Fig 2: ZEB2 Controls Tissue Identity of KCs and AMs(A) Schematic of experimental set up.(B) Expression of CD45.1 (donor) and Tim4 in total Clec4F+ KCs in Clec4f-crexZeb2fl/fl chimeras. Percentage of total chimerism of KCs in Zeb2fl/fl and Clec4f-crexZeb2fl/fl mice. Data are pooled from two experiments with n = 6–10 per group.***p < 0.001 Student’s t-test.(C) Expression of CD45.1 (donor) and CD45.2 (host) in total lung AMs in Itgax-crexZeb2fl/fl mice. Percentage of total chimerism of AMs in Zeb2fl/fl and Itgax-crexZeb2fl/fl mice. Data are pooled from two experiments with n = 5–8 per group. NS; non-significant. Student’s t-test. Percentage total chimerism calculated as ratio over the chimerism in blood Ly6Chi monocytes in the same mouse.(D and E) Heatmap of expression of cell death-associated genes per group of KCs (D) or AMs (E) from SC-RNA-seq data.(F) Representative western blots (n = 2) for RIPK3, pMLKL, and Tubulin expression by total BAL cells isolated from CD45.1 mice that were irradiated (8 Gy) and reconstituted with Rosa26-creert2xZeb2fl/fl BM. 33 weeks post reconstitution, mice were fed 5 mg tamoxifen or corn oil as a control for 5 days. 27 days after the last dose, mice were sacrificed and BAL fluid isolated. BAL fluid from 3 or 4 mice was pooled per replicate and 150,000 cells were used per lane. Ratio of band intensity was calculated using ImageJ. Data are pooled from two experiments with n = 6–8 per group. Student’s t-test was used to calculate indicated p values.(G) Venn diagram showing DE genes specific to Zeb2-/- KCs (group 3), Zeb2-/- AMs (group 3), or shared between both mac populations.(H and I) Heatmap showing expression of top core genes across KC (H) or AM (I) groups from SC-RNA-seq data. Genes in red are significantly differentially expressed. See also Figure S3.

Fig 3: ZEB2 Controls Mac Number and Surface Phenotype(A) Expression of CD64 and F4/80 by live CD45+Ly6G-Ly6C- liver cells and Clec4F and Tim4 by total liver macs in Clec4f-crexZeb2fl/fl and Zeb2fl/fl mice. Absolute number of liver macs per gram of liver and % of total macs expressing Clec4F and Tim4. Data are pooled from four experiments with n = 11–13 per group. ***p < 0.001 Student’s t test.(B) Expression of SiglecF, F4/80, and CD11b by live CD45+CD64+CD11c+ Lung macs in Itgax-crexZeb2fl/fl and Zeb2fl/fl mice. AMs as a percentage of total live CD45+ cells, absolute number, and percentage of CD11b+ and CD11b- AMs in Itgax-crexZeb2fl/fl or Zeb2fl/fl mice. Data are pooled from two experiments with n = 7–8 per group. *p < 0.05, ***p < 0.001 Student’s t test.(C and D) t-SNE plot of SC-RNA-seq data of KCs from Clec4f-crexZeb2fl/fl or Zeb2fl/fl mice (C) or AMs from Itgax-crexZeb2fl/fl or Zeb2fl/fl mice (D), showing clusters, assigned groups, and CRE- (Red) and CRE+ (Teal) overlay.(E) tSNE plots showing expression of Zeb2, Siglecf, and Ms4a1 in KCs.(F) tSNE plots showing expression of Zeb2, Epcam, and Cd101 in AMs.(G and H) Top 15 DE genes per group based on LogFC per group of KCs (G) or AMs (H). See also Figure S1.

Fig 4: Loss of TIM4+ Resident KCs and Replacement from the BM in MAFLD(A) Gating strategy used to identify monocyte and mac populations in all figures (for full gating strategy, see Figure S3A).(B and C) Absolute cell numbers per liver of indicated cell types from mice fed the diets for 12 (blue), 24 (green), or 36 (red) weeks, excluding mice that developed HCC. Data are pooled from 3–7 independent experiments with n = 9–38. **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA compared with pooled SD.(D) Schematic showing generation of protected chimeras.(E and F) % chimerism (compared with blood monocytes) in indicated hepatic populations was assessed in protected chimeras 18 (E) or 24 (F) weeks after feeding the SD or WD. Data are pooled from 2 independent experiments with n = 5–9 per group.(G) % of Ki-67+ cells among Clec4F+ KCs in mice fed the WD for indicated time points, together with pooled results from SD-fed mice (left panel) and representative flow cytometry plots show Ki67 expression by Clec4F+ KCs from a mouse fed SD or WD for 24 weeks (right panels). Data are from 1 (36 weeks) or pooled from 3 (12 and 24 weeks) independent experiments with n = 3–24.(H) Confocal microscopy of livers of SD or WD-fed mice (24 weeks), showing expression of Clec4F (green) and Ki-67 (red). White arrows indicate Ki-67+ KCs. Images are representative of 2 mice per diet.(I) Absolute number of CLEC4F+TIM4+ ResKCs and CLEC4F+TIM4- moKCs in SD- or WD-fed (24 weeks) mice injected with 1 mg/kg CSF1-Fc or PBS subcutaneously for 4 days before being sacrificed at day 6. Data are pooled from 2 independent experiments, with n = 4–6 per group. *p < 0.05, **p < 0.01, unpaired Student’s t test.(J and K) qPCR analysis for indicated genes in LSECs (J) and HSCs (K) sorted from SD- (black) or WD-fed (red) mice (36 weeks). Data are from 1 experiment, with n = 4–6 per group. *p < 0.05, Student’s t test. All error bars indicate SEM. See also Figure S3.

Fig 5: Hepatic LAMs in MAFLD Are Identified by Spp1 Expression(A) Heatmap showing DEGs between Mac2, Mac1, and moKC populations from mice fed the WD for 24 and 36 weeks pooled and their expression by indicated populations.(B) KEGG pathway analysis on DEGs for each indicated subset (see Tables S1 and S3).(C) The adipose tissue LAM signature (Jaitin et al., 2019) was mapped onto the liver mac UMAP to identify cells with a similar profile using the Signature Finder algorithm (Pont et al., 2019).(D and E) Expression of Spp1 by CLEC4F- macrophages (D) and moKCs and ResKCs (E) at 24 and 36 weeks on WD as measured by Prime Flow (left panels, representative plots), and right, proportions of indicated populations (T4 = TIM4). Data are pooled from 2 experiments with 6 mice per group. *p < 0.05, ***p < 0.001, ****p < 0.0001. One-way ANOVA. Error bars indicate ±SEM.(F) Confocal microscopy (2x2 Tiles) showing expression of F4/80 (blue), SPP1 (green), EPCAM (yellow), CD31 (red), Desmin (cyan), and tissue autofluorescence (gray) in livers of SD- and WD-fed mice (36 weeks). Scale bar, 100 µm. White arrows identify SPP1+ macrophages. Inset shows zoomed in images showing colocalization of SPP1 and F4/80 signal (36 weeks WD). Scale bar, 10 µm. Images are representative of 6 mice from 2 independent experiments. See also Figure S6.