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Analysis of the interplay between hepatitis B virus-positive hepatocytes and Kupffer cells ex vivo using mice as a model.

ABSTRACT: Kupffer cells play critical roles in both hepatitis B virus (HBV) persistence and clearance. Here, we provide a protocol for studying the interplay between Kupffer cells and HBV-positive hepatocytes ex vivo using mice as a model. This protocol includes hydrodynamic injection of HBV DNA into mouse hepatocytes, liver perfusion for isolating hepatocytes and Kupffer cells, and Seahorse metabolic analysis of Kupffer cells. This protocol allows the detailed analysis of how HBV-positive hepatocytes and Kupffer cells impact each other ex vivo. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).

SUBMITTER: Li Y

PROVIDER: S-EPMC9097500 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Analysis of the interplay between hepatitis B virus-positive hepatocytes and Kupffer cells ex vivo using mice as a model.

Li Yumei Y Lee Jiyoung J Helou Doumet Georges DG Akbari Omid O Ou Jing-Hsiung James JJ

STAR protocols 20220506 2

Kupffer cells play critical roles in both hepatitis B virus (HBV) persistence and clearance. Here, we provide a protocol for studying the interplay between Kupffer cells and HBV-positive hepatocytes ex vivo using mice as a model. This protocol includes hydrodynamic injection of HBV DNA into mouse hepatocytes, liver perfusion for isolating hepatocytes and Kupffer cells, and Seahorse metabolic analysis of Kupffer cells. This protocol allows the detailed analysis of how HBV-positive hepatocy ...[more]

PMID: 35573478

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Project description:Background & aimsComplex communications between hepatocytes and Kupffer cells (KCs) are known to drive or suppress hepatocarcinogenesis, with controversial data in the literature. In previous experiments that aimed to decipher hepatocyte/KC interactions, we unexpectedly unveiled a tumor-suppressing effect of polyinosinic-polycytidylic acid, a widely used inducer of MX dynamin like GTPase 1 (Mx1)-cre expression, which questioned a theory of interleukin 1a/6 cytokine circuit in hepatocyte/KC communication. The goal of this study was to clarify the controversy and decipher unique functions of KCs and non-KC macrophages in liver tumorigenesis.MethodsWe used the C-type lectin domain family 4 member F (Clec4f)-cre system to delete Src-homology 2 domain-containing tyrosine phosphatase 2 (Shp2)/protein tyrosine phosphatase nonreceptor 11 (Ptpn11) in KCs, and a combination of Clec4f-cre and adeno-associated virus-cre to delete Shp2 in KCs and hepatocytes to investigate the effects on hepatocellular carcinoma development and immune cell compositions/activities.ResultsAblating Shp2 in KCs generated a tumor-promoting niche, which was exacerbated further by concurrent removal of Shp2 in both KCs and hepatocytes. Shp2 deficiency induced KC apoptosis and decreased its numbers, which induced compensatory recruitment of bone marrow-derived monocytes into liver. These newly recruited monocytes differentiated into non-KC macrophages with tumor-associated macrophage function, leading to aggravated tumor progression through down-regulation of CD8 T cells. Tumor-associated macrophage blockade by anti-chemokine (C-C motif) ligand 2 (CCL2) antibody inhibited hepatocellular carcinoma progression, while depletion of all macrophages had a tumor-promoting effect by increasing myeloid-derived suppressor cells (M-MDSCs) and decreasing CD8 T cells.ConclusionsShp2 loss in KCs or hepatocytes generated a protumorigenic microenvironment, which was exacerbated by its removal in both cell types. These results show the complexity of intercellular signaling events in liver tumorigenesis and raises caution on the use of specific Shp2 inhibitor in liver cancer therapy. Transcript profiling: RNA sequencing data are available at Gene Expression Omnibus (GSE222594).

Dataset Information

Analysis of the interplay between hepatitis B virus-positive hepatocytes and Kupffer cells ex vivo using mice as a model.

Publications

Analysis of the interplay between hepatitis B virus-positive hepatocytes and Kupffer cells <i>e</i><i>x vivo</i> using mice as a model.

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