Expansion of macrophage and liver sinusoidal endothelial cell subpopulations during non-alcoholic steatohepatitis progression

Figures

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  Figure 1. Transcriptional analysis of liver NPCs on the bulk and single-cell level in healthy and NASH mice (A) Body weight and liver weight of chow and HFD mice. Mice were fed with chow diet (Chow) and HFD diet supplemented with fructose and sucrose in the drinking water (HFD), respectively for 20 weeks. (B) Hepatic steatosis, lipid accumulation, inflammation, and fibrosis in each group were evaluated by hematoxylin-eosin (H&E), oil red O, picrosirius red (PSR), and NAFLD activity score (NAS). Scale bar, 100 μm. The statistics were shown on the right. (C) Differentially expressed genes between chow and HFD group on the bulk level. Genes up- or downregulated by > 1.5-fold were shown in orange and blue, respectively, while those ≤ 1.5-fold were shown in gray. (D) t-SNE visualization of scRNA-seq results. NPCs were classified into 10 distinct types. Cell counts for each cell type were shown in parentheses. (E) Heatmap of cell type specific marker genes. (F) Representative marker gene expression in each cell type. (G) Percentage of each cell type in NPCs isolated from livers of chow and HFD mice. (H) Differences in the percentage of hepatic NK cells, DCs, and HSCs between chow and HFD group.…

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  Figure 2. Identification of Itgadhi/Fcrl5hi macrophages in mice liver (A) Violin plots showing marker gene expression for each cluster (cluster 7, 12, 19, 21, 23, and 25) of macrophages. (B) t-SNE visualization of clusters of macrophages in chow and HFD group. (C) The percentage of each cluster in liver NPCs in chow and HFD group. (D) Expression of marker genes for cluster 21(Trem2, Gpnmb) and cluster 23 (Itgad, Fcrl5) in liver NPCs of chow and HFD group at mRNA level. (E) Dynamic changes in gene expression along pseudotime in macrophages. (F) Pseudotime trajectory of macrophages as a whole. (G) Pseudotime trajectory of each cluster of macrophages. (H) Detection of Itgad+ macrophages in chow and HFD mice liver by immunofluorescence staining. Scale bar, 50 μm. (I) Percentage of Fcrl5+ cells in F4/80+CD11b+ double-positive macrophages isolated from chow and HFD mice liver. The statistics were shown on the right. n = 3–5 per group. The data are expressed as mean ± SEM. Statistical significance is shown as ∗ for p < 0.05, ∗∗ for p < 0.01, ∗∗∗ for p < 0.001, and ∗∗∗∗ for p < 0.0001, as evaluated by Student’s t test.

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  Figure 3. Heterogeneity of ECs in mice liver (A) t-SNE visualization of re-clustered hepatic ECs. (B) Vwfhi and Vwflow ECs in the periportal area in chow and HFD mice liver. PV, portal vein. BD, bile duct. Arrowheads indicated Vwfhi ECs, and arrows indicated Vwflow ECs. Scale bar, 50 μm. (C) Vwfhi and Vwflow ECs in the pericentral area in chow and HFD mice liver. CV: Central vein. Arrowheads indicate Vwfhi ECs, and arrows indicated Vwflow ECs. Scale bar, 50 μm. (D) The percentage of each cluster in hepatic ECs in chow and HFD group. (E) Detection of Egr1+ ECs (ECs-3) in chow and HFD mice liver by immunofluorescence staining. Scale bar, 100 μm. (F) Detection of Ly6a+ ECs (ECs-7) in chow and HFD mice liver by immunofluorescence staining. Scale bar, 100 μm. (G) The statistics of CD31+Egr1+ double-positive and CD31+Ly6a+ double-positive areas in chow and HFD mice liver. (H) Expression of marker genes for ECs-3 (Egr1, Plk2) and ECs-7 (Ly6a, Ifit1, Ifit3, Isg15) in liver NPCs of chow and HFD group at mRNA level. (I) Detection of Ly6ahi ECs (ECs-7) in chow and HFD mice liver by flow cytometry. Ly6ahi Vegfr2hi ECs were located on the top right panel of each plot, and their percentages in liver…

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  Figure 4. Dynamic changes of Itgadhi/Fcrl5hi macrophages in NASH progression (A) Representative images for H&E, oil red O and PSR stained liver tissues of chow and HFD (week 8, 16 and 32) mice. The statistics of NAS and the area of oil red O and PSR were shown on the right. Scale bar, 100 μm. (B) Immunofluorescence staining of Itgad+ macrophages in liver tissues of chow and HFD (week 8, 16, and 32) mice. Scale bar, 50 μm. (C) The statistics of Itgad+ macrophages in liver tissues of chow and HFD (week 8, 16, and 32) mice. (D) Relative mRNA levels of Itgad and Fcrl5 in liver tissues of chow and HFD (week 8, 16, and 32) mice. (E) Percentage of Fcrl5+ cells in F4/80+CD11b+ double-positive macrophages isolated from liver tissues of chow and HFD (week 16 and 32) mice. The statistics were shown on the right. n = 3–5 per group. The data are expressed as mean ± SEM. Statistical significance is shown as ∗ for p < 0.05, ∗∗∗ for p < 0.001, and ∗∗∗∗ for p < 0.0001, as evaluated by one-way ANOVA.

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  Figure 5. Dynamic changes of Egr1hi LSECs and Ly6ahi LSECs in NASH progression (A) Detection of Egr1hi ECs in chow and HFD (week 8, 16, and 32) mice liver by immunofluorescence staining. Scale bar, 100 μm. (B) Detection of Ly6ahi ECs in chow and HFD (week 8, 16, and 32) mice liver by immunofluorescence staining. Scale bar, 100 μm. (C) The statistics of CD31+Egr1+ double-positive and CD31+Ly6a+ double-positive areas in chow and HFD (week 8, 16, and 32) mice liver. (D) Relative mRNA levels of Itgad, Fcrl5 and Ly6a, Ifit1, Ifit3, Isg15 in liver tissues of chow and HFD (week 8, 16, and 32) mice. (E) Detection of Ly6ahi ECs in chow and HFD (week 16 and 32) mice liver by flow cytometry. Ly6ahi Vegfr2hi ECs were located on the top right panel of each plot, and their percentages in liver NPCs were shown in red. The statistics were shown on the right. n = 3–5 per group. The data are expressed as mean ± SEM. Statistical significance is shown as ∗ for p < 0.05, ∗∗ for p < 0.01, ∗∗∗ for p < 0.001, and ∗∗∗∗ for p < 0.0001, as evaluated by one-way ANOVA.

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  Figure 6. Cell-cell communications among NPCs in mice liver (A) Visualization of interacting pairs among different NPCs in chow and HFD mice. (B) Visualization of the intensity of cell-cell communication among different NPCs in chow and HFD mice. (C) Correlation of mRNA expression level of Col1α1 and Itgad, Fcrl5 in chow and HFD (week 8, 16, and 32) mice liver. (D) Correlation of mRNA expression level of Col1α1 and Egr1, Plk2 in chow and HFD (week 8, 16, and 32) mice liver. (E) Correlation of mRNA expression level of Col1α1 and Ly6a, Ifit1, Ifit3, Isg15 in chow and HFD (week 8, 16, and 32) mice liver. n = 3 per group.