Dynamic Shifts in the Composition of Resident and Recruited Macrophages Influence Tissue Remodeling in NASH

Abstract

Macrophage-mediated inflammation is critical in the pathogenesis of non-alcoholic steatohepatitis (NASH). Here, we describe that, with high-fat, high-sucrose-diet feeding, mature TIM4^pos Kupffer cells (KCs) decrease in number, while monocyte-derived Tim4^neg macrophages accumulate. In concert, monocyte-derived infiltrating macrophages enter the liver and consist of a transitional subset that expresses Cx3cr1/Ccr2 and a second subset characterized by expression of Trem2, Cd63, Cd9, and Gpmnb; markers ascribed to lipid-associated macrophages (LAMs). The Cx3cr1/Ccr2-expressing macrophages, referred to as C-LAMs, localize to macrophage aggregates and hepatic crown-like structures (hCLSs) in the steatotic liver. In C-motif chemokine receptor 2 (Ccr2)-deficient mice, C-LAMs fail to appear in the liver, and this prevents hCLS formation, reduces LAM numbers, and increases liver fibrosis. Taken together, our data reveal dynamic changes in liver macrophage subsets during the pathogenesis of NASH and link these shifts to pathologic tissue remodeling.

Keywords: CCR2; Cx3cr1; Kupffer cells; crown-like structures; diabetes; fibrosis; inflammation; lipid-associated macrophages; liver.

Figure 1.. Accumulation of TIM4^neg Macrophages in a Mouse Model of NASH

(A) Body weight of C57BL/6 male mice fed a HFD for the indicated time. (B–D) Liver triglyceride (B) and mRNA markers of steatosis (C) and fibrosis (D) were quantified. (E) H&E (top panels) and picrosirius red (bottom panels) from liver tissue. (F) Flow-cytometry plots are shown for CD45⁺, live, singlets (top panels). Representative plots of F4/80^hi cells (red gate) were analyzed for expression of TIM4 (bottom panels). (G) Quantification of flow data per gram of liver tissue. (H) Percentage of F4/80^hi cells that are TIM4^neg over course of HFD feeding. In (A), each dot represents the mean ± SEM and in (B)–(D). In (G), each dot represents a single mouse, and the mean is shown (n = 7–8 mice/group). The data represent the results of one independent experiment with n = 7–8 mice per group. *p < 0.05 for HFD versus STD. Scale bars, 250 μm

Figure 2.. TIM4^neg Macrophages in the Kupffer Cell Gate Are Monocyte Derived

(A) CD115-CreER mice were crossed with Rosa26^TdT mice to for fate mapping. Male mice were fed tamoxifen (2 weeks) to induce labeling. After a 1 week washout period, the mice were started on STD/HFD. (B) Representative flow plots are shown for CD45⁺, singlet, live cells that were F4/80^hi, CD11b^int. TIM4^pos (black) and TIM4^neg (green) macrophages are shown, and TdT expression was assessed (red). (C) F4/80^hi macrophages from Cre negative Rosa26^TdT treated with tamoxifen for 2 weeks. (D) TdT label present in Ly6C^hi monocytes at 20 weeks. (E) Percentage of TdT-labeled cells in the total F4/80^hi gate (white bars); F4/80^hi, TIM4^pos (black bars); and F4/80^hi, TIM4^neg (gray bars) at baseline or following STD/HFD. (F) Quantitation of total (black dots) or TdT-labeled (red dots) TIM4^pos and TIM4^neg F4/80^hi macrophages in the liver following the indicated diet interventions. (G) The percentage of labeled ATMs (CD11b^hi, F4/80^hi, CD64^hi) is shown in the left panel with the total number of labeled and unlabeled macrophages shown in the right panel. (H) Flt3-Cre;Rosa26^TdT mice were fed a STD or HFD and percentage of labeled TIM4^pos and TIM4^neg macrophages cells was determined by flow cytometry. All dots represent data from one mouse with the mean shown in the bar. The bar graphs represent the mean of the group. The data represent the results of three independent experiments with n = 3–8 mice per groups. *p < 0.05.

Figure 3.. Transcriptional Profiling of Hepatic Macrophages during NASH

(A) CD45⁺, live, singlet cells were prepared and pooled together from 3 individual mice fed HFD for 16 weeks and the hepatic non-parenchymal cells were flow sorted into CD11b^hi, F4/80^int (green gate) and F4/80^hi, CD11b^int (red gate) populations with removal of PMNs and eosinophils form the CD11b^hi gate. The remaining cells were pooled 1:1 to create the hepatic macrophage/monocyte subsets and the sample was subjected to 10× scRNA-seq. The cluster-based t-SNE plot of the cell clusters is shown. (B) Expression of the indicated genes as shown using t-SNE visualization. (C) K means clustering analysis of the top 5 genes from each leukocyte population. TIM4^neg macrophages are represented by clusters 1 (green) and 2 (blue), whereas cluster 3 represents TIM4^pos KCs (red). (D) mRNA expression of genes associated with TIM4^neg macrophages in liver tissue from mice fed a STD or HFD for 16 weeks. Bars represent the means, and each dot represents a single mouse. The data represent the results of one independent experiment with n = 4–5 mice per groups. The p values are shown.

