Abstract
Background & aimsLimited 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.
Highlights
Macrophages are a heterogeneous and plastic cell population that respond to environmental signals in various cholestatic liver diseases [1,2,3]
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
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
Summary
Macrophages are a heterogeneous and plastic cell population that respond to environmental signals in various cholestatic liver diseases [1,2,3]. In the setting of liver injury, tissue-resident macrophages can adopt a pro-inflammatory state and additional monocyte-derived macrophages may be recruited from the peripheral circulation to the liver [5,6,7,8]. This leads to a heterogeneous population of macrophages that may have distinct functions in disease. C-C chemokine receptor type 2 (CCR2)-mediated recruitment of monocyte-derived macrophages in a murine model of primary sclerosing cholangitis has been implicated in the mechanism of liver injury and fibrosis [10]. We aim to further characterize the transcriptional profile of macrophages in pediatric cholestatic liver disease
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