Thymic Stromal Lymphopoietin–Driven B-Cell Hyperactivation Impairs Intestinal Epithelial Homeostasis Predisposing to Enterocolitis in Hirschsprung Disease

Background & Aims Hirschsprung-associated enterocolitis (HAEC), a lethal complication of Hirschsprung disease (HSCR)—a prevalent congenital disorder with high morbidity—has a poorly understood pathogenesis. We investigated how aberrant immune-stromal crosstalk drives mucosal injury in HSCR. Methods Integrated single-cell RNA sequencing, spatial transcriptomics, bulk RNA-seq, and proteomics were performed using human HSCR tissues. Validation was conducted using murine HAEC ( Ednrb -/- ) models, flow cytometry, multiplex immunohistochemistry, in vitro B cell stimulation, epithelial co-culture, rescue experiments with the thymic stromal lymphopoietin TSLP inhibitor baicalein, and ex vivo intestinal organoid assays. Results We identified significant expansion and hyper-activation of B cells that co-localized with epithelial barrier defects in HSCR segments. Multi-omics analysis revealed an activated phenotype in these naive and memory B cells, characterized by the up-regulation of BLK, BLNK, and PCNA, which may contributes to impaired epithelial homeostasis. Crucially, we identified a pro-fibrotic TSLP+ fibroblasts enriched in HSCR. Spatial mapping and in vitro assays demonstrated that thymic stromal lymphopoietin (TSLP) potently activated B cells, synergizing with classical stimuli to enhance proliferation. Activated B cells, in turn, secreted pro-inflammatory factors, such as, ICAM1 and CCL5 , which induced epithelial apoptosis, suppressed proliferation, and disrupted tight junctions–effects that were amplified by TSLP co-stimulation. Activated B cells and epithelial dysfunction were conserved in HAEC ( Ednrb -/- ) mouse models. Co-culture and ex vivo organoid experiments confirmed that TSLP-activated B cells directly destabilized epithelial homeostasis; crucially, baicalein treatment rescued epithelial proliferation, attenuated apoptosis, and reduced inflammation-associated gene expression. Conclusions Our study reveals a novel pathogenic mechanism whereby TSLP instigates pathogenic B cell hyper-activation, thereby disrupting epithelial integrity and increasing susceptibility to HAEC. Inhibition of TSLP signaling with baicalein ameliorates epithelial injury, highlighting this axis as a promising therapeutic target for HAEC prevention in patients with HSCR.