Fig 2: 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.

Fig 3: 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 4: Identification of bona fide Kupffer cells across species(A) Cells/nuclei were isolated from liver biopsies (~1–2 mm3; 14 cells, 5 nuclei) from patients undergoing either liver resection, cholecystectomy or gastric bypass. Live cells/intact nuclei were FACS-purified. Either total live, live CD45+, and live CD45- or live CD45+, CD3-, and CD19- cells were sorted. 7 cell samples were stained with a panel of 198 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC, 167,598 cells/nuclei were analyzed using TotalVI.(B) UMAP of sc/snRNA-seq data.(C) UMAP of Visium data from 4 patient biopsy samples.(D) Split of Visium spots based on % steatosis.(E) Healthy and steatotic Visium liver tissue with clusters overlaid and H+E staining to identify steatotic zones.(F) Zonation of Visium data (top) with zonation pattern mapped onto liver tissue (bottom).(G) Indicated cell signatures from sc/snRNA-seq mapped onto Visium zonation trajectory, healthy (top), steatotic (bottom).(H) Myeloid cells (40,821 cells) were isolated from Figure 4B and re-clustered with TotalVI.(I) Expression of VSIG4 protein (top) and CD5L mRNA (bottom).(J) Expression of VSIG4, F4/80, FOLRB, and GLUL combined with Cd5l/CD5L on murine (left) and human (H25; right) livers. Scale bars, 50 µm. Inset in bottom panels. Scale bars, 20 µm. Images are representative of 2–4 livers.(K) Livers (2/species) were isolated from healthy macaque, pig, chicken, hamster, and zebrafish. Cells were isolated by ex vivo digestion for CITE-seq (pig; 198 human antibodies) or scRNA-seq (hamster, chicken, and zebrafish), or nuclei were isolated for snRNA-seq (macaque). Total live cells (hamster, chicken, and pig), DsRed+GFP+ cells (zebrafish) or nuclei (macaque) were FACS-purified. Following QC, 8,483 nuclei (macaque) or 21,907 (pig), 5,965 (hamster), 7,457 (chicken), and 4,957 (zebrafish) cells were analyzed using TotalVI (pig) or scVI (macaque, hamster, chicken, and zebrafish) (top). KCs were identified using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019) (bottom). See also Figures S6 and S7 and Tables S1, S2,S3, S5, S6,S8, and S9.

Fig 5: Evolutionarily conserved signals regulate LAM and KC development, related to Figures 5 and 7(A) Confocal microscopy of healthy human liver showing expression of indicated markers. Scale bars, 200 µm.(B and C) Expression of conserved human-murine bile-duct LAM signature in human (B) and mouse (C) hepatic myeloid cells.(D) Proportion of indicated myeloid cell populations as a % of total myeloid cells in human liver biopsies profiled by scRNA-seq when divided based on presence of steatosis.(E) Mice were fed a western diet (WD) or standard diet (SD) for 36 weeks to induce NAFLD and Visium analysis was performed. Analysis is pooled from 1 liver slice from the SD condition and 3 liver slices from the WD condition. Shown are cluster and sample annotations.(F) Zonation of all cell types from Figure S5J in murine NAFLD map (SD&WD).(G) Differential NicheNet highlighting prioritized conserved (human-mouse) ligand-receptor (LR) pairs between indicated macs and their niche cells. LR pairs are grouped according to the niche cell type with highest ligand expression.(H) Expression of ALK1 (ACVRL1), BMP9 (GDF2), and BMP10 in human, mouse, and macaque livers where both KCs and stellate cells were profiled.(I) Livers were harvested from Clec4f-CrexAcvrl1fl/fl mice or Acvrl1fl/fl controls and KCs examined (top) and quantified (middle) using VSIG4 expression. Expression of indicated KC markers by mac populations in Clec4f-CrexAcvrl1fl/fl or Acvrl1fl/fl control mice (bottom). Data are pooled from 2 independent experiments with n = 14 per group. Student’s t test. ****p < 0.0001.(J) Expression of CD31 (ECs), DESMIN (stromal cells), F4/80 (Macs), and EPCAM (cholangiocytes) by confocal microscopy in Fcgr1-CrexAcvrl1fl/fl mice and Acvrl1+/+ controls. PV, portal vein; CV, central vein. Images are representative of 2 mice per group.