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PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
18 samples (7 ex vivo, 11 in vivo) were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Scale bars, 50 μm.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
PV, portal vein; CV, central vein.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Arrows indicate specific cell types, colors correspond to cell type/markers.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Images are representative of 2–4 mice.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
See also Figure S2 and Tables S1, S2, S3, and S5.Figure S2Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables identification of all hepatic cell types including bona fide cell doublets, related to Figure 1(A and B) Top DEGs (A) and DEPs (B) for cell types from Figure 1B.(C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocols; 71,162 cells from ex vivo digestions, 96,066 cells from in vivo digestions, and 18,666 nuclei.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Numbers on plots represent numbers of cells/nuclei per population.(D) Correlation plots showing genes captured within the KC, B cell and neutrophil populations with and without addition of CITE-seq antibodies.(E) Expression of VSIG4, CD206, and ESAM (protein, top) and Vsig4, Mrc1, and Esam (mRNA, bottom).(F) UMAP showing clusters of cells when only minimal QC for gene number and % mitochondrial genes is performed; 17,669 cells pooled from 3 samples.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Expression of Cd5l, Cd19, and Kdr by the clusters facilitating identification of cell types per annotation.(G) CITE-seq data from (F) in Flow-Jo showing expression of CD206 and ESAM in total KCs (left) and total B cells (middle).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Numbers represent % of entire KC or B cell population.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Identified populations were then mapped back onto the original UMAP (right).(H) Expression of CD31, CD26, and CD38 by indicated populations.(I) Heatmaps showing expression of top DEGs between KC1s and LSECs (left), KC2s and KC1s + LSECs (middle) and B cell2s and B cell1s + LSECs (right).(J) 3D reconstruction of murine liver following perfusion with antigen fix to inflate endothelial cells and staining with antibodies against CD31, CD206, and F4/80.(K) UMAP showing clusters generated from Visium analysis of liver tissue (4 samples) and liver capsule (1 sample).(L) Top unbiased genes defining zonation trajectory from portal to central vein in Visium.(M) Expression of Glul and Epcam by confocal microscopy (left), annotation of portal, periportal, mid, and central regions on same tissue section (middle) and overlay of both datasets (right).(N) Identification of cholangiocyte (left) and cDC (right) signatures on zonated Visium spots.(P) Molecular Cartography showing expression of indicated zonated hepatocyte mRNAs in liver tissue.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Data are representative of 2 mice.(O) Expression of Itgae (encoding CD103) in the UMAP of the total liver (left) and flow cytometric analysis of total cDC1s for CD103 and MHCII expression in the healthy murine liver (right).Figure 2A 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.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Capsule macs identified by white arrows.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Scale bars, 100 μm.(H) Molecular Cartography of indicated genes at portal triad.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Arrows indicate specific cell types, where color corresponds to markers.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Data are pooled from multiple fields of view in 2 mice.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
KCs and LAMs were FACS-purified and expression of Il1b, Tnf, IL10, and Il18 was examined by qPCR, compared with b-actin.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
p < 0.05, p < 0.01, p < 0.001, p < 0.0001, one-way ANOVA with Bonferroni post-test.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
See also Figure S3 and Table S2.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Cell types identified in transcriptomic studies depend upon cell/nuclei isolation technique used, related to Figure 1 Cells were isolated from livers of healthy C57B/l6 mice by either ex vivo or in vivo enzymatic digestion.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Alternatively, livers were snap frozen and nuclei subsequently isolated following tissue homogenization by a sucrose gradient (3 mice per isolation method).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Live cells/intact nuclei were identified and purified using flow cytometry.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
For the cells, either live CD45, live CD45 or live hepatocytes were sorted.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
1 ex vivo digested sample and 1 in vivo digested sample were also stained with a panel of 107 (ex vivo cells) or 161 (in vivo cells) oligo-conjugated antibodies for CITE-seq analysis.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
FACS-purified cells/nuclei were loaded onto the 10× chromium platform and scRNA-seq, CITE-seq, or snRNA-seq performed.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Following clean up and QC, cells from the same mice were pooled together in the same ratios (CD45:CD45:Heps) as found in the tissue as a whole before sorting, different mice were then pooled together and the data were analyzed using scVI. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
A–C) UMAPs showing annotations of cell types and proportions of each cell type as a % of total cells in the UMAP isolated using (A) ex vivo digestion; 13,144 cells, (B) in vivo digestion; 24,014 cells and (C) nuclei; 8,583 nuclei. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
D) Average number of genes/cell in the annotated mac, B cell, hepatocyte, endothelial, and stromal cell populations following each isolation method.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
p < 0.05 one-way ANOVA with Bonferroni post-test per cell type. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
E) Correlation plot showing genes captured within the mac population when the liver is digested with the in vitro versus the in vivo digestion protocol. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
F) Correlation plots showing genes captured within the mac, endothelial cell, and hepatocyte populations when cells are isolated using the in vivo digestion protocol or nuclei are isolated. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
G–L) Confocal microscopy images to determine true abundance of (G) stromal cells and cholangiocytes (H) endothelial cells, (I) macs, (J) dendritic cells, (K) B cells, and (L) T cells in vivo.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Scale bars, 200 μm. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
M) The percentage of each population was calculated based on the percentage of a given population divided by the total number of nuclei.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
A threshold was applied to the DAPI channel (picture 1) in ImageJ (picture 2) and nuclei were automatically counted based on the ImageJ “analyze particles” plugin (size ).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Due to the density of some liver zones, some nuclei were not automatically counted (arrow, picture 3).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Those were then manually counted and added to the total number of nuclei.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
