Fig 2: Localization and Heterogeneity of Macrophages in MAFLD(A) Confocal microscopy showing cells expressing CLEC4F (red), F4/80 (green), TIM4 (blue), and CD31 (gray) in the livers of SD- or WD-fed mice at the indicated time points. Smaller images show results for individual channels; the larger image is merged from all channels. Scale bar, 50 µm. Images are representative of 5–6 mice per time point and are extracted from 4 × 4 tiled images. White arrows point to CLEC4F+TIM4- moKCs. Dashed line highlights the zones enriched for CLEC4F- macs.(B) Tile scans (4x4) of livers from SD- and WD-fed mice (36 weeks) showing annotation of identified macs per subset in indicated colors. Indicated regions (dashed lines) identify the areas from the WD-fed mouse used in (A). Shaded gray boxes identify large vessels (portal or central veins) that were excluded from quantification analysis in (C) and (E). Images are representative of 6 mice.(C) Quantification of indicated populations shown in (B) as a % of total F4/80+ macs. **p < 0.01, ****p < 0.0001, Student’s t test compared with SD control. Error bars indicate ±SEM.(D) Tile scan of liver from a WD-fed mouse (36 weeks; same mouse as from A and B) with identified Desminhi regions demarcated in blue. The rest of the tissue is classified as Desminlo, excluding the larger vessels, for quantification in (E).(E) Quantification of indicated populations in Desminhi and Desminlo zones (from D) as a % of each mac subset. *p < 0.05, Student’s t test compared with Desminlo area. Quantification data in (C) and (E) are pooled from 2 independent experiments with n = 6. Error bars indicate ±SEM.(F–I) Monocyte- and mac-containing clusters (based on expression of Mafb, Ly6c2, Ccr2, Fcgr1, Adgre1) were isolated from the CITE-Seq data (18,241 cells) and re-clustered.(F) UMAP showing annotated monocyte and macrophage clusters.(G) Distribution of cells on SD or WD at indicated time points, with SD data coming from cells pooled after 12, 24, and 36 weeks.(H and I) Expression of indicated genes by the different clusters (SD + WD pooled). See also Figure S5.

Fig 3: 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 4: Hepatic Immune Cell Transcriptome and Surface Proteome in MAFLDC57BL/6 mice were fed either an SD or WD for 12, 24, or 36 weeks, and livers were harvested. Total live CD45+ cells were sorted (1 mouse per time point per diet), stained with total-seq A antibodies, and loaded onto the 10X Chromium platform. After QC, 56407 cells remained.(A) UMAP showing distinct clusters among total CD45+ live cells.(B) Expression of indicated proteins based on CITE-Seq antibody binding.(C) Expression of indicated genes across the 25 clusters.(D) Annotation of the cell types within the UMAP based on both transcriptome and surface proteome.(E) Distribution of clusters from SD or WD, with SD data obtained from cells pooled after 12, 24, and 36 weeks.(F–H) Heatmaps showing top DEGs for Monocytes (F), KCs (G), and Clec4f- Macrophages (H) as assessed by comparing SD and WD samples pooled from all time points. Genes in red are conserved across multiple cell types.(I) MEM heatmap showing surface proteins whose expression was altered in at least 1 cell type during MAFLD.(J and K) CITE-Seq data were exported into FlowJo software, and (J) the KC cluster was gated and TIM4 and MerTK expression were examined at indicated time points on WD and in pooled SD-fed mice or (K) the T cell cluster was gated and CD8a and CD8ß expression were examined at 36 weeks on WD and in pooled SD-fed mice. See also Figure S2.