Single Cell Sequencing of a Novel Model of Neonatal Bile Duct Ligation in Mice Identifies Etiology-Specific Macrophage Function Compared to Murine Biliary Atresia
Background & Aims: Macrophages (MF) play a role in neonatal etiologies of obstructive cholestasis, however, the role for precise MF subsets remains poorly defined.  We developed a neonatal murine model of bile duct ligation (BDL) to characterize etiology-specific differences in neonatal cholestatic MF polarization.  Approach & Results: Neonatal BDL surgery was performed on female BALB/c mice at 10 days of life (DOL) with sham laparotomy as controls.  Comparison was made to the Rhesus Rotavirus (RRV)-induced murine model of biliary atresia (BA).  Evaluation of changes at day 7 after surgery (BDL and sham groups) and murine BA (DOL14) included laboratory data, histology (H&E, anti-CD45 and anti-CK19 staining), flow cytometry of MF subsets by MHCII and Ly6c expression, and single cell RNA-sequencing (scRNA-seq) analysis.  Neonatal BDL achieved a 90% survival rate; mice had elevated bile acids, bilirubin, and alanine aminotransferase (ALT) versus controls (p < 0.05 for all).  Histology demonstrated hepatocellular injury, CD45+ portal infiltrate, and CK19+ bile duct proliferation in neonatal BDL.  Comparison to murine BA showed increased ALT in neonatal BDL despite no difference in histology Ishak score.  Neonatal BDL had significantly lower MHCII-Ly6c+ MF versus murine BA, however, scRNA-seq identified greater etiology-specific MF heterogeneity with increased endocytosis in neonatal BDL MF versus cellular killing in murine BA MF.  Conclusions: We generated an innovative murine model of neonatal obstructive cholestasis with low mortality.  This model enabled comparison to murine BA to define etiology-specific cholestatic MF function.  Further comparisons to human data may enable development of immune modulatory therapies to improve patient outcomes. Overall design: CITE-seq (Cellular Indexing of  Transcriptomes and Epitopes) on CD45+CD3-CD19-Siglec-F-Ly6g-  cells from neonatal BDL, BA and control mouse models.