The transcriptomic immune profiling across the different Ileal Layers Highlights the Relevant Role of Neutrophils and the active involvement of Healthy Mucosa in Human Crohn’s Disease

Background and aims: Crohn's disease (CD) is a chronic inflammatory disorder characterized by discontinuous and transmural inflammation of the gastrointestinal tract. While most studies have focused on mucosal immunity and individual immune cell subsets, little is known about the coordinated immune activity across the different ileal layers. This study aimed to characterize and compare, for the first time, the transcriptomic and immune landscape of the mucosa, submucosa, and serosa in diseased and adjacent healthy ileal tissue from CD patients. Methods: Matched healthy and damaged ileal samples from 17 CD patients were dissected into three layers. Expression of 579 immune-related genes was quantified using the NanoString nCounter® GX Human Immunology V2 kit. Results: Distinct transcriptional profiles were found between diseased and healthy tissues across all layers, with the mucosa showing the most pronounced dysregulation. Damaged mucosa exhibited upregulation of innate immunity-related genes, while neutrophil-associated transcripts and chemokines predominated in the serosa. Interestingly, healthy ileal tissue mirrored, layer by layer, the same distribution of innate (neutrophils) and adaptive (T and B) immune cells as diseased areas. A vertical immune gradient from mucosa to serosa was identified in both tissue types. Conclusions: This study provides novel insights into the compartmentalized immune architecture of the ileum in CD. The findings highlight a potential role of healthy tissue and serosal neutrophils in CD pathogenesis and progression. Layer-resolved transcriptomic profiling could aid early diagnosis, reveal new biomarkers, and support the development of personalized therapeutic strategies targeting specific ileal compartments.