C/EBPβ-VCAM1 axis in Kupffer cells promotes hepatic inflammation in MASLD

Abstract

Background & aims: Kupffer cells (KCs) can promote hepatic inflammation in metabolic dysfunction-associated steatotic liver disease (MASLD), but the underlying molecular mechanisms are not fully understood. C/EBPβ in macrophages can mediate metabolic and immune dysregulations. Therefore, we aimed to explore its role in KCs in MASLD pathogenesis.

Methods: A 12-week high-fat and high-cholesterol diet (HFHCD) model was used in wild-type or KC-specific Cebpb heterozygous knockout mice (n = 10 per group), followed by liver evaluation using histopathology, flow cytometry, and RNA-seq. RNA-seq of liver tissue (n = 3 per group) and C/EBPβ CUT&Tag-seq of sorted KCs were comprehensively analyzed to elucidate the transcriptional regulatory network. Flow cytometry and immunofluorescence were used to detect the expression or distribution of key proteins.

Results: HFHCD induced prominent immune cell infiltration and a concomitant increase in C/EBPβ in KCs. KC-specific Cebpb heterozygous knockout significantly reduced HFHCD-induced lobular inflammation (p <0.05) and inflammation-related gene expression (p <0.05) in the liver. Multi-omics analysis revealed increased C/EBPβ activity in KCs in MASLD, leading to a selective promotive effect on MASLD-induced genes. Further integrated analysis identified Vcam1 as a key direct downstream gene of C/EBPβ in KCs in MASLD, which involves C/EBPβ-mediated activation of the Vcam1 promoter. VCAM1 was predominantly expressed in KCs in the hepatic tissue of MASLD mice and patients. KC-expressed VCAM1 was significantly increased in MASLD compared with healthy controls (p <0.01), and it promoted immune cell infiltration into the liver.

Conclusions: Increased C/EBPβ in KCs promotes pathogenic transcriptional activation, leading to increased VCAM1 expression and inflammatory cell infiltration in MASLD. Inhibition of C/EBPβ in KCs might be a potential therapeutic strategy against hepatic inflammation in MASLD.

Impact and implications: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, but its pathogenesis remains elusive. In this study, we investigated the critical role of CCAAT/enhancer binding protein β (C/EBPβ) in Kupffer cells and its implications in MASLD pathogenesis. We found that an increased C/EBPβ level in Kupffer cells promotes hepatic inflammation in MASLD by upregulating VCAM1 expression. Our findings provide valuable insights into the molecular mechanisms driving MASLD and propose a potential novel therapeutic target to mitigate hepatic inflammation in MASLD.

Keywords: C/EBPβ; Kupffer cell; MASH; MASLD; VCAM1.

Graphical abstract

Fig. 1

Presence of hepatic inflammation and changes in hepatic macrophages in the HFHCD-induced MASLD model. (A and B) Representative H&E staining (A) (scale bar = 100 μm) and NAS score (B) based on liver sections. (C) Body weight, liver index (the ratio of liver weight to body weight), and epidymal fat weight at Week 12 (n = 5 for each group). (D) Representative immunofluorescence images showing the distribution of CLEC4F and CD45 in liver sections (scale bar = 50 μm). (E) Flow cytometry analysis of KCs and non-KC myeloid cells from ND and HFHCD mice with relative quantification (n = 4 for the ND group and n = 5 for the HFHCD group). (F) Intracellular flow cytometry analysis showing the protein level of C/EBPβ in KCs (n = 4 for each group). Results were compared by Wilcoxon Rank Sum test (B) or unpaired two-tailed Student’s t-test (D-F). Bars represent mean ± SEM. ∗p <0.05, ∗∗p <0.01. C/EBPβ, CCAAT/enhancer binding protein β; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Fig. 2

KC-specific Cebpb knockdown alleviates hepatic inflammation in MASLD. (A and B) Representative H&E staining (A) (scale bar = 100 μm) and NAS score (B) based on liver sections (n = 10 for each group from two independent experiments). (C–E) Representative immunofluorescence images of liver sections showing the distribution of CLEC4F and CD45 (C) and CLEC4F and IBA1 (D) (scale bar = 50 μm); the number of CD45⁺ leukocyte aggregates per 20 × field was additionally counted (E). (F) Relative mRNA levels of EmKC marker genes in livers. (G) Flow cytometry analysis of KCs and non-KC myeloid cells with relative quantification (n = 5 for each group). Results were compared by Wilcoxon Rank Sum test (B, E) or unpaired two-tailed Student’s t-test (F, G). Bars represent mean ± SEM. ∗p <0.05, ∗∗p <0.01. EmKC, embryonic KC; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease.

Fig. 3

KC-specific Cebpb knockdown reduces hepatic immune cell infiltration by inhibiting leukocyte migration in MASLD. (A–D) RNA-seq was performed on RNA isolated from liver tissue (n = 3 for each group). (A) Principal component analysis. (B) Volcano plot showing upregulated/downregulated DEGs between two groups. (C) Top 10 GO biological processes in GSEA analysis of DEGs. (D) Heatmap showing indicated cell subset marker expression. (E) Relative mRNA levels of hepatic immune cell marker genes in livers. (F–H) Representative immunofluorescence images of liver sections showing the distribution of CD8α (F) and Ly6G (G) with quantification of CD8α⁺ or Ly6G⁺ cells per high-power field (HPF) (H) (scale bar = 50 μm). Results were compared using unpaired two-tailed Student’s t test; n = 7 for the Clec4f-iCre;Cebpb^+/+ group and n = 6 for the Clec4f-iCre;Cebpb^fl/+ group. Bars represent mean ± SEM. ∗p <0.05, ∗∗p <0.01. DEG, differentially expressed gene; GO, gene ontology; GSEA, gene set enrichment analysis; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MoMF, monocyte-derived macrophage; PC, principal component.

