Thymic Stromal Lymphopoietin-Driven B-Cell Hyperactivation Impairs Intestinal Epithelial Homeostasis Predisposing to...

Hirschsprung-associated enterocolitis, a lethal complication of Hirschsprung's disease-a prevalent congenital disorder with high morbidity-has a poorly understood pathogenesis. We investigated how aberrant immune-stromal crosstalk drives mucosal injury in Hirschsprung's disease.

Integrated single-cell RNA sequencing, spatial transcriptomics, bulk RNA sequencing, and proteomics were performed using human Hirschsprung's disease tissues. Validation was conducted using murine Hirschsprung-associated enterocolitis (Ednrb^-/-) models, flow cytometry, multiplex immunohistochemistry, in vitro B-cell stimulation, epithelial coculture, rescue experiments with the thymic stromal lymphopoietin inhibitor baicalein, and ex vivo intestinal organoid assays.

We identified significant expansion and hyperactivation of B cells that colocalized with epithelial barrier defects in Hirschsprung's disease segments. Multiomics analysis revealed an activated phenotype in these naive and memory B cells, characterized by the upregulation of B lymphocyte kinase, B cell linker, and proliferating cell nuclear antigen, which may contribute to impaired epithelial homeostasis. Crucially, we identified profibrotic thymic stromal lymphopoietin+ fibroblasts enriched in Hirschsprung's disease. Spatial mapping and in vitro assays demonstrated that thymic stromal lymphopoietin potently activated B cells, synergizing with classical stimuli to enhance proliferation. Activated B cells, in turn, secreted proinflammatory factors, such as, ICAM1 and CCL5, which induced epithelial apoptosis, suppressed proliferation, and disrupted tight junctions-effects that were amplified by thymic stromal lymphopoietin costimulation. Activated B cells and epithelial dysfunction were conserved in Hirschsprung-associated enterocolitis (Ednrb^-/-) mouse models. Coculture and ex vivo organoid experiments confirmed that thymic stromal lymphopoietin-activated B cells directly destabilized epithelial homeostasis; crucially, baicalein treatment rescued epithelial proliferation, attenuated apoptosis, and reduced inflammation-associated gene expression.

Our study reveals a novel pathogenic mechanism whereby thymic stromal lymphopoietin instigates pathogenic B-cell hyperactivation, thereby disrupting epithelial integrity and increasing susceptibility to Hirschsprung-associated enterocolitis. Inhibition of thymic stromal lymphopoietin signaling with baicalein ameliorates epithelial injury, highlighting this axis as a promising therapeutic target for Hirschsprung-associated enterocolitis prevention in patients with Hirschsprung's disease.