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IntroductionMacrophages play an important role in disease progression of pediatric cholestatic liver disease, particularly biliary atresia (BA); however, the restorative versus pathogenic role for precise macrophage subsets remains poorly defined. We aimed to distinguish the transcriptional profiles and roles of defined macrophage subset(s) in murine BA.MethodsWe used multiparameter flow cytometry and RNA-sequencing analysis to profile recruited CD11bhiCD64+ hepatic macrophages by cell surface expression of MHCII and Ly6c in the Rhesus rotavirus (RRV)-induced murine model of BA versus saline controls. Modulation of macrophage numbers via intra-peritoneal injections of clodronate-loaded liposomes was performed to determine the association between macrophage numbers and histologic injury (Ishak score).ResultsLy6c+ macrophages demonstrated the greatest increase in numbers and percent of total macrophages in murine BA versus saline controls whereas MHCII+ macrophages decreased. Transcriptional changes in murine BA MHCII+ macrophages included reduced expression of the Kupffer cell gene signature, lower expression of genes involved in homeostatic processes, and increased expression of genes involved in inflammatory processes. Ly6c+ macrophages in murine BA showed increased expression for Hif1a and other genes involved in the cellular response to hypoxia. Among all subsets, the number of Ly6c+ macrophages exhibited the strongest correlation with severity of histologic liver injury by Ishak score.ConclusionsOur data identify specific pathways upregulated in Ly6c vs MHCII+ macrophage subsets in murine BA. Transcriptional similarities between murine BA and human cholestatic macrophages may enable translation of future mechanistic studies to new macrophage subset-specific therapies.

Abstract There are limited murine models of cholestatic liver diseases characterized by chronic biliary obstruction and resumption of bile flow. While murine bile duct ligation (BDL) is a well‐established model of obstructive cholestasis, current models of BDL reversal (BDLR) alter biliary anatomy. We aimed to develop a more physiologic model of BDLR to evaluate the time course and mechanism for resolution of hepatic injury after biliary obstruction. In the present study, we restored bile flow into the duodenum without disruption of the gall bladder after murine BDL using biocompatible PE‐50 tubing. After establishing the technique, overall survival for BDLR at 7 or 14 days after BDL was 88%. Sham laparotomy was performed in control mice. Laboratory data, liver histology, and hepatic gene expression were compared among BDL, BDLR, and controls. Laboratory evidence of cholestatic liver injury was observed at day 7 after BDL and rapid improvement occurred within 48 hr of BDLR. After BDLR there was also enhanced gene expression for the bile acid transporter Abcb11, however, bile duct proliferation persisted. Assessment of the immune response showed increased gene and protein expression for the general immune cell marker Cd45 in BDLR versus BDL mice suggesting a reparative immune response after BDLR. In summary, we have established a novel murine model of BDLR that allows for the investigation into bile acid and immune pathways responsible for hepatic repair following obstructive cholestasis. Future studies with our model may identify targets for new therapies to improve outcome in pediatric and adult cholestatic liver disease.

Introduction: Macrophages play an important role in disease progression of pediatric cholestatic liver disease, particularly biliary atresia (BA); however, the restorative versus pathogenic role for precise macrophage subsets remains poorly defined. We aimed to distinguish the transcriptional profiles and roles of defined macrophage subset(s) in murine BA. Methods: We used multiparameter flow cytometry and RNA-sequencing analysis to profile recruited CD11bhiCD64+ hepatic macrophages by cell surface expression of MHCII and Ly6c in the Rhesus rotavirus (RRV)-induced murine model of BA versus saline controls. Modulation of macrophage numbers via intra-peritoneal injections of clodronate-loaded liposomes was performed to determine the association between macrophage numbers and histologic injury (Ishak score). Results: Ly6c+ macrophages demonstrated the greatest increase in numbers and percent of total macrophages in murine BA versus saline controls whereas MHCII+ macrophages decreased. Transcriptional changes in murine BA MHCII+ macrophages included reduced expression of the Kupffer cell gene signature, lower expression of genes involved in homeostatic processes, and increased expression of genes involved in inflammatory processes. Ly6c+ macrophages in murine BA showed increased expression for Hif1a and other genes involved in the cellular response to hypoxia. Among all subsets, the number of Ly6c+ macrophages exhibited the strongest correlation with severity of histologic liver injury by Ishak score. Conclusions: Our data identify specific pathways upregulated in Ly6c vs MHCII+ macrophage subsets in murine BA. Transcriptional similarities between murine BA and human cholestatic macrophages may enable translation of future mechanistic studies to new macrophage subset-specific therapies. [ABSTRACT FROM AUTHOR]

Macrophages (MΦ) play a role in neonatal etiologies of obstructive cholestasis, however, the role for precise MΦ subsets remains poorly defined. We developed a neonatal murine model of bile duct ligation (BDL) to characterize etiology-specific differences in neonatal cholestatic MΦ polarization. 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 MΦ 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+ MΦ versus murine BA, however, scRNA-seq identified greater etiology-specific MΦ heterogeneity with increased endocytosis in neonatal BDL MΦ versus cellular killing in murine BA MΦ. 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 MΦ function. Further comparisons to human data may enable development of immune modulatory therapies to improve patient outcomes. [ABSTRACT FROM AUTHOR]

There are limited murine models of cholestatic liver diseases characterized by chronic biliary obstruction and resumption of bile flow. While murine bile duct ligation (BDL) is a well‐established model of obstructive cholestasis, current models of BDL reversal (BDLR) alter biliary anatomy. We aimed to develop a more physiologic model of BDLR to evaluate the time course and mechanism for resolution of hepatic injury after biliary obstruction. In the present study, we restored bile flow into the duodenum without disruption of the gall bladder after murine BDL using biocompatible PE‐50 tubing. After establishing the technique, overall survival for BDLR at 7 or 14 days after BDL was 88%. Sham laparotomy was performed in control mice. Laboratory data, liver histology, and hepatic gene expression were compared among BDL, BDLR, and controls. Laboratory evidence of cholestatic liver injury was observed at day 7 after BDL and rapid improvement occurred within 48 hr of BDLR. After BDLR there was also enhanced gene expression for the bile acid transporter Abcb11, however, bile duct proliferation persisted. Assessment of the immune response showed increased gene and protein expression for the general immune cell marker Cd45 in BDLR versus BDL mice suggesting a reparative immune response after BDLR. In summary, we have established a novel murine model of BDLR that allows for the investigation into bile acid and immune pathways responsible for hepatic repair following obstructive cholestasis. Future studies with our model may identify targets for new therapies to improve outcome in pediatric and adult cholestatic liver disease. [ABSTRACT FROM AUTHOR]