Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder characterized by discontinuous and transmural inflammation of the gastrointestinal tract. Most previous studies focus on mucosal immunity and mainly on individual immune cells or specific subsets thereof, often through longitudinal studies. However, the immune system operates as a complex and dynamic network, in which immune cells interact extensively with one another and with more molecular and environmental factors shared between the different ileal layers and adjacent non-damaged tissue; but currently this part of CD development remains poorly understood. In this scenario, this study aimed to characterize and compare for the first time the transcriptomic and immune landscape across the different ileal layers (mucosa, submucosa, and serosa) of both diseased and adjacent healthy tissue from CD patients. Methods Matched healthy and damaged ileal samples obtained from patients with diagnosed CD were dissected into mucosa, submucosa, and serosa layers. The relative mRNA copies number of 579 human genes were quantified as per the manufacturer’s recommendations (NanoString Technologies, USA) using a nCounter® GX Human Immunology V2 kit (NanoString Technologies, USA). Results The gene expression profiles differed between diseased and adjacent healthy tissues in the different three layers, with the mucosa showing the most significant transcriptional dysregulation. In detail, upregulation of innate immunity-related genes was observed in the damaged mucosa, while neutrophil-related transcripts and chemokines were prominent in the damaged serosa. Notably, for the first time, we documented that the healthy ileal parts of the CD patients mirror, layer by layer, the same functional distribution of innate (neutrophils) and adaptative (T and B cells) immune cells in the ileum, highlighting a potential involvement of the healthy gut in the CD pathogenesis. Mainly, we documented that neutrophils were highly represented in the serosa ileum of both parts (diseased and healthy). Finally, vertical analysis revealed a gradient of immune activity from mucosa to serosa in both healthy and diseased tissue. Conclusion This study provides novel insights into the ileal and compartmentalized immune responses in human CD. Our findings highlight the potential and currently underappreciated role of healthy ileal tissue and serosal neutrophils in CD development and, additionally suggest that resolved transcriptomics can uncover new biomarkers and therapeutic targets. Understanding the layered immune architecture of the ileum may refine the existing models of CD pathogenesis, supporting early diagnosis and guiding personalized treatment strategies. Conflict of interest: Dr. Nannini, Giulia: No conflict of interest Giudici, Francesco: No conflict of interest Fink, Dorian: No conflict of interest Scaringi, Stefano: No conflict of interest Cei, Francesco: No conflict of interest Bertorello, Sara: No conflict of interest Staderini, Fabio: No conflict of interest Cianchi, Fabio: No conflict of interest Niccolai, Elena: No conflict of interest Amedei, Amedeo: No conflict of interest
Nannini et al. (Thu,) studied this question.