CD14+CD16- monocytes exhibit NF-κB hyperactivation in biliary atresia: Clinical association and murine therapeutic validation.

BACKGROUND: Classical CD14+CD16- monocytes are elevated in biliary atresia (BA); however, their specific role in bile duct injury and the underlying regulatory mechanisms remain unclear. This study aimed to define their contribution to BA pathogenesis, focusing on the NF-κB signaling pathway. METHODS: Liver tissues and blood samples from patients with BA and controls were analyzed by single-cell RNA sequencing, flow cytometry, and immunofluorescence. A rhesus rotavirus-induced BA mouse model was used for anti-Ly6C monocyte depletion and NF-κB inhibition (dehydroxymethylepoxyquinomicin). Transcriptomic profiling and cytokine analysis revealed key molecular mechanisms. RESULTS: Classical monocytes were significantly enriched near the damaged bile ducts in patients with BA and positively correlated with liver injury severity. These monocytes exhibited NF-κB hyperactivation, marked by the upregulation of TNF, IL-1β, Cxcl2, and NLRP3 inflammasome components. RNA-seq revealed BA-specific monocyte clusters with enriched NF-κB signatures. The depletion of classical monocytes (anti-Ly6C) in rhesus rotavirus-induced BA mice reduced biliary inflammation, restored bile duct patency, and improved survival. Pharmacological NF-κB inhibition (dehydroxymethylepoxyquinomicin) similarly attenuated inflammation and liver dysfunction and improved survival in rhesus rotavirus-induced BA mice. CONCLUSIONS: Classical CD14+CD16- monocytes are spatially enriched and exhibit NF-κB hyperactivation in BA. Targeting these cells or their NF-κB axis represents a promising therapeutic strategy to mitigate disease progression.