Single-cell transcriptome atlas revealed liver immune features in biliary atresia

While immune dysfunction is recognized as pivotal in biliary atresia (BA) pathogenesis, comprehensive characterization of disease-specific immune cell subtypes and stromal components remains incomplete. This study aims to define the altered hepatic immune-stromal landscape and aberrant intercellular communication networks associated with BA progression using single-cell transcriptomics.

We performed single-cell RNA sequencing (scRNA-seq) on BA liver tissues to profile immune and stromal cell populations. Cellular subclustering, differential gene expression, pathway enrichment, and cell-cell communication analyses were employed to identify potential pathogenic drivers and predicted possible signaling networks. Multiplex immunofluorescence staining was used to verify receptor-ligand interaction in BA liver.

BA livers exhibited profound expansion of pro-inflammatory neutrophil subsets - Neutrophils with interferon-stimulated genes (Neu_ISG), Aged neutrophils (Neu_Aged) - with transcriptional signatures suggestive of enhanced reactive oxygen species (ROS) production, extracellular matrix (ECM) modulation, and cytokine activity. Concurrent cytotoxic/immunoregulatory lymphocyte populations - Effector memory CD4⁺ T Cell (CD4⁺ TEM), Tregs, cytotoxic NK cells- showed upregulated inflammatory and oxidative stress pathways. Periportal and lymphatic liver endothelial cells (LECs) demonstrated significant perturbations. Central venous LECs displayed signatures of oxidative stress and dysregulated proliferation/migration. Cell-cell communication analysis highlighted a possibly dominant "CXCL12-CXCR4" axis mediating neutrophil recruitment across endothelia, NK&T/B, and myeloid cells. Enhanced "MIF-CD74/CXCR4" signaling between endothelia and immune cells, "VEGFB-VEGFR1" interactions between hepatocyte-cholangiocyte compartments and central venous/periportal endothelia were observed, suggesting that these interactions are associated with BA progression.

Our scRNA-seq atlas uncovers coordinated innate/adaptive immune responses and endothelial-stromal dysfunction that shape a hepatic niche in BA associated with ROS, unchecked inflammation, and fibrotic remodeling. Key cellular and molecular contributors include pathogenic neutrophils, cytotoxic lymphocytes, and injured endothelia, which interact via "CXCL12-CXCR4" and "VEGFB-VEGFR1" signaling axes. These findings from the present exploratory study point to potential novel therapeutic targets (e.g., neutrophil subsets, chemokine pathways) for mitigating BA pathogenesis.