Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	All images are representative of 2–6 mice.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Protein markers of murine CD45 cell subsets, related to Figure 3 (A) CITE-seq data from the murine CD45 cells in Figure 3A were exported as an FCS file and an in silico gating strategy identified in FlowJo. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	B) Gated cell overlay of populations identified using strategy in (A). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	C) Expression of CD90, CD204, CD73, and CD29 markers by indicated cell types. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	D) Expression of indicated protein markers in 60-plex MICS analysis in endothelial cells. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	E) Expression of DESMIN, EPCAM, LYVE1, and CD31 at a portal triad (left) with inset (right). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	F) Expression of indicated protein markers in 60-plex MICS analysis in stromal cells. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	G) Molecular Cartography of indicated genes and cell types at portal vein.
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, where color corresponds to cell type/markers.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	All images are representative of 2–6 mice.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	We next sought to investigate the differences between KCs and LAMs.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Analysis of GO terms associated with biological processes for these cells suggested that KCs may play a role in regulating humoral responses, while LAMs were more broadly associated with immune responses (Figures 2I and 2J).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Consistent with this, in the 100-plex protein microscopy data we noted that a significant proportion of the B cells present were interacting with KCs, which was not observed with T cells (Figure 2K).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This suggests cross-talk between these two populations, potentially linked to the high expression of the B cell chemokine Cxcl13 by murine KCs (Table S2).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To assess the inflammatory nature of LAMs compared with KCs, we FACS-purified the cells and performed qPCR analysis to examine expression of various cytokines.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To enable LAM purification, we eliminated the capsule prior to digesting the tissue.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Fitting with the GO analysis, LAMs expressed more Il1b at steady state compared with KCs (Figure 2L).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, despite this, upon in vivo TLR4 stimulation, they were less responsive than KCs, both in terms of pro- and anti-inflammatory cytokines (Figure 2L), possibly indicative of LPS tolerance.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This may result from their location at the PV and hence exposure to blood from the intestine, although this remains to be tested.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Taken together this highlights the distinct nature and functions of these cells; however, further research is required to determine the precise roles of these cells.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	As all the mac populations are in close contact with CD45 cells in their local environment (Bonnardel et al., 2019; Figure S3M), we further analyzed the CD45 cells, identifying multiple subsets of ECs and SCs and a gating strategy to distinguish them (Figures 3A–3C and S4A–S4C; Table S3).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	ECs could be further subdivided into 4 distinct clusters and analysis of their locations allowed them to be identified as CV ECs (cluster10), LSECs (cluster9), PV ECs (cluster11), and lymphatic ECs (LECs; cluster12) (Figures 3D, 3E, S4D, and S4E).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	As Visium found fibroblasts at both the PVs and CVs (Figure 3D), and as a previous report has suggested the presence of distinct subsets within these cells (Dobie et al., 2019), we further zoomed in on the SCs to better assess their heterogeneity (Figures 3F, 3G, S4A–S4C, and S4F; Table S4).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This revealed subsets of mesothelial cells and fibroblasts restricted to the capsule (Figures 3H and 3I).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Myh11 vascular smooth muscle cells (VSMCs) were localized around hepatic arteries, PVs and CVs (Figures 3F–3H, 3J, and S4G) and Mfap4Svep1Clic5Reln fibroblasts were found to be CV fibroblasts (Figures 3G, 3H, 3J, and S4G).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Finally, we identified a subset of Clic5Reln fibroblasts (cluster3), which were localized around the cholangiocytes, that we termed bile-duct fibroblasts (Figures 3J and S4G).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Taken together, the presence of these spatially distinct subsets of ECs and SCs highlights the uniqueness of the specific microenvironments in which the distinct mac populations reside.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Figure 3Hepatic macrophage populations reside in distinct niches(A) UMAP of murine CD45 cells (83,410 cells/nuclei) isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) between cell types.(D) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data.(E) Molecular Cartography of indicated genes at central vein (left) and 2 different portal triads (center and right).(F) UMAP of murine stromal cells (5,430 cells/nuclei) isolated from the UMAP in Figure 3A and re-clustered with scVI.(G) Top DEGs between different cell types identified.(H) Identification of mesothelial cell (top) and VSMC (bottom) signatures on zonated Visium data.(I and J) Molecular Cartography of indicated genes at the liver capsule (I) or the central vein (J; left) and portal triad (J; right).
