Background:
Although a gluten-free diet (GFD) is the standard therapy in celiac disease (CeD), a substantial proportion of patients have incomplete recovery. The cytokine and cell-cell signaling circuitry that drives villous damage and whether this circuitry persists despite GFD remains incompletely defined.
Methods:
We evaluated epithelial- immune interactions in the duodenal biopsies from patients with active CeD, GFD-treated CeD, and normal healthy controls were profiled using single-cell RNA sequencing and high-plex spatial transcriptomics (CosMx) and RNAscope. Analyses focused on cell-type resolution; ligand–receptor inference; spatial mapping of interferon-stimulated gene programs along the crypt–villus axis; and pseudotime-based assessment of epithelial differentiation trajectories.
Results:
Across platforms, IFN stimulated gene signature and incomplete restoration of epithelial enterocyte trajectories emerged as the dominant organizing signal in active CeD. Spatial profiling recovered major epithelial, stromal, and immune populations, confirming immune cell expansion and loss of mature APOA4+ enterocytes. Independently with RNAscope we confirmed strong IFNG production by T cells in the duodenal epithelial layer. This IFNG stimulated gene signature initiated in crypt stem and transit-amplifying cells and was strengthened along the crypt–villus axis, with highest expression in mature APOA4+ enterocytes. In GFD patients, antigen-presentation and interferon-response genes remain upregulated along the crypt–villus axis versus controls, even with full histologic recovery (Marsh 0). Epithelial trajectory analysis shows a reduction in mature-enterocyte density, and metabolic/nutrient-absorption gene programs remain decreased compared with controls despite reported avoidance of gluten.
Conclusion:
An IFN-centered epithelial–T cell circuit spatially reprograms villous enterocytes to promote epithelial injury in CeD. Residual interferon and metabolic defects despite GFD suggest molecular non-resolution beyond histology, informing biomarker development and therapeutic targeting of IFN-stimulated gene pathways to improve complete mucosal recovery.