Myeloid-specific ablation of Basp1 ameliorates diet-induced NASH in mice by attenuating pro-inflammatory signaling

Abstract

Background and aims: NASH represents a severe stage of fatty liver disease characterized by hepatocyte injury, inflammation, and liver fibrosis. Myeloid-derived innate immune cells, such as macrophages and dendritic cells, play an important role in host defense and disease pathogenesis. Despite this, the nature of transcriptomic reprogramming of myeloid cells in NASH liver and its contribution to disease progression remain incompletely defined.

Approach and results: In this study, we performed bulk and single-cell RNA sequencing (sc-RNA seq) analysis to delineate the landscape of macrophage and dendritic cell transcriptomes in healthy and NASH livers. Our analysis uncovered cell type-specific patterns of transcriptomic reprogramming on diet-induced NASH. We identified brain-abundant membrane-attached signal protein 1 (Basp1) as a myeloid-enriched gene that is markedly induced in mouse and human NASH liver. Myeloid-specific inactivation of Basp1 attenuates the severity of diet-induced NASH pathologies, as shown by reduced hepatocyte injury and liver fibrosis in mice. Mechanistically, cultured macrophages lacking Basp1 exhibited a diminished response to pro-inflammatory stimuli, impaired NLRP3 inflammasome activation, and reduced cytokine secretion.

Conclusions: Together, these findings uncover Basp1 as a critical regulator of myeloid inflammatory signaling that underlies NASH pathogenesis.

Figure 1.. Transcriptomic signatures of the resident macrophage and recruited myeloid cell compartments in NASH liver.

A) UMAP visualization of liver cell clusters based on single-cell RNA sequencing analysis of liver non-parenchymal cells isolated from mice fed chow or NASH diet (GSE129516) (up). Bar plots of cell number and percentage of KDM cells in chow and NASH mouse livers (down). B) Heatmap of genes exhibiting enriched expression in Kupffer cells (KC), Monocyte-derived macrophages (MDM), and Dendritic cells (DC) based on averaged expression levels in individual cell types. A total of 608 genes exhibited highest mRNA expression in one of the KDM cell types with average normalized Unique Molecular Identifiers (UMIs) > 1. C) Heatmap of differentially expressed macrophage/myeloid-enriched genes based on bulk RNAseq data from mice fed with chow or NASH diet for 20 weeks (left, GSE119340), and chow or CDA-HFD for 12 weeks (right, GSE120977). 234 and 270 genes passed the filter of adjusted P-value < 0.05 and Log2 Fold Change (NASH/chow) > 1 in two dietary NASH models, respectively. D) Venn diagram of differentially expressed macrophage/myeloid-enriched genes in two NASH models. E) Bubble plot of Gene Ontology analysis of differentially expressed macrophage/myeloid-enriched genes in NASH liver.

Figure 2.. Regulation of Basp1 expression during NASH and in cultured macrophages.

A) Heatmap of differentially expressed genes in KDM cells based on sc-RNAseq data from mice fed with chow or NASH diet for 20 weeks. 91 genes passed the filter of average UMI > 1 and Log2 Fold Change (NASH/chow) > 1 in at least one of three cell types. B) Feature plot illustrating mRNA expression of Basp1 in individual liver cells from mice fed chow or NASH diet. C) Violin plot illustrating mRNA expression of Basp1 in KDM clusters. D) qPCR analysis of gene expression in cultured BMDMs treated with PBS or LPS (5 ng/mL) plus IFN-γ (5 ng/mL), IL-4 (5 ng/mL) plus IL-10 (5 ng/mL), or TGF-β (2.5 ng/mL) for 24 hours, as indicated. Data in D) represent mean ± SEM; ***p < 0.001, ****p < 0.0001. Two-tailed unpaired Student’s t-test.

Figure 3.. Regulation of BASP1 expression during NASH and NASH resolution.