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.	See also Figure S4 and Tables S3 and S4.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Hepatic macrophage populations reside in distinct niches (A) UMAP of murine CD45 cells (83,410 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) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	E) Molecular Cartography of indicated genes at central vein (left) and 2 different portal triads (center and right). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	F) UMAP of murine stromal cells (5,430 cells/nuclei) isolated from the UMAP in Figure 3A and re-clustered with scVI. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	G) Top DEGs between different cell types identified. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	H) Identification of mesothelial cell (top) and VSMC (bottom) signatures on zonated Visium data. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	I and J) Molecular Cartography of indicated genes at the liver capsule (I) or the central vein (J; left) and portal triad (J; right).
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.	See also Figure S4 and Tables S3 and S4.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Finally, in addition to providing gating strategies for myeloid and CD45 cells, we also wanted to investigate if the CITE-seq data would allow us to develop similar strategies for the lymphoid populations.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To examine this, we re-clustered the T cells, NK cells, ILCs, B cells, and pDCs from Figure 1B identifying 12 distinct populations (Figure S5A).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Analysis of the DEGs highlighted that while B cells, NK cells, ILC1s, and pDCs were distinct populations, there was considerable overlap between the transcriptomic profiles of the T cells (Figure S5B; Table S5).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, by analyzing the DEPs we were able to define distinct subsets including naive CD4 and CD8 T cells, TRegs, TH1s, CTLs, and TH17s (Figures S5A and S5C; Table S5).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Moreover, we were able to design a gating strategy to isolate the distinct populations (Figure S5D).Figure S5Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas, related to Figure 4(A) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 27,398 cells) were isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) for the cell types from Figure S5A.(D) CITE-seq data from Figure S5A were exported as an FCS file and an in silico gating strategy identified in FlowJo.(E) Human lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 105,790 cells) were isolated from Figure 4B and re-clustered with TotalVI.(F and G) Top DEGs (F) and DEPs (G) for the cell types from Figure S5E.(H) CITE-seq data from Figure S5E were exported as an FCS file and an in silico gating strategy identified in FlowJo.(I) Proportion of indicated cell types arising from patients with <10% (purple) or >10% steatosis (yellow).(J) Hepatic cells were isolated from 22 C57B/l6 mice fed either a standard diet (SD) or a western diet (WD) for 24 or 36 weeks to induce NAFLD and NASH by ex vivo (10 samples) or in vivo (12 samples) enzymatic digestion.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Alternatively, livers were snap frozen and nuclei isolated by tissue homogenization (14 samples).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Live cells/intact nuclei were purified using FACS.
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.	10 samples 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 121,980 cells/nuclei were clustered using TotalVI.(K) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 21,322 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI.(L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow).(M) Top DEGs between CTLs isolated from mice fed the SD (purple) or WD (yellow).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas, related to Figure 4 (A) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 27,398 cells) were 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) for the cell types from Figure S5A. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	D) CITE-seq data from Figure S5A were exported as an FCS file and an in silico gating strategy identified in FlowJo. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	E) Human lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 105,790 cells) were isolated from Figure 4B and re-clustered with TotalVI. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	F and G) Top DEGs (F) and DEPs (G) for the cell types from Figure S5E. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	H) CITE-seq data from Figure S5E were exported as an FCS file and an in silico gating strategy identified in FlowJo. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	I) Proportion of indicated cell types arising from patients with <10% (purple) or >10% steatosis (yellow). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	J) Hepatic cells were isolated from 22 C57B/l6 mice fed either a standard diet (SD) or a western diet (WD) for 24 or 36 weeks to induce NAFLD and NASH by ex vivo (10 samples) or in vivo (12 samples) enzymatic digestion.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Alternatively, livers were snap frozen and nuclei isolated by tissue homogenization (14 samples).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Live cells/intact nuclei were purified using FACS.
