial types and multiple immune populations. Enterocyte subclusters showed distinct nutrient-related functions: Ent_C1, C4, C7, and C8 were enriched for vitamin A absorption; Ent_C0, C1, C2 and C7 for carotenoid metabolism; and Ent_C1, C4, C7, C8, and C9 for vitamin B12 absorption. Coexpression of β-carotene oxygenase 2 (Bco2) and IL 18 (Il18) occurred across enterocytes, stem cells, and goblet cells, whereas noncanonical goblet cells exhibited high Bco2-Il18 expression together with signatures of fatty acid metabolism and stress responses. Plasmacytoid dendritic cells were identified as central regulators of immune-epithelial interactions.

This study provides the first integrated single-cell atlas of the mouse ileum, profiling both epithelial and immune cells and revealing nutrient metabolism programs and epithelial-immune crosstalk relevant to intestinal health and disease.