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Single-cell RNA sequencing to reveal non-parenchymal cell heterogeneity and immune network of acetaminophen-induced liver injury in mice

ABSTRACT: The role of non-parenchymal cells (NPCs) in the early phase of acetaminophen (APAP)-induced liver injury (AILI) remains unclear. Therefore, single-cell sequencing (scRNA-seq) was performed to explore the heterogeneity and immune network of NPCs in the livers of mice with AILI. Mice were challenged with saline, 300 mg/kg APAP, or 750 mg/kg APAP (n=3 for each group). After 3 h, the liver samples were collected, digested, and subjected to scRNA-seq. Immunohistochemistry and immunofluorescence were performed to confirm the expression of Makorin ring finger protein 1 (Mkrn1). We identified 14 distinct cell subtypes among the 120,599 cells. A variety of NPCs were involved even in the early stages of AILI, indicating highly heterogeneous transcriptome dynamics. Cholangiocyte cluster 3 with high Deleted in malignant brain tumors 1 (Dmbt1) expression was found to perform drug metabolism and detoxification functions. Liver sinusoidal endothelial cells displayed fenestrae loss and angiogenesis. Macrophage cluster 1 displayed an M1 polarization phenotype, whereas cluster 3 tended to exhibit M2 polarization. Kupffer cells (KCs) particularly highly expressed Cxcl2 exhibiting pro-inflammatory effects. qRT-PCR and western blotting verified that LIFR-OSM axis might promote the activation of MAPK signaling pathway in RAW264.7 macrophages. Mkrn1 was highly expressed in the liver macrophages of AILI mice and patients with AILI. Interaction patterns between macrophages/KCs and other NPCs were complex and diverse. NPCs were highly heterogeneous and involved in the immune network in the early phase of AILI. In addition, we proposed that Mkrn1 may serve as a potential biomarker of AILI.

ORGANISM(S): Mus musculus

PROVIDER: GSE228305 | GEO | 2023/09/30

REPOSITORIES: GEO

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Project description:There is an evident, unmet need to develop a commercially available in vitro system that can model inflammatory states of the liver and predict immune-mediated hepatotoxicity of drugs and xenobiotics taken under inflamed conditions. Hepatocyte-Kupffer cell co-cultures can model inflammation-mediated hepatotoxicity; however, Kupffer cell (KC) source remains an important bottleneck for the development of such models. Primary human Kupffer cells (PHKCs) are costly, limited in availability and exhibit donor variability. An alternative cell source for KCs has not been reported. Important paradigm shift from the classical dogma of adult blood-circulating monocyte-derived macrophages to intrahepatic precursor/fetal monocyte-derived macrophages has shed new light into the origin of KCs in vivo. Based on these recent findings, we report here, a novel method to generate human KCs in vitro from stem cells (hPSC-KCs) via fetal monocytes. hPSC-KCs expressed macrophage markers, CD11, CD14, CD68, CD163 and CD32 at gene and protein level and exhibited functional properties such as phagocytosis and Interleukin-6 and Tumor Necrosis Factor-4alpha production upon activation. Importantly, molecular signature, liver-macrophage specific CLEC-4F expression and cytokines production levels of hPSC-KCs were similar to PHKCs but different from non-liver macrophages. We used an inflammatory liver co-culture model to demonstrate that activated hPSC-KCs, but not non-liver macrophages, were able to recapitulate effects of PHKCs when stimulated with paradigm hepatotoxicants. hPSC-KCs developed in this study offer a renewable human cell source for liver-specific macrophages which can be used to develop in vitro systems for modelling the inflammatory state of the liver. Gene expression profiles of 9 samples were determined using Human Gene 2.0 ST Array. These 9 samples included three replicates each of PHKCs (primary human Kupffer cells), human pluripotent stem cell-dervied Kupffer cells (hPSC-KCs) and non-liver macrophages (NL-Mφ).