Single-Cell Atlas Maps the Inflammatory Immune Landscape of Biliary Atresia Livers

Biliary atresia, a devastating inflammatory disease of the bile ducts in infants, remains one of the most challenging conditions in pediatric hepatology. Although the Kasai portoenterostomy can extend survival of the native liver, liver transplantation ultimately remains the only curative option for most patients. Now, a team of researchers in China has produced one of the most detailed single-cell views yet of what happens inside the liver during the disease, and their findings point to a coordinated storm of innate and adaptive immune activity, oxidative stress, and vascular injury that may drive the fibrotic destruction characteristic of the disorder.

The study, published in Frontiers in Pediatrics, was led by Xuelai Liu and colleagues at the Capital Center for Children’s Health, Capital Medical University, together with collaborators at the Capital Institute of Pediatrics and Heilongjiang Hospital of Beijing Children’s Hospital. The researchers performed single-cell RNA sequencing on liver tissue collected from two infants with biliary atresia undergoing Kasai operations, comparing the cellular landscape against histologically normal liver tissue obtained from a pediatric hepatoblastoma patient. After rigorous quality control, the analysis retained 24,882 individual cells, including 17,608 from the biliary atresia samples and 7,274 from the control tissue, allowing the team to profile ten major cell types spanning immune, epithelial, and stromal compartments.

Among the most striking findings was a profound expansion of pro-inflammatory neutrophil subsets within the biliary atresia liver. The researchers sub-clustered neutrophils into seven distinct subtypes, including mature, homeostatic, interferon-stimulated, and aged populations. Two interferon-stimulated gene-expressing subsets, designated Neu_ISG, and an aged subset, Neu_Aged, stood out for their transcriptional signatures suggesting enhanced production of reactive oxygen species, modulation of the extracellular matrix, and heightened cytokine activity. Genes such as COL17A1, VTN, ITIH2, and CHADL, all linked to fibrosis and extracellular matrix remodeling, were elevated in these subsets, while SLC25A34-AS1, an acute phase protein, and AGTR1, previously associated with immune activation, were upregulated in the Neu_ISG_01 population.

The aged neutrophil subset deserves particular attention. These cells, characterized by elevated expression of CXCR4, PI3, and SIGLEC10, are known from prior research to originate from the bone marrow, liver, and spleen, to exhibit high phagocytic activity during acute inflammation, and to induce CD4-positive T cells that aggravate inflammation and promote neutrophil extracellular trap formation. In the biliary atresia livers analyzed here, the proportion of aged neutrophils was markedly higher than in controls, suggesting that these cells may be actively contributing to the inflammatory processes that destroy bile ducts and drive fibrosis. Gene ontology analysis of the homeostatic neutrophil subsets in biliary atresia also revealed enrichment for responses to hydrogen peroxide and oxygen transport, mirroring the oxidative stress signatures seen in the interferon-stimulated populations.

Beyond neutrophils, the study uncovered a coordinated adaptive immune response. Among the nine NK and T cell subclusters identified, the proportions of CD4-positive effector memory T cells and CD4-positive regulatory T cells were both significantly expanded in biliary atresia. The effector memory T cells showed elevated expression of IL7R, CD40LG, ANXA1, FOS, and JUN, and their differentially expressed genes were enriched for pathways involving positive regulation of leukocyte activation and immune effector processes. Regulatory T cells, characterized by FoxP3, SAT1, IL2RA, and CTLA4 expression, displayed enrichment for cytokine production and T cell receptor signaling, consistent with previous reports of an activated Treg phenotype in the peripheral blood of biliary atresia patients.

Intriguingly, the researchers also found evidence of immune suppression within the CD8 compartment. Interferon-stimulated genes such as IFIT1, MX2, OAS1, STAT1, MX1, IFI6, and ISG15 were downregulated in CD8-positive effector memory T cells from biliary atresia livers, suggesting an inactivation of the interferon response in this cytotoxic population. The authors interpret this as a potentially protective mechanism, noting that prior work has shown upregulation of inhibitory T cell receptors on effector memory cells can limit T cell-mediated liver damage. Meanwhile, oxidative stress response terms, including response to reactive oxygen species and integrated stress response signaling, were enriched in CD8-experienced cells, and natural killer cells displayed pro-inflammatory characteristics with enrichment for cytokine-mediated signaling and response to oxidative stress.

