S1P/S1PR4 promotes the differentiation of CD8+ tissue-resident memory T cells aggravating bile duct injury in biliary atresia

Abstract

Background & aims: Biliary atresia (BA) is a severe neonatal cholangiopathy characterized by progressive inflammation and fibrosis. We aimed to systematically investigate the pathogenesis of BA using integrated multi-omics.

Methods: We performed multi-omics analysis of BA and control livers. CX3CR1⁺ CD8⁺ effector T (Teff) cells were isolated via flow cytometry and subjected to functional assays, including sphingosine-1-phosphate (S1P)-gradient Transwell migration, tissue-resident memory T (TRM) differentiation, and cholangiocyte co-culture apoptosis analysis. We then studied the effect of sphingosine-1-phosphate receptor 4 (S1PR4) inhibition in mice with rhesus rotavirus (RRV)-induced BA, validating our findings through histopathology, flow cytometry, and serum biochemistry.

Results: Multi-omics data showed that sphingolipid metabolism was pathologically activated in the S1 subtype of BA, a molecular subtype marked by abnormal immune inflammation and poor prognosis. Single-cell RNA profiling identified S1PR4 as primarily expressed in CX3CR1⁺ CD8⁺ Teff cells. In vitro, S1P/S1PR4 signaling promoted CX3CR1⁺ CD8⁺ Teff migration and facilitated their differentiation into CD8⁺ TRM cells. Co-culture of CD8⁺ TRM cells with cholangiocytes induced apoptosis. In vivo, S1PR4 inhibition alleviated liver inflammation and fibrosis by limiting CD8⁺ TRM accumulation.

Conclusions: We identified an S1 subtype of BA characterized by dysregulated immune pathways and poor prognosis. S1P/S1PR4 signaling promotes cholangiocyte injury by driving CX3CR1⁺ CD8⁺ Teff migration and their differentiation into apoptosis-inducing CD8⁺ TRM cells.

Impact and implications: Our study addresses a critical knowledge gap in biliary atresia (BA) pathogenesis by showing that sphingolipid-driven CD8⁺ TRM differentiation through S1P/S1PR4 signaling worsens bile duct injury, establishing a mechanistic link between immunometabolic dysregulation and BA progression. These findings are particularly relevant to pediatric hepatologists and immunologists, as they identify those with the S1 molecular subtype - characterized by poor prognosis and immune hyperactivation - as a high-risk population warranting precision intervention. In preclinical BA mouse models, S1PR4 inhibition (CYM50358) reduced biliary obstruction and improved survival, supporting its prioritization for clinical trials, especially in S1-subtype BA.

Keywords: Biliary atresia; CD8(+)TRM; CX3CR1(+)CD8(+)Teff; Multi-omics; S1P; S1PR4.