Abstract Background Crohn’s disease (CD) is a highly heterogeneous inflammatory disorder of the gastrointestinal tract characterised by exacerbated immune responses to the gut microbiota in genetically susceptible individuals. While microbiota has been implicated in CD pathogenesis, it remains unclear whether distinct microbiome profiles contribute to disease heterogeneity by triggering diverse inflammatory responses in the intestinal mucosa. It is also unknown how the presence of specific microbiome profiles in CD associates with unique mucosal antibody repertoires. Methods We analysed the composition of intestinal mucus-embedded bacterial microbiota through 16S rRNA sequencing in a cohort of non-IBD controls and CD patients undergoing surgery (n = 108). We applied single-cell RNA sequencing (scRNA-seq) and single-cell V(D)J sequencing (scV(D)J-seq) on cryopreserved mucosal cells obtained from the same surgically resected ileal tissues used for bacterial characterisation (n = 22) to identify cellular modules and inflammatory pathways associated with the inflammation status and the microbiome composition. To further profile the mucosal B cell compartment we combined scRNA-seq with high-dimensional spectral flow cytometry of cryopreserved mucosal cells. Differential cellular composition results on study groups were also validated using immunofluorescence staining on paraffin embedded tissue slides. Results Our analysis revealed a significant reduction in alpha diversity within mucus-associated microbiota in CD (both in inflamed and not inflamed areas), along with a marked expansion of mucus-resident Escherichia coli (E. coli) in a subset of patients with ileal involvement. Building on these findings, we selected an exploratory cohort comprising non-IBD controls, and CD patients with or without mucus-embedded E. coli expansion for scRNA-seq and scV(D)J-seq sequencing of gut tissues. Our data showed that gut inflamed areas of CD patients display a reconfiguration of the B cell compartment and the emergence of unique inflammatory cell subsets across both immune and non-immune cellular compartments. Notably, CD patients with E. coli expansion showed distinct cellular modules driving inflammation within the myeloid compartment, alongside a profound remodelling of mucosa humoral responses marked by extensive IgG clonal expansion. Conclusion Altogether, our findings suggest that the expansion of E. coli in the gut mucosa might be promoting a unique inflammatory signature in CD and may play a role in shaping mucosal humoral immunity. Conflict of interest: Gumà Vique, Núria: No conflict of interest Sánchez-Gaona, Nerea: No conflict of interest Suárez-García, Leticia: No conflict of interest Tejedor Vaquero, Sonia: No conflict of interest Berenguer-Molins, Pau: No conflict of interest Perera-Bel, Julia: No conflict of interest Martinez Medina, Margarita: No conflict of interest Iglesias, Mar: No conflict of interest Cerutti, Andrea: No conflict of interest Márquez Mosquera, Lucía: None Magri, Giuliana: No conflict of interest
Vique et al. (Thu,) studied this question.