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.	10 samples 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 121,980 cells/nuclei were clustered using TotalVI. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	K) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 21,322 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	M) Top DEGs between CTLs isolated from mice fed the SD (purple) or WD (yellow).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To determine the degree of conservation between the mac subsets and their different microenvironmental niches between the mouse and the human liver, we next generated a proteogenomic atlas of the human liver using sc/snRNA-seq and CITE-seq on 19 liver biopsies (Figures 4A, 4B, S6A, and S6B; Tables S1 and S5).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Of these, most were histologically healthy with only 5 patients showing >10% hepatic steatosis in the absence of any significant fibrosis (Table S6).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Cellular proportions varied according to the isolation technique used, and while there was some variability between patients, this was not linked to the surgery (Figures S6C–S6E).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Further confirming the lack of fibrosis in the steatotic livers, we did not detect any increase in CTLs (Figures S5E–S5I; Table S5), which have been shown to correlate with non-alcoholic steatohepatitis (NASH) (Haas et al., 2019).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A significant increase in CTLs was detected in the setting of murine NASH induced by feeding a western diet (WD) for up to 36 weeks, demonstrating that this is not due a limitation in detecting these differences using CITE-seq (Figures S5J–S5M; Table S7).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	As Visium reliably located murine hepatic cells, we used this to locate the cells of the human liver in 4 biopsies (Figure 4C).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, the Visium spots from patients with >10% steatosis were found to cluster separately from the healthy samples (<10% steatosis; Figures 4D and 4E).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	We therefore used the healthy samples to calculate a baseline zonation and then transferred this trajectory onto the steatotic samples (Figures 4F and S6F).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This identified the steatosis to be predominantly present in regions expressing peri-central zonation genes like CYP2E1.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This zonation pattern was further validated using Molecular Cartography (Figure S6G).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This fits with previous clinical studies demonstrating peri-central steatosis to be most common in non-alcoholic fatty liver disease (NAFLD) patients, especially in early disease (Chalasani et al., 2008; Kleiner and Makhlouf, 2016).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, as these peri-central regions are larger than in the healthy controls, it could also imply that the presence of steatosis alters expression of the zonated hepatocyte genes, but this remains to be tested.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Notably, the overall cellular distribution was not impacted by the presence of steatosis, although neutrophils and monocytes and monocyte-derived cells were preferentially localized peri-centrally in the steatotic patients correlating with the presence of steatosis (Figure 4G).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	MICS 100-plex protein analysis further validated the cellular distributions predicted by Visium and confirmed the increase in neutrophils in the steatotic livers (Figures S6H and S6I).Figure 4Identification of bona fide Kupffer cells across species(A) Cells/nuclei were isolated from liver biopsies (∼1–2 mm; 14 cells, 5 nuclei) from patients undergoing either liver resection, cholecystectomy or gastric bypass.
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.	Either total live, live CD45, and live CD45 or live CD45, CD3, and CD19 cells were sorted.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	7 cell samples were stained with a panel of 198 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, 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.
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.	Inset in bottom panels.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Scale bars, 20 μm.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Images are representative of 2–4 livers.(K) Livers (2/species) were isolated from healthy macaque, pig, chicken, hamster, and zebrafish.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	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).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Total live cells (hamster, chicken, and pig), DsRedGFP cells (zebrafish) or nuclei (macaque) were FACS-purified.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	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).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	KCs were identified using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019) (bottom).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	See also Figures S6 and S7 and Tables S1, S2,S3, S5, S6,S8, and S9.Figure S6Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas and identification of bona fide human KCs, related to Figure 4(A and B) Top DEGs (A) and DEPs (B) for the cell types from Figure 4B.(C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocol used; 152,535 cells from ex vivo digestions and 15,063 nuclei.(D) Proportion of each cell type per patient profiled.(E) Proportion of indicated cell types as a % of total CD45 cells calculated from ex vivo digested samples per surgery type.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Ch; cholecystectomy, Re; resection, GB; gastric bypass.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	p < 0.05; one-way ANOVA with Bonferroni post-test.(F) Mapping of Visium UMAP zonation patterns onto tissue sections from patient H35 and H37.(G) Expression of indicated zonation genes in patients H35–H38 assessed by Molecular Cartography.(H and I) Expression of indicated proteins by MICS 100-plex protein analysis in the healthy (H) and steatotic (I) human liver.(J) Murine myeloid cells (cDC1s, cDC2s, Mig.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