Figure 4.. TIM4^neg Macrophage Subsets Accumulate with NASH and Form Macrophage Aggregates in the Liver

(A) Violin plots of macrophage markers in monocyte/macrophage clusters where the macrophages are clusters 1: Cx3cr1^hi,Timd4^neg (green), 2: Cx3cr1^lo,Timd4^neg (blue), 3: KCs (red). (B) F4/80^hi, CD11b^int cells (too panels) assessing TIM4 and Cx3cr1 expression (bottom panels) from indicated conditions. The boxes indicate cells corresponding to the clusters shown above. (C) Histogram of F4/80, CD11c, and MHCII on the indicated macrophage populations. (D) IF analysis of F4/80 and Cx3cr1-GFP with lipid stain monodansylpentane (MDH). Arrows indicate aggregates around lipid droplets. (E) Images of macrophage aggregates using IF to assess expression of markers identified in (A), including CLEC4F, MHCII, CD63, and Gpnmb. The results are from three independent experiments n = 4–8 mice/group. Scale bars, 30 μm.

Figure 5.. Loss of Ccr2 Prevents Accumulation of C-LAMs with NAFLD

(A) Image of a macrophage aggregate stained for F4/80, CLEC4F, and Ccr2-GFP. (B) Image of macrophage clusters from mice co-expressing Ccr2-RFP and Cx3cr1-GFP. (C–F) Body weight (C), liver TAG (D), and serum ALT (E) from WT and KO mice fed a STD/HFD for 20 weeks. (F) ITT from WT or KO mice fed a HFD. (G) Ly6C^hi monocyte counts in the liver. (H) Flow plot of F4/80^hi, CD11b^int macrophages where red and green text indicate two phenotypes observed in Ccr2 KO mice. (I) Quantification of indicated macrophage populations in WT and KO mice. The red and green symbols indicate the phenotype of the mice from (H). (J and K) Quantification of C-LAM number as an absolute value (J) and as a percentage of the Tim4^neg macrophages (K). Each dot represents an individual mouse, and the bar reflects the mean. The data represent three independent experiments with n = 3–17 mice/group. The p values are shown. Scale bars, 30 μm.

Figure 6.. Ccr2 Deficiency Leads to Reduced Macrophage Aggregate Formation and Increased Fibrosis

(A and B) IF images (A) of Ccr2 WT and KO mice stained for F4/80 and Ccr2-GFP and (B) quantification of macrophage aggregates, aggregate size, and number with Ccr2-GFP-expressing cells. (C and D) Images of macrophage aggregates from WT and KO mice stained for expression of CD63 (C) and Gpnmb (D). (E) Liver tissue from humans with NAFLD stained with antibodies to CD68 (red) and CCR2 (green). (F) Representative picrosirius red staining (PRS) of liver sections from WT and KO mice at low (top panels) and high power (bottom panels) with quantification of PRS area (right plot). Each dot represents an individual mouse, and the bar reflects the mean. The data represent two independent experiments with n = 6–8 mice/group. The p values are shown. Scale bars, 30 μm (A and C–E), 250 μm (F).

Figure 7.. Ccr2 KO Mice Have Impaired Formation of hCLSs and Increased Fibrosis on a NASH Diet

(A) Flow-cytometry assessment of macrophages isolated from WT and KO mice fed FPC diet for 16 weeks. Images shown are gated on CD45⁺, F4/80^hi, CD11b^int cells. The green box indicates C-LAMs. (B) Quantification of the indicated macrophage subsets. (C) Representative flow plot and quantification of VSIG4 staining for the indicated macrophage populations in WT (white bars) and KO mice (green bars). (D) Liver sections were stained with antibodies to GFP and F4/80 to identify hCLSs (arrows). Quantification of hCLSs per high-powered field (hpf; top graph) and percentage of hCLSs containing GFP-expressing cells (bottom graph) are shown. (E) Imaging of hCLSs and macrophage subsets from WT and KO mice stained with antibodies against F4/80, CLEC4F, MHCII, CD63, and Gpnmb as indicated. (F) Representative images of PRS imaging from WT and KO mice on FPC diet. Quantification of PRS area is shown in the right panel. (G) IF montage of Ccr2-GFP, F4/80, and desmin. The orange line indicates the separation of a desmin-rich area (below) from more typical stellate cell distribution (above). The data represent two independent experiments with n = 5–6 group. The bars represent with mean and each dot is an individual mouse. *p < 0.05. Scale bars, 30 μM (D, E, and G) and 250 μm (F).