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, which had high deleted in malignant brain tumors 1 (Dmbt1) expression, was found to perform drug metabolism and detoxification functions. Liver sinusoidal endothelial cells exhibited fenestrae loss and angiogenesis. Macrophage cluster 1 displayed a M1 polarization phenotype, whereas cluster 3 tended to exhibit M2 polarization. Kupffer cells (KCs) exhibited pro-inflammatory effects due to the high expression of Cxcl2. qRT-PCR and western blotting verified that the 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 AILI patients. Interaction patterns between macrophages/KCs and other NPCs were complex and diverse. NPCs were highly heterogeneous and were involved in the immune network during the early phase of AILI. In addition, we propose that Mkrn1 may serve as a potential biomarker of AILI.
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Data and materials used and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Abbreviations
- NPCs:
-
Non-parenchymal cells
- APAP:
-
N-acetyl-p-aminophenol, acetaminophen
- AILI:
-
Acetaminophen-induced liver injury
- scRNA-seq:
-
Single-cell RNA sequencing
- Mkrn1:
-
Makorin ring finger protein 1
- Dmbt1:
-
Deleted in malignant brain tumor 1
- KCs:
-
Kupffer cells
- DILI:
-
Drug-induced liver injury
- NAPQI:
-
N-acetyl-p-benzoquinone imine
- LSECs:
-
Liver sinusoidal endothelial cells
- HSCs:
-
Hepatic stellate cells
- ALT:
-
Alanine aminotransferase
- AST:
-
Aspartate aminotransferase
- DAMPs:
-
Damage-associated molecular patterns
- CCR2:
-
Chemokine (C–C motif) receptor 2
- APCs:
-
Antigen-presenting cells
- NASH:
-
Non-alcoholic steatohepatitis
- UMI:
-
Unique molecular identifiers
- DEGs:
-
Differentially expressed genes
- GO:
-
Gene ontology
- KEGG:
-
Kyoto encyclopedia of genes and genomes
- HILI:
-
Herbal-induced liver injury
- NAFLD:
-
Non-alcoholic fatty liver disease
- CHB:
-
Chronic hepatitis B
- PBC:
-
Primary biliary cholangitis
- OSM:
-
Oncostatin M
- TUNEL:
-
Terminal dUTP nick‑end labeling
- ECs:
-
Endothelial cells
- pDCs:
-
Plasmacytoid dendritic cells
- Mps:
-
Mononuclear phagocytes
- SMCs:
-
Smooth muscle cells
- HepSCs:
-
Hepatic stellate cells
- UMAP:
-
Uniform manifold approximation and projection
- LIFR:
-
Leukemia inhibitory factor receptor
- MAPK:
-
Mitogen-activated protein kinase
- Spp1:
-
Secreted phosphoprotein 1
- OPN:
-
Osteopontin; VEGF, vascular endothelial growth factor
- CDKN1A:
-
Cyclin-dependent kinase inhibitor 1A
- LDH:
-
Lactate dehydrogenase
- Epcs:
-
Epithelial progenitors
- GSVA:
-
Gene set variation analysis
- ALP:
-
Alkaline phosphatase
- GGT:
-
γ-Glutamyl transferase
- TBIL:
-
Total bilirubin
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (NSFC 81970513, NSFC 82270619, and NSFC 32171175), National Key R&D Program of China (2022YFC3502101), Clinical Research Innovation and Development Fund of Renji Hospital, School of Medicine, Shanghai Jiao Tong University (PY120-05).
Funding
This work was supported by the National Natural Science Foundation of China (NSFC 81970513, NSFC 82270619, and NSFC 32171175), the National Key R&D Program of China (2022YFC3502101), and the Clinical Research Innovation and Development Fund of Renji Hospital, School of Medicine, Shanghai Jiao Tong University (PY120-05).
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YXL, YZ, JL, and BXL performed the experiment and wrote and drafted the manuscript. HXL, YJ, TYZ, and FYZ retrieved the data and performed the statistical analysis. MXK, FX, WZ, and YXC performed the bioinformatics analysis. TJT, BXL, and MYM acquired funding. BXL and MYM revised the manuscript. All authors read and approved the final manuscript.
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All animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University Renji Hospital. Animal research was conducted in accordance with international guidelines. The clinical research was approved by the Ethics Committee of Shanghai Jiaotong University Renji Hospital in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.
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Li, X., Zhi, Y., Li, J. et al. Single-cell RNA sequencing to reveal non-parenchymal cell heterogeneity and immune network of acetaminophen-induced liver injury in mice. Arch Toxicol 97, 1979–1995 (2023). https://doi.org/10.1007/s00204-023-03513-4
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DOI: https://doi.org/10.1007/s00204-023-03513-4