Transcriptional profiling of pediatric cholestatic livers identifies three distinct macrophage populations

Abstract

Background & aims Limited understanding of the role for specific macrophage subsets in the pathogenesis of cholestatic liver injury is a barrier to advancing medical therapy. Macrophages have previously been implicated in both the mal-adaptive and protective responses in obstructive cholestasis. Recently two macrophage subsets were identified in non-diseased human liver; however, no studies to date fully define the heterogeneous macrophage subsets during the pathogenesis of cholestasis. Here, we aim to further characterize the transcriptional profile of macrophages in pediatric cholestatic liver disease. Methods We isolated live hepatic immune cells from patients with biliary atresia (BA), Alagille syndrome (ALGS), and non-cholestatic pediatric liver by fluorescence activated cell sorting. Through single-cell RNA sequencing analysis and immunofluorescence, we characterized cholestatic macrophages. We next compared the transcriptional profile of pediatric cholestatic and non-cholestatic macrophage populations to previously published data on normal adult hepatic macrophages. Results We identified 3 distinct macrophage populations across cholestatic liver samples and annotated them as lipid-associated macrophages, monocyte-like macrophages, and adaptive macrophages based on their transcriptional profile. Immunofluorescence of liver tissue using markers for each subset confirmed their presence across BA (n = 6) and ALGS (n = 6) patients. Cholestatic macrophages demonstrated reduced expression of immune regulatory genes as compared to normal hepatic macrophages and were distinct from macrophage populations defined in either healthy adult or pediatric non-cholestatic liver. Conclusions We are the first to perform single-cell RNA sequencing on human pediatric cholestatic liver and identified three macrophage subsets with distinct transcriptional signatures from healthy liver macrophages. Further analyses will identify similarities and differences in these macrophage sub-populations across etiologies of cholestatic liver disease. Taken together, these findings may allow for future development of targeted therapeutic strategies to reprogram macrophages to an immune regulatory phenotype and reduce cholestatic liver injury.

Identification of Liver Immune Microenvironment-Related Hub Genes in Liver of Biliary Atresia

“…However, sights were increasingly focused on the immune response to figure this puzzle out. Recently, Wang et al ( 7 ) and Taylor et al ( 29 ) used single-cell RNA sequencing to demonstrate the abnormality of immune cells in BA patients, which further proved the role of immune response in BA pathogenesis. Here, we utilized the public gene expression data set and deconvolution algorithm of BA liver to explore its immune-related genes and immune microenvironment.…”

Clinical implementation of single-cell RNA sequencing using liver fine needle aspirate tissue sampling and centralized processing captures compartment specific immuno-diversity

“…Liver macrophages have been characterized in studies from healthy livers 19 , livers with cholestatic liver disease 83 , and cirrhosis 20 , all of which required tissue digestion. The FNA sampling approach captured macrophages without the need for tissue digestion, minimizing processing time outside of the liver to preserve their in vivo transcriptional profiles.…”

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1

“…Tissue digestion and cell isolation for flow cytometry was performed as previously described [8]. Briefly, cardioperfusion was performed with PBS and whole liver tissue was finely cut into small pieces and added to a c-tube with 2.5 ml digestion buffer per tube composed of DNase I (Sigma), Liberase TL (Sigma), and HBSS (Gibco).…”