Integrated single-cell and bulk-tissue transcriptomic analysis reveals an epithelial-stromal inflammatory microenvironment in biliary atresia

Background: Biliary atresia is a severe fibroinflammatory cholangiopathy of infancy. Its pathological process involves biliary epithelial injury, immune activation, and intrahepatic stromal remodeling, but the cellular interactions linking these processes remain incompletely defined. Using public single-cell and bulk-tissue transcriptomic cohorts, this study systematically characterized disease-associated alterations across liver cell populations in biliary atresia and further evaluated their consistency in independent cohorts. Methods: Public single-cell liver data (GSE176189) and three independent bulk-tissue cohorts (GSE122340, GSE300457, and GSE46960) were analyzed. The single-cell cohort contained 119,538 cells after filtering, including 89,118 cells from biliary atresia samples and 30,420 cells from choledochal cyst samples. Disease-associated genes were identified within major cell populations, used to construct cell-origin microenvironment signatures, and evaluated across bulk cohorts. Results: Broad cell-type annotation identified cholangiocytes, macrophages, T cells, endothelial cells, fibroblasts, and other liver-resident or immune populations. Based on biliary atresia-upregulated genes from five key cell populations, a 219-gene pathological microenvironment signature was constructed. Cell-type-specific enrichment analysis highlighted immune, antiviral, migration, and matrix-related biological processes, although the fibroblast component was based on fewer genes and was interpreted cautiously. In three bulk-tissue cohorts, the composite microenvironment score was elevated in biliary atresia samples, with areas under the receiver operating characteristic curve of 0.993, 0.782, and 0.798, respectively. Cell-communication analysis suggested candidate MIF, GALECTIN, CLEC, PTN, NOTCH, and CXCL signaling axes, which require spatial or experimental validation. Conclusion: These findings support a multi-cellular inflammatory microenvironment in biliary atresia liver and nominate epithelial-immune-stromal communication axes for future validation, while recognizing that the single-cell comparison used choledochal cyst rather than normal liver as the control condition.