S1P/S1PR4 Promotes the Differentiation of CD8+TRM Cells Aggravating Bile Duct Injury in Biliary Atresia

BACKGROUND AND AIMS: Biliary atresia (BA) is the most common and serious neonatal biliary disease, characterized by progressive biliary inflammation and fibrosis. We aim to comprehensively and systematically investigate the complex pathological process of BA from multiple molecular dimensions, combining transcriptomics, proteomics, metabolomics, and single-cell RNA sequencing. METHODS: Integration of multi-omics data detected and localized aberrant metabolite regulation in immune cells of BA liver tissue. By flow cytometry sorting of specific immune cell subsets and in vitro experiments, we verified the role and mechanism of the aberrant metabolite signaling pathway in the progression of BA. In vivo experiments confirmed the effect of inhibiting the aberrant signaling pathway in alleviating bile duct injury and fibrosis. RESULTS: Based on multi-omics data, the sphingolipid metabolism pathway was abnormally activated in the S1 subtype of BA, a molecular subtype characterized by abnormal immune inflammation and poor prognosis. By integrating single-cell RNA profiling, Sphingosine-1-Phosphate Receptor 4 (S1PR4), a receptor of Sphingosine-1-Phosphate (S1P), was primarily expressed in CX3CR1+CD8+effector T (Teff) cells. In vitro, S1P/S1PR4 signaling promoted the migration of CX3CR1+CD8+Teff cells, and facilitated the differentiation of CX3CR1+CD8+Teff cells into CD8+ tissue-resident memory T (TRM) cells. Co-culture of CD8+TRM cells and cholangiocytes could induce the apoptosis of cholangiocytes. In vivo, S1PR4 inhibitor could alleviate liver inflammation and fibrosis by inhibiting the accumulation of CD8+TRM cells. CONCLUSIONS: Our study revealed that activation of the S1P/S1PR4 signaling pathway promotes the differentiation of CD8+ TRM cells, which subsequently induces cholangiocyte injury. Targeting the activation of the S1P/S1PR4 signaling pathway is a promising therapeutic strategy for BA, especially for patients with the S1 subtype.