[Single-cell RNA sequencing reveals the driving mechanism of CXCL9꞉SPP1 polarization in the progression of non-alcoholic fatty liver disease].

Authors

Yao Lu, Wenjuan Ni, Rongcui Chen, Wei Wang

Published in

Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences. Volume 51. Issue 5. Pages 924-936. May 28, 2026.

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a globally prevalent chronic liver disease, and its progression is closely associated with Kupffer cells (KCs). This study aims to characterize C-X-C motif chemokine ligand 9꞉secreted phosphoprotein 1 (CS) polarization in KCs and investigate its intercellular communication with endothelial cells (ECs) in NAFLD.
Single-cell RNA sequencing data of liver tissues from 3 NAFLD mice and 3 control mice were obtained from the Gene Expression Omnibus (GEO) dataset GSE129516. Seurat was used for data integration, quality control, clustering, and annotation. KCs and ECs were divided into subgroups, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. A mouse liver single-cell dataset (GSE270583) was further included for cross-validation of CS polarization. CellChat was used to infer intercellular communication networks between KC and EC subgroups. Twenty male C57BL/6J mice were assigned to a control group (n=10) and a high-fat diet (HFD) group (n=10). Body weight was measured weekly. At 20 weeks of feeding, glucose tolerance and insulin tolerance were assessed, after which the mice were sacrificed, and serum and liver tissues were collected for subsequent experiments. Hematoxylin and eosin (HE) staining and Oil Red O staining were used to observe pathological changes in the liver. Immunofluorescence staining of liver tissues was performed to validate the characteristics of CS polarization and M1/M2 polarization.
A total of 29 cell clusters belonging to 8 cell types were identified. Compared with the control group, the proportion of KCs was increased and the proportion of ECs was decreased in the NAFLD group. Under M1/M2 and CS polarization frameworks, subgroups including CD80⁺ KCs, CD206⁺ KCs, S100A4⁺ KCs, secreted phosphoprotein 1 (SPP1)⁺ KCs, and C-X-C motif chemokine ligand 9 (CXCL9)⁺ KCs were identified. Among these, CD80⁺ KCs and SPP1⁺ KCs were significantly expanded, and their characteristic genes were mainly enriched in nuclear factor-kappa B (NF-κB) and tumor necrosis factor (TNF) signaling pathways. Peroxiredoxin 3 (PRDX3) was upregulated in CD80⁺ KCs and downregulated in CD206⁺ KCs. ECs were divided into 3 subgroups: Apoa2⁺ ECs, PRDX3⁺ ECs, and Col4a1⁺ ECs. In the NAFLD group, the PRDX3⁺ EC subgroup was abnormally active, accompanied by high expression of PRDX1 and downregulation of glutathione peroxidase 4 (GPX4). Further external validation using the single-cell dataset GSE270583 showed that SPP1⁺ KCs and CXCL9⁺ KCs could be clearly separated, and the proportion of SPP1⁺ KCs was increased in the NAFLD group, supporting the cross-cohort reproducibility of CS polarization in NAFLD. CellChat analysis suggested activation of the TNF receptor superfamily member 1A (TNFRSF1A) signaling pathway from CD80⁺ KCs and SPP1⁺ KCs to PRDX3⁺ ECs. After 20-week HFD feeding, mice displayed significant weight gain, impaired glucose tolerance, and reduced insulin sensitivity compared with controls (all P<0.05). The levels of alanine aminotransferase (ALT), aspartate transferase (AST), total cholesterol (TC), and triglyceride (TG) were significantly higher in the HFD group than in the control group (all P<0.05). HE staining and Oil Red O staining showed marked disordered hepatic architecture, lipid vacuoles, and lipid deposition in the livers of HFD mice. Immunofluorescence staining showed increased expression of PRDX3 and CD80 in liver tissues of the HFD group. Compared with the control group, SPP1 expression was increased, whereas CXCL9 expression was decreased, in hepatic KCs of the HFD group. These results were consistent with the single-cell transcriptome sequencing findings.
KCs in NAFLD exhibit characteristics of CS polarization, with increased proportions of SPP1⁺ KCs and CD80⁺ KCs, as well as enhanced TNF-TNFRSF1A communication with PRDX3⁺ ECs. Changes in the expression of SPP1, CXCL9, CD80, and PRDX3 in the liver tissues of HFD mice were generally consistent with the single-cell analysis results. CS polarization may participate in alterations of the hepatic inflammatory microenvironment in NAFLD and may provide a new research direction for understanding the inflammation-fibrosis progression of NAFLD.

PMID:
42565569
Bibliographic data and abstract were imported from PubMed on 07 Aug 2026.

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