Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Abstract

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c⁺/Ly6C^- and CD11c^-/Ly6C⁺ cells. CD11c⁺ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c⁺ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C⁺ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c⁺/Ly6C^- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c^-/Ly6C⁺ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

Keywords: Kupffer cell; fibrosis; inflammation; macrophage; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis.

Figure 1

An iHFC (high-fat/cholesterol/cholate-based) diet induces steatohepatitis in the liver of Tsumura-Suzuki non-obese (TSNO) mice. (A) Representative photos of the livers from TSNO mice fed with the normal diet (ND) or iHFC diet for the indicated time periods. Scale bars, 1 cm. (B) Liver weights of TSNO mice fed with the ND or iHFC diet for the indicated time periods (n = 6 per group). (C) Left, plasma alanine aminotransferase (ALT) levels were measured for TSNO mice fed with the ND or iHFC diet for the indicated time periods (n = 6 per group). Right, plasma ALT levels per liver weight (g) were also calculated (n = 6 per group). (D) Representative images of hematoxylin and eosin-stained sections of the livers from TSNO mice fed with the ND or iHFC diet for the indicated time periods. Scale bars, 100 μm. (E) RT-qPCR of TNF-α (Tnf), iNOS (Nos2), CCL2 (Ccl2), CD11c (Itgax), and IL-1β (Il1b) mRNA in the livers from TSNO mice fed with the ND or iHFC diet for the indicated time periods (n = 3 per group). Data are shown as means ± SD. * p < 0.05, *** p < 0.001. Statistical significance was evaluated by 2-way ANOVA followed by post hoc Sidak test.

Figure 2

The iHFC diet induces advanced fibrosis in the liver of TSNO mice. (A) Representative images of Sirius red-stained sections of the livers from TSNO mice fed with the ND or iHFC diet for the indicated time periods. Scale bars, 100 μm. (B) Liver fibrosis (0 to 4) was assessed according to the criteria proposed by Kleiner et al., as described in the Materials and Methods (n = 3 per group). (C) Five locations were photographed per three sections per each group. Then, positive areas for Sirius red were measured at 15 locations using ImageJ software, and the mean and SD were calculated. (D) RT-qPCR of collagen type 1 (Col1a1), αSMA (Acta2), TIMP-1(Timp1), and TGF-β (Tgfb1) mRNA in the livers from TSNO mice fed with the ND or iHFC diet for the indicated time periods (n = 3 per group). Data are shown as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance was evaluated by 2-way ANOVA followed by post hoc Sidak test.

Figure 3

The iHFC diet induces the accumulation of CD45⁺ leukocytes in the liver of TSNO mice. (A) Cell number of live non-parenchymal cells of the livers from TSNO mice fed with the ND or iHFC diet for the indicated time periods (n = 3 or 4 per group). (B) Representative flow cytometry data of CD45 expression on live non-parenchymal cells of the livers from TSNO mice fed with the ND or iHFC diet for the indicated time periods. (C) Percentage (Left) and cell number (Right) of CD45+ live non-parenchymal cells were determined by flow cytometry analysis done in (B) (n = 3 or 4 per group). (D) Percentage (Left) and cell number (Right) of CD45- live non-parenchymal cells were determined by flow cytometry analysis done in (B) (n = 3 or 4 per group). (E) RT-qPCR of F4/80 (Adgre1), CD11c (Itgax), Desmin (Des), TNF-a (Tnf), iNOS (Nos2), collgen type 1 (Col1a1), and TIMP-1 (Timp1) mRNA in sorted CD45+ and CD45- cells from TSNO mice fed with the ND or iHFC diet for 8 weeks (n = 3 to 5 per group). Data are shown as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance was evaluated by 2-way ANOVA followed by post hoc Sidak test.

Figure 4

The iHFC diet accumulates F4/80^Int/CD11b^Int-Hi recruited macrophages which constitute hCLS. (A) Representative flow cytometry data of F4/80 and CD11b expression on CD45⁺ non-parenchymal cells of the livers from TSNO mice on the ND or iHFC diet for the indicated time periods. (B) Percentage (Left) and cell number (Right) of F4/80^Hi/CD11b^Int KCs were determined by flow cytometry analysis done in (A) (n = 3 or 4 per group). (C) Percentage (Left) and cell number (Right) of F4/80^Int/CD11b^Int-Hi recruited macrophages were determined by flow cytometry analysis done in (A) (n = 3 or 4 per group). (D) Representative histological images of F4/80 immunostaining of the livers from TSNO mice on the ND or iHFC diet for the indicated time periods. Arrow heads depict hCLSs. Scale bars, 100 μm. (E) Five locations were photographed per three sections per each group. Then, positive areas for F4/80 were measured at 15 locations per each group using ImageJ software, and the mean and SD were calculated. Data are shown as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance was evaluated by two-way ANOVA followed by post hoc Sidak test.

Figure 5

The iHFC diet accumulates two types of recruited macrophage subsets, CD11c⁺/Ly6C⁻ and CD11c⁻/Ly6C⁺ cells. (A) Representative flow cytometry data of CD11c and Ly6C expression on F4/80⁺ non-parenchymal cells excluding KCs of the livers from TSNO mice on the ND or iHFC diet for the indicated time periods. (B) Percentage and cell number of CD11c⁺/Ly6C⁻ or CD11c⁻/Ly6C⁺ were determined by flow cytometry analysis done in (A) (n = 3 or 4 per group). (C) Representative histological images of CD11c and Ly6C immunostaining of the livers from TSNO mice on the ND or iHFC diet for the indicated time periods. Scale bars, 100 μm. (D) Representative magnified images of CD11c and Ly6C immunostaining of the livers from TSNO mice on the iHFC diet for 24 weeks. Scale bars, 50 μm. (E) Positive areas for CD11c or Ly6C in the livers from TSNO mice on the iHFC diet for the indicated time periods were measured using ImageJ software. Data are shown as means ± SD. ** p < 0.01, *** p < 0.001. Statistical significance was evaluated by two-way ANOVA followed by post hoc Sidak test.

Figure 6

The iHFC diet accumulates CD11c⁺ cells which are colocalized with collagen fibers. (A) Representative histological images (20× or 40× magnification) of fluorescent immunohistochemistry for CD11c, collagen type 1, and DAPI of the livers from TSNO mice on the ND or iHFC diet for 12 weeks. White scale bars, 100 μm. Gray scale bars, 50 µm. (B) Colocalization areas were calculated as described in Figure S10 and the Materials and Methods. Data are shown as means ± SD. * p < 0.05. Statistical significance was evaluated by unpaired Student’s t-test.

Figure 7

RNA sequence analysis of CD11c⁺/Ly6C⁻ and CD11c⁻/Ly6C⁺ cells in the liver from iHFC diet-fed TSNO mice. (A) PCA of two subsets of recruited macrophages from TSNO mice fed with the iHFC diet for 8 weeks. PCA was performed using Gene Spring. (B) Heatmap showing relative expression levels of upregulated or downregulated genes in two subsets of recruited macrophages from TSNO mice fed with the iHFC diet for 8 weeks. (C) The changes of the genes were analyzed using Ingenuity Pathways Analysis tools. The networks were displayed graphically as nodes (genes or proteins) and edges (the biological relationships between the nodes). Nodes and edges are displayed by various shapes and labels that represent the functional class of genes and the nature of the relationship between the nodes, respectively.