A) qPCR analysis of hepatic Basp1 expression in mice fed chow (n = 7), NASH diet for 20 weeks (n = 7), or CDA-HFD for 8 weeks (n = 7). Age-matched mice were divided into three dietary cohorts and harvested at the same time following indicated feeding time. B) BASP1 immunofluorescence staining of liver sections. Scale bars, 100μm. C) qPCR analysis of human hepatic Basp1 expression in non-NASH (n = 6) or NASH patients (n = 7). D) qPCR analysis of hepatic Basp1 expression in mice fed chow (n = 4), NASH diet for 6 months (n = 8), or 4 months NASH diet followed by switching to chow for 1 month (n = 9) or 2 months (n = 8). Data in C) represent mean ± SEM; *p < 0.05, analyzed by two-tailed unpaired Student’s t-test. Data in A) and D) represent mean ± SEM; **p < 0.01, ****p < 0.0001, analyzed by one-way ANOVA with post hoc analysis using Tukey’s test.

Figure 4.. Correlation of Basp1 expression with metabolic liver disease.

A) Correlation of hepatic Basp1 mRNA level with plasma ALT and AST levels, and hepatic mRNA levels of Col1a1, Adgre1, Ccl2, and H2-Ab1 in mice fed NASH (n = 11) or chow (n = 4) diet for 12 weeks. B) Correlation of hepatic Basp1 mRNA level with body weight, liver TAG content, and hepatic Col1a1 mRNA level in mice fed HFD for 8 weeks (n = 55).

Figure 5.. Myeloid-specific ablation of Basp1 ameliorates liver injury during NASH.

A) A schematic outline of the NASH liver study. B) Body weight, liver weight, and liver/body weight ratio of control (n = 9) and MKO (n = 9) mice following 20 weeks of NASH diet feeding. C) Plasma ALT and AST levels in control and MKO mice after 20 weeks of NASH diet feeding. D) Metabolic parameters of NASH diet-fed mice. E) H&E histology of liver sections from NASH diet-fed mice. Scale bars, 100 μm. Data represent mean ± SEM. Two-tailed unpaired Student’s t-test.

Figure 6.. Basp1 ablation in myeloid cells improves diet-induced NASH pathologies in mice.

A) Sirius Red staining of liver sections from NASH diet-fed mice. Scale bars, 100 μm. B) Decorin immunofluorescence staining. Scale bars, 100 μm. C) qPCR analysis of hepatic gene expression from NASH diet-fed mice. Data represent mean ± SEM. Two-tailed unpaired Student’s t-test.

Figure 7.. Effects of myeloid-specific Basp1 inactivation on liver inflammation.

A) Plasma CCL-2 and TNFα levels in control and MKO mice after 20 weeks of NASH diet feeding. B) qPCR analysis of hepatic expression of inflammation- and NLRP3 inflammasome-related genes in NASH diet-fed mice. C) F4/80 (left) and MHC-II (right) immunofluorescence staining. Scale bars, 100 μm. D) qPCR analysis of hepatic gene expression in NASH diet-fed mice. E) Immunoblots of liver lysates from NASH diet-fed mice. Data represent mean ± SEM. Two-tailed unpaired Student’s t-test.

Figure 8.. Basp1 inactivation attenuates proinflammatory signaling and NLRP3 inflammasome activation in cultured macrophages.

A) qPCR analysis of gene expression in cultured BMDMs treated with PBS or LPS (5 ng/mL) plus IFN-γ (5 ng/mL) for 24 hours. B) qPCR analysis of gene expression in cultured liver macrophages isolated from NASH livers treated with PBS or LPS (5 ng/mL) plus IFN-γ (5 ng/mL) for 24 hours. C) Immunoblots of total protein lysates from cultured BMDMs isolated from control and MKO mice. Differentiated macrophages were treated with PBS or LPS (200 ng/mL) for indicated time followed by ATP (3 mM) treatment for 30 minutes. D) Immunoblots of total protein lysates from cultured liver macrophages isolated from NASH diet-fed control and MKO mice. Primary macrophages were treated with PBS or LPS (200 ng/mL) for 3 hours followed by ATP (3 mM) treatment for 30 minutes. Data in A) and B) represent mean ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-tailed unpaired Student’s t-test.