All images are representative of 2–6 mice.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Protein markers of murine CD45 cell subsets, related to Figure 3 (A) CITE-seq data from the murine CD45 cells in Figure 3A were exported as an FCS file and an in silico gating strategy identified in FlowJo. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

B) Gated cell overlay of populations identified using strategy in (A). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

C) Expression of CD90, CD204, CD73, and CD29 markers by indicated cell types. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

D) Expression of indicated protein markers in 60-plex MICS analysis in endothelial cells. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

E) Expression of DESMIN, EPCAM, LYVE1, and CD31 at a portal triad (left) with inset (right). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

F) Expression of indicated protein markers in 60-plex MICS analysis in stromal cells. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

G) Molecular Cartography of indicated genes and cell types at portal vein.

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, where color corresponds to cell type/markers.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

All images are representative of 2–6 mice.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

We next sought to investigate the differences between KCs and LAMs.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Analysis of GO terms associated with biological processes for these cells suggested that KCs may play a role in regulating humoral responses, while LAMs were more broadly associated with immune responses (Figures 2I and 2J).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Consistent with this, in the 100-plex protein microscopy data we noted that a significant proportion of the B cells present were interacting with KCs, which was not observed with T cells (Figure 2K).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This suggests cross-talk between these two populations, potentially linked to the high expression of the B cell chemokine Cxcl13 by murine KCs (Table S2).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To assess the inflammatory nature of LAMs compared with KCs, we FACS-purified the cells and performed qPCR analysis to examine expression of various cytokines.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To enable LAM purification, we eliminated the capsule prior to digesting the tissue.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Fitting with the GO analysis, LAMs expressed more Il1b at steady state compared with KCs (Figure 2L).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, despite this, upon in vivo TLR4 stimulation, they were less responsive than KCs, both in terms of pro- and anti-inflammatory cytokines (Figure 2L), possibly indicative of LPS tolerance.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This may result from their location at the PV and hence exposure to blood from the intestine, although this remains to be tested.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Taken together this highlights the distinct nature and functions of these cells; however, further research is required to determine the precise roles of these cells.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

As all the mac populations are in close contact with CD45 cells in their local environment (Bonnardel et al., 2019; Figure S3M), we further analyzed the CD45 cells, identifying multiple subsets of ECs and SCs and a gating strategy to distinguish them (Figures 3A–3C and S4A–S4C; Table S3).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

ECs could be further subdivided into 4 distinct clusters and analysis of their locations allowed them to be identified as CV ECs (cluster10), LSECs (cluster9), PV ECs (cluster11), and lymphatic ECs (LECs; cluster12) (Figures 3D, 3E, S4D, and S4E).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

As Visium found fibroblasts at both the PVs and CVs (Figure 3D), and as a previous report has suggested the presence of distinct subsets within these cells (Dobie et al., 2019), we further zoomed in on the SCs to better assess their heterogeneity (Figures 3F, 3G, S4A–S4C, and S4F; Table S4).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This revealed subsets of mesothelial cells and fibroblasts restricted to the capsule (Figures 3H and 3I).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Myh11 vascular smooth muscle cells (VSMCs) were localized around hepatic arteries, PVs and CVs (Figures 3F–3H, 3J, and S4G) and Mfap4Svep1Clic5Reln fibroblasts were found to be CV fibroblasts (Figures 3G, 3H, 3J, and S4G).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Finally, we identified a subset of Clic5Reln fibroblasts (cluster3), which were localized around the cholangiocytes, that we termed bile-duct fibroblasts (Figures 3J and S4G).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Taken together, the presence of these spatially distinct subsets of ECs and SCs highlights the uniqueness of the specific microenvironments in which the distinct mac populations reside.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Figure 3Hepatic macrophage populations reside in distinct niches(A) UMAP of murine CD45 cells (83,410 cells/nuclei) isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) between cell types.(D) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data.(E) Molecular Cartography of indicated genes at central vein (left) and 2 different portal triads (center and right).(F) UMAP of murine stromal cells (5,430 cells/nuclei) isolated from the UMAP in Figure 3A and re-clustered with scVI.(G) Top DEGs between different cell types identified.(H) Identification of mesothelial cell (top) and VSMC (bottom) signatures on zonated Visium data.(I and J) Molecular Cartography of indicated genes at the liver capsule (I) or the central vein (J; left) and portal triad (J; right).