For the populations of interest, cells were counted manually based on specific markers (for example, CD3 for T cells, picture 4).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Counting was performed blinded prior to analysis of the sequencing results. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
N) Proportion of indicated cell types as a % of total cells identified in confocal microscopy images.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Data are from 3–7 images per cell type taken from 2–4 mice.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Alternatively, nuclei were isolated by tissue homogenization (4 mice, 12 samples).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Live cells/intact nuclei were FACS-purified.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
For cells, total live, live CD45, live CD45, live hepatocytes, or myeloid cells (live CD45, CD3, CD19, B220, NK1.1) were sorted.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
18 samples (7 ex vivo, 11 in vivo) were also stained with a panel of 107–161 barcode-labeled antibodies for CITE-seq analysis.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
All datasets were pooled together and after QC 185,894 cells/nuclei were clustered using TotalVI. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
B) UMAP of sc/snRNA-seq data. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
C) Tissue and capsule images from Visium analysis with clusters overlaid. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
D) UMAP of zonation of Visium spots (left) and origin of the cells (right). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
E) Zonation pattern mapped onto tissue slice. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
F and G) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
H) mRNA zonation pattern in Visium highly multiplexed protein analysis and VSIG4-ADT expression pattern (left) and zonated expression patterns of indicated antibodies (right). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
I) MICS analysis of indicated proteins. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
J) Molecular Cartography of indicated genes and cell types. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
K) mRNA (Xcr1, Flt3l, Mafb, and Clec10a) and protein (MHCII and F4/80) expression in the same tissue slice.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Scale bars, 50 μm.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
PV, portal vein; CV, central vein.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Arrows indicate specific cell types, colors correspond to cell type/markers.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Images are representative of 2–4 mice.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
See also Figure S2 and Tables S1, S2, S3, and S5.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables identification of all hepatic cell types including bona fide cell doublets, related to Figure 1 (A and B) Top DEGs (A) and DEPs (B) for cell types from Figure 1B. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocols; 71,162 cells from ex vivo digestions, 96,066 cells from in vivo digestions, and 18,666 nuclei.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Numbers on plots represent numbers of cells/nuclei per population. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
D) Correlation plots showing genes captured within the KC, B cell and neutrophil populations with and without addition of CITE-seq antibodies. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
E) Expression of VSIG4, CD206, and ESAM (protein, top) and Vsig4, Mrc1, and Esam (mRNA, bottom). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
F) UMAP showing clusters of cells when only minimal QC for gene number and % mitochondrial genes is performed; 17,669 cells pooled from 3 samples.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Expression of Cd5l, Cd19, and Kdr by the clusters facilitating identification of cell types per annotation. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
G) CITE-seq data from (F) in Flow-Jo showing expression of CD206 and ESAM in total KCs (left) and total B cells (middle).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Numbers represent % of entire KC or B cell population.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Identified populations were then mapped back onto the original UMAP (right). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
H) Expression of CD31, CD26, and CD38 by indicated populations. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
I) Heatmaps showing expression of top DEGs between KC1s and LSECs (left), KC2s and KC1s + LSECs (middle) and B cell2s and B cell1s + LSECs (right). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
J) 3D reconstruction of murine liver following perfusion with antigen fix to inflate endothelial cells and staining with antibodies against CD31, CD206, and F4/80. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
K) UMAP showing clusters generated from Visium analysis of liver tissue (4 samples) and liver capsule (1 sample). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
L) Top unbiased genes defining zonation trajectory from portal to central vein in Visium. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
M) Expression of Glul and Epcam by confocal microscopy (left), annotation of portal, periportal, mid, and central regions on same tissue section (middle) and overlay of both datasets (right). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
N) Identification of cholangiocyte (left) and cDC (right) signatures on zonated Visium spots. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
P) Molecular Cartography showing expression of indicated zonated hepatocyte mRNAs in liver tissue.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Data are representative of 2 mice. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
O) Expression of Itgae (encoding CD103) in the UMAP of the total liver (left) and flow cytometric analysis of total cDC1s for CD103 and MHCII expression in the healthy murine liver (right).
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
B and C) Top DEGs (B) and DEPs (C) between cell types. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
D) Expression of Gpnmb and Cd207. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
E) Expression of VSIG4 and F4/80 (left) or MHCII, CD11c, and DAPI (right) by confocal microscopy.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Capsule macs identified by white arrows.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Scale bars, 50 μm. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
F) Molecular Cartography of indicated genes at liver capsule. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
G) Expression of VSIG4, F4/80, GLUL, and DAPI (left) or F4/80 or CCR2 (right, inset) by confocal microscopy.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Scale bars, 100 μm. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
H) Molecular Cartography of indicated genes at portal triad.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Arrows indicate specific cell types, where color corresponds to markers.
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
Images are representative of 2–4 mice. (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
I and J) Top GO terms for KCs (I) and bile-duct LAMs (J). (
PMC8809252
Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
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).