Fig. 4

C/EBPβ activity in KCs is increased in MASLD and mediates selective modulation of ATF3/p300-related transcriptional regulation. (A) Immunofluorescence showing the intracellular distribution of C/EBPβ (green) in murine liver immune cells (scale bar = 10 μm). (B) Average enrichment heatmap and profile of C/EBPβ CUT&Tag signals around genic regions. (C) Spearman correlation heatmap with hierarchical clustering showing the correlation in overall genomic distribution between C/EBPβ CUT&Tag signals and ChIP-seq (LXR, ATF3, and p300) or ATAC-seq signals. (D) Barplot of C/EBPβ CUT&Tag peak distribution in genomic features. (E) Venn plots showing the overlaps between AMLN-induced upregulated/downregulated DEGs and four gene sets annotated from C/EBPβ peak sets in promoter regions. (F) Average enrichment heatmap and profile of ATF3 and p300 signals ± 3 kb around four C/EBPβ peak sets. (G) Log ratio of ATF3 and p300 ChIP-seq signal (AMLN vs. ND) at four C/EBPβ peak sets. Results were compared using Dunn’s test after the Kruskal–Wallis test (G). ∗p <0.05, ∗∗p < 0.01. C/EBPβ, CCAAT/enhancer binding protein β; DEG, differentially expressed gene; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Fig. 5

C/EBPβ-mediated transcriptional activation promotes the expression of VCAM1 in KCs of MASLD mice. (A) Activating/repressive function prediction of HFHCD-induced upregulated C/EBPβ peaks in KCs from MASLD mice, with the top five genes in prediction. (B) Expression of indicated genes in scRNA-seq of murine MASLD liver. (C) Relative mRNA levels of indicated genes in the liver. (D) Representative genomic track showing C/EBPβ, ATF3, and p300 distribution at the Vcam1 gene loci in KCs. (E and F) Flow cytometry analysis of the VCAM1 protein level (E) with quantification (F) (n = 3 for each group). (G) Relative mRNA level of Vcam1 in sorted KCs (n = 4 for the “Clec4f-iCre;Cebpb^+/+ HFHCD” group and n = 3 for the other two groups). (H) Relative luciferase activity of the Vcam1 promoter in AML12 cells transfected with murine C/EBPβ overexpression plasmid (left panel, n = 4 for each group) or siRNA targeting C/EBPβ (right panel, n = 3 for each group). Results were compared using an unpaired two-tailed Student’s t test (C, F, and H) or one-way ANOVA and least significant difference (LSD) post hoc test (G). Bars represent mean ± SEM. ∗p <0.05, ∗∗p <0.01. C/EBPβ, CCAAT/enhancer binding protein β; HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Fig. 6

KC-expressed VCAM1 is upregulated and promotes immune cell infiltration in MASLD. (A, D–F) Wild-type C57BL/6J mice were administered with HFHCD or ND. (A) Relative mRNA levels of Vcam1 in liver tissue. (D and E) Representative immunofluorescence images of liver sections showing the distribution of VCAM1 with CLEC4F and IBA1 (D), and VCAM1 with CD8α (E) (scale bar = 20 μm). (F) Flow cytometry analysis showing the VCAM1 protein level in KCs. (B and C) Vcam1 expression in RNA-seq of liver tissue (B) or FCM-sorted KCs (C) from mice in different dietary MASLD models. (G) Cell adhesion assay in vitro showing the adhesion of RAW264.7 cells to murine primary KCs after administration of murine TNF-α and/or anti-VCAM1 antibody. Results were compared using an unpaired two-tailed Student’s t test (A, F) or one-way ANOVA and an LSD post hoc test (G). Bars represent mean ± SEM. ∗p <0.05, ∗∗p <0.01. HFHCD, high-fat and high-cholesterol diet; KC, Kupffer cell; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal diet.

Fig. 7

Increased VCAM1 in KCs is associated with hepatic inflammation in patients with MASH. (A and B) VCAM1 expression in RNA-seq of liver tissue from patients with MASLD or healthy controls (A) and patients with MASH before and after RYGB or LI (B). (C) Representative immunofluorescence images of VCAM1 and IBA1 (C) in liver sections of patients with MASH or healthy controls (scale bar = 20 μm). (D) Expression of VCAM1 in liver scRNA-seq of patients with different hepatic steatosis degrees. (E) Relative luciferase activity of the VCAM1 promoter in HUVECs transfected with human C/EBPβ overexpression plasmid or control plasmid (n = 3 for each group). Results were compared using one-way ANOVA and an LSD post hoc test (A), a paired two-tailed Student’s t test (B), Wilcoxon rank sum test (D), or an unpaired two-tailed Student’s t test (E). Bars represent mean ± SEM. ∗p <0.05, ∗∗p <0.01. C/EBPβ, CCAAT/enhancer binding protein β; HUVEC, human umbilical vein endothelial cell; KC, Kupffer cell; LI, lifestyle intervention; MASL, metabolic dysfunction-associated steatotic liver; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; RYGB, Roux-en-Y gastric bypass.