The study’s most novel contribution may lie in its characterization of liver endothelial cells, which have received comparatively little attention in biliary atresia research. The team identified four endothelial subtypes: periportal liver sinusoidal endothelial cells, central venous liver sinusoidal endothelial cells, venous endothelial cells, and lymphatic endothelial cells. Periportal and lymphatic endothelial populations were significantly expanded in biliary atresia. Central venous endothelial cells showed signatures of apoptosis, oxidative phosphorylation, and response to oxidative stress, along with dysregulated proliferation and migration involving genes such as APOE, MAP4K4, CCL2, ITGB1, HMGB1, NR2F2, NRP2, EGFL7, and APLNR. These findings suggest that endothelial injury is not merely a passive consequence of inflammation but may actively contribute to disease progression.

To map how these cellular players communicate, the researchers employed CellChat, a computational framework for inferring ligand-receptor interactions from single-cell data. The analysis revealed a dominant CXCL12-CXCR4 signaling axis mediating neutrophil recruitment across endothelial cells, NK and T cells, B cells, mononuclear phagocytes, and neutrophils themselves. This chemokine pathway, well known from studies of myocardial infarction and hepatocellular carcinoma, had not previously been implicated in biliary atresia pathogenesis. The team also identified enhanced MIF-CD74/CXCR4 signaling between endothelial cells and immune populations, a pathway linked to macrophage survival and cytokine amplification in atherosclerosis, and VEGFB-VEGFR1 interactions between hepatocyte-cholangiocyte compartments and central venous and periportal endothelia, suggesting a role for compensatory angiogenesis in response to vascular injury.

Critically, the researchers did not rely solely on computational inference. They validated the CXCL12-CXCR4 interaction using multiplex immunofluorescence staining with tyramide signal amplification on additional liver samples from four biliary atresia patients and four controls. By quantifying adjacent CXCL12-positive endothelial cells and CXCR4-positive T cells across twenty random fields of view per group, they demonstrated statistically significant enrichment of these interacting pairs in biliary atresia tissue. They also cross-validated their findings against a public single-cell dataset (GSE176189) containing nine biliary atresia samples and three controls, confirming the widespread presence of MIF-CD74/CXCR4 interactions between non-parenchymal and immune cells, although the CXCL12-CXCR4 axis was not significantly enriched in that independent dataset, possibly reflecting heterogeneity in clinical samples or disease stage.

The authors are candid about the limitations of their exploratory study. With only two biliary atresia patients and one control in the sequencing cohort, and with the control tissue derived from histologically normal liver adjacent to hepatoblastoma rather than truly healthy pediatric liver, the findings should be interpreted as hypothesis-generating rather than definitive. The use of paracancerous tissue as a comparator reflects practical constraints, as truly normal pediatric liver tissue is exceedingly scarce, though the authors acknowledge that a field effect from the tumor cannot be entirely excluded. Nevertheless, the high-resolution atlas they have produced identifies pathogenic neutrophil subsets, cytotoxic lymphocytes, and injured endothelial cells as key cellular mediators orchestrating a hepatic microenvironment characterized by reactive oxygen species, unchecked inflammation, and fibrotic remodeling. By highlighting the CXCL12-CXCR4 and VEGFB-VEGFR1 axes as potential therapeutic targets, the study opens a path toward interventions that could modulate these signaling pathways and potentially alter the trajectory of a disease that currently offers few options beyond surgery and transplantation.

Subject of Research: Single-cell transcriptomic profiling of liver immune and endothelial cell alterations in biliary atresia

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

Article References: Liu, X., Liu, Z., Wang, H., Chen, Y., Ye, M., Li, X., Geng, Y., Cui, X., & He, F. (2026). Single-cell transcriptome atlas revealed liver immune features in biliary atresia. Frontiers in Pediatrics, 14, Article 1995199. https://doi.org/10.3389/fped.2026.1995199

Image Credits: AI Generated

DOI: 10.3389/fped.2026.1995199

Keywords: biliary atresia, single-cell RNA sequencing, neutrophils, CXCL12-CXCR4, liver endothelial cells, oxidative stress, liver fibrosis, T cells, NK cells, VEGFB-VEGFR1, pediatric liver disease, cell-cell communication