In its second reporting period, FIBROTARGET has advanced the molecular, preclinical and imaging work needed to address intestinal fibrosis in Crohn's disease. The consortium analysed single cell RNA sequencing datasets from non inflamed, inflamed and stenotic tissues, and showed that inflammation and stenosis share overlapping transcriptional profiles, a finding that refined the biomarker strategy towards spatial and protein level validation. Spatial transcriptomics using the Xenium platform generated a high resolution dataset of more than 2.8 million cells and mapped 41 cell types, including 10 fibroblast subtypes, across intestinal layers. The project identified fibroblast populations linked to inflammation and tissue remodelling, including CHI3L1 positive, MMP1 positive, GREM1 positive and PDGFRA positive fibroblasts. In parallel, a multiplexed spatial proteomics strategy was prepared to validate candidate pro fibrotic markers at protein level. The pharmacological work established in vitro, ex vivo and in vivo platforms to evaluate C3, TYK2 and NLRP3 pathway inhibitors. NLRP3 inhibition reduced inflammatory cytokine production in ex vivo systems, but did not show therapeutic efficacy in chronic colitis models. TYK2 inhibition reduced collagen related responses in fibroblasts and showed promising effects in the chronic TNBS model, while C3 evaluation remains ongoing. WP3 completed data collection and statistical analyses to support magnetic resonance enterography models for stricturing Crohn's disease, and MOTILENT developed and validated an artificial intelligence segmentation and feature extraction pipeline. WP5 maintained stakeholder engagement through industry meetings at major gastroenterology congresses, outreach activities, a project training academy and public communication channels.
