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

Background: 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.

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

Methods: scRNA-seq was performed on ileal cells from 8-week-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.

Results: A total of 32,076 ileal cells were identified, including six 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. Co-expression of β-carotene oxygenase 2 (Bco2) and interleukin 18 (Il18) occurred across enterocytes, stem cells, and goblet cells, whereas non-canonical 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.

Conclusions: 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.