Fig 2: 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 3: A proteogenomic atlas of the healthy murine liver(A) Hepatic cells were isolated from healthy C57B/l6 mice by ex vivo (5 mice, 15 samples) or in vivo (5 mice, 19 samples) enzymatic digestion. Alternatively, nuclei were isolated by tissue homogenization (4 mice, 12 samples). Live cells/intact nuclei were FACS-purified. For cells, total live, live CD45+, live CD45−, live hepatocytes, or myeloid cells (live CD45+, CD3−, CD19−, B220−, NK1.1−) were sorted. 18 samples (7 ex vivo, 11 in vivo) were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis. All datasets were pooled together and after QC 185,894 cells/nuclei were clustered using TotalVI.(B) UMAP of sc/snRNA-seq data.(C) Tissue and capsule images from Visium analysis with clusters overlaid.(D) UMAP of zonation of Visium spots (left) and origin of the cells (right).(E) Zonation pattern mapped onto tissue slice.(F and G) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data.(H) mRNA zonation pattern in Visium highly multiplexed protein analysis and VSIG4-ADT expression pattern (left) and zonated expression patterns of indicated antibodies (right).(I) MICS analysis of indicated proteins.(J) Molecular Cartography of indicated genes and cell types.(K) mRNA (Xcr1, Flt3l, Mafb, and Clec10a) and protein (MHCII and F4/80) expression in the same tissue slice. Scale bars, 50 μm. PV, portal vein; CV, central vein. Arrows indicate specific cell types, colors correspond to cell type/markers. Images are representative of 2–4 mice. See also Figure S2 and Tables S1, S2, S3, and S5.

Fig 4: A population of macrophages reside around the bile duct in the healthy murine liver(A) UMAP of murine myeloid cells (71,261 cells/nuclei) isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) between cell types.(D) Expression of Gpnmb and Cd207.(E) Expression of VSIG4 and F4/80 (left) or MHCII, CD11c, and DAPI (right) by confocal microscopy. Capsule macs identified by white arrows. Scale bars, 50 µm.(F) Molecular Cartography of indicated genes at liver capsule.(G) Expression of VSIG4, F4/80, GLUL, and DAPI (left) or F4/80 or CCR2 (right, inset) by confocal microscopy. Scale bars, 100 µm.(H) Molecular Cartography of indicated genes at portal triad. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to markers. Images are representative of 2–4 mice.(I and J) Top GO terms for KCs (I) and bile-duct LAMs (J).(K) Representative image showing expression of VSIG4 (red) CD19 (yellow) and CD3E (magenta) by MICS analysis (left) and % of B or T cells found with/without a KC per field of view (right). Data are pooled from multiple fields of view in 2 mice. ***p < 0.001 Student’s t test.(L) Mice (29-week-old) were treated with 3.5 mg/kg LPS or PBS and 2 h later, livers were harvested without the capsule. KCs and LAMs were FACS-purified and expression of Il1b, Tnf, IL10, and Il18 was examined by qPCR, compared with b-actin. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Bonferroni post-test. See also Figure S3 and Table S2.

Fig 5: Validated flow cytometry gating strategy for murine myeloid cells, related to Figure 2(A) CITE-seq data from the murine myeloid cells in Figure 2A were exported as an FCS file and an in silico gating strategy identified in FlowJo software.(B) Application of the in silico gating strategy with a 21-color flow cytometry panel. Myeloid cells were pre-gated as live CD45+ lineage cells (Ly6G-CD19-NK1.1-B220-CD3-). Data are representative of 3 experiments with 3–6 mice per experiment.(C) cDC1s, cDC2s, migratory cDCs (Mig. cDCs), peritoneal macs (Peri. Macs), KCs, and non-KC macs (non-KCs) were FACS-purified using gating strategy in (B), mRNA was isolated and qPCR performed to examine expression of indicated genes defining each population to validate their identity. Data are representative of 2 experiments with n = 3–6.(D) Putative peritoneal macs were FACS-purified using gating strategy in (B) and expression of Gata6 was examined by qPCR compared with other hepatic myeloid populations. Data are from a single experiment with n = 6.(E) Peritoneal macs as a % of total macs recovered from the liver using different digestion techniques (in vivo, ex vivo, or capsule) or in supernatants in which livers were washed following removal from the mouse but prior to digestion (wash). Data are from a single experiment with n = 4. *p < 0.05, **p < 0.01 one-way ANOVA with Bonferroni post-test compared with wash data.(F) Expression of CD14 and CD207 within the non-KC mac population from (B) (left) and % of CD207+ and CD207- populations among total macs in livers digested using the ex vivo or in vivo protocols or in dissected and digested liver capsule (right). Data are representative of two experiments with n = 4–5 mice per experiment. ****p < 0.0001 mixed effects analysis with Tukey’s multiple comparison test.(G) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy. Insets represent zones featured in Figures 2E, 2G, and S3I.(H) Molecular Cartography of indicated genes and cell types. Insets represent zones featured in Figures 2F, 2H, and S3J.(I) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy at the central vein. Scale bars, 50 µm.(J) Molecular Cartography of indicated genes and cell types at central vein.(K) Expression of F4/80, EPCAM, CCR2, GPNMB, and DAPI by confocal microscopy at a portal vein (top) or F4/80 or GPNMB alone (bottom). Scale bars, 25 µm.(L) Quantification of % of Gpnmb & Trem2 counts over Adgre1 counts in indicated regions of tissue as assessed using Molecular Cartography data. Each dot represents an individual region. *p < 0.05, ****p > 0.0001 one-way ANOVA with Bonferroni post-test.(M) Expression of DESMIN and F4/80 at the liver capsule and underlying parenchyma (left) or EPCAM, DESMIN and F4/80 at the bile duct by confocal microscopy. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to cell type/markers. All images are representative of 2–6 mice.