IL1β+ macrophages promote intestinal fibrosis in Crohn’s disease by secreting AREG to activate PI16+ fibroblasts

Excessive extracellular matrix deposition is a hallmark of intestinal fibrosis in Crohn’s disease (CD), with fibroblasts playing a central pathogenic role. This study aimed to elucidate how macrophages regulate fibroblast activity and contribute to intestinal fibrosis development in CD. Single-cell RNA sequencing (scRNA-seq) data from the Cleveland and MGH cohorts were integrated to identify IL1β+ macrophages and PI16+ fibroblasts involved in CD-associated fibrosis. Subsequently, above cell–cell interactions were validated by in vitro experiments such as flow cytometry, co-culture assays, ELISA and western blotting. The functional role of amphiregulin (AREG) and its neutralizing antibody AR37 was further assessed in the DSS-induced chronic intestinal fibrosis mice models. PI16+ fibroblasts exhibited the highest ECM score, indicating their prominent role in extracellular matrix production. IL1β+ macrophages showed the highest pro-fibrotic signature score, suggesting strong pro-fibrotic potential. The ligand–receptor analysis revealed AREG–EGFR interactions between IL1β+ macrophages and PI16+ fibroblasts. The scRNA-seq data identified IL1β+ macrophages as the predominant cellular source of pro-fibrotic factor AREG. Moreover, the in-vitro assays revealed that IL1β+ macrophages promoted proliferation and activation of PI16+ fibroblasts by secreting AREG. The animal experiment results revealed that AREG neutralizing antibody AR37 attenuated PI16 + fibroblasts expansion and intestinal fibrosis in DSS-induced chronic fibrosis models. IL1β+ macrophages promote intestinal fibrosis of CD by increasing proliferation and activation of PI16 + fibroblasts via AREG-EGFR signaling. Targeting the AREG-EGFR signaling could disrupt IL1β+ macrophages-PI16+ fibroblasts crosstalk to mitigate intestinal fibrosis progression in CD.