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.

See also Figure S4 and Tables S3 and S4.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Hepatic macrophage populations reside in distinct niches (A) UMAP of murine CD45 cells (83,410 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) Indicated cell signatures from sc/snRNA-seq mapped onto the Visium zonation data. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

E) Molecular Cartography of indicated genes at central vein (left) and 2 different portal triads (center and right). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

F) UMAP of murine stromal cells (5,430 cells/nuclei) isolated from the UMAP in Figure 3A and re-clustered with scVI. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

G) Top DEGs between different cell types identified. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

H) Identification of mesothelial cell (top) and VSMC (bottom) signatures on zonated Visium data. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

I and J) Molecular Cartography of indicated genes at the liver capsule (I) or the central vein (J; left) and portal triad (J; right).

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.

See also Figure S4 and Tables S3 and S4.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Finally, in addition to providing gating strategies for myeloid and CD45 cells, we also wanted to investigate if the CITE-seq data would allow us to develop similar strategies for the lymphoid populations.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To examine this, we re-clustered the T cells, NK cells, ILCs, B cells, and pDCs from Figure 1B identifying 12 distinct populations (Figure S5A).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Analysis of the DEGs highlighted that while B cells, NK cells, ILC1s, and pDCs were distinct populations, there was considerable overlap between the transcriptomic profiles of the T cells (Figure S5B; Table S5).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, by analyzing the DEPs we were able to define distinct subsets including naive CD4 and CD8 T cells, TRegs, TH1s, CTLs, and TH17s (Figures S5A and S5C; Table S5).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Moreover, we were able to design a gating strategy to isolate the distinct populations (Figure S5D).Figure S5Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas, related to Figure 4(A) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 27,398 cells) were isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) for the cell types from Figure S5A.(D) CITE-seq data from Figure S5A were exported as an FCS file and an in silico gating strategy identified in FlowJo.(E) Human lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 105,790 cells) were isolated from Figure 4B and re-clustered with TotalVI.(F and G) Top DEGs (F) and DEPs (G) for the cell types from Figure S5E.(H) CITE-seq data from Figure S5E were exported as an FCS file and an in silico gating strategy identified in FlowJo.(I) Proportion of indicated cell types arising from patients with <10% (purple) or >10% steatosis (yellow).(J) Hepatic cells were isolated from 22 C57B/l6 mice fed either a standard diet (SD) or a western diet (WD) for 24 or 36 weeks to induce NAFLD and NASH by ex vivo (10 samples) or in vivo (12 samples) enzymatic digestion.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Alternatively, livers were snap frozen and nuclei isolated by tissue homogenization (14 samples).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Live cells/intact nuclei were purified using FACS.

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.

10 samples 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 121,980 cells/nuclei were clustered using TotalVI.(K) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 21,322 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI.(L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow).(M) Top DEGs between CTLs isolated from mice fed the SD (purple) or WD (yellow).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas, related to Figure 4 (A) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 27,398 cells) were 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) for the cell types from Figure S5A. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

D) CITE-seq data from Figure S5A were exported as an FCS file and an in silico gating strategy identified in FlowJo. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

E) Human lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 105,790 cells) were isolated from Figure 4B and re-clustered with TotalVI. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

F and G) Top DEGs (F) and DEPs (G) for the cell types from Figure S5E. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

H) CITE-seq data from Figure S5E were exported as an FCS file and an in silico gating strategy identified in FlowJo. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

I) Proportion of indicated cell types arising from patients with <10% (purple) or >10% steatosis (yellow). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

J) Hepatic cells were isolated from 22 C57B/l6 mice fed either a standard diet (SD) or a western diet (WD) for 24 or 36 weeks to induce NAFLD and NASH by ex vivo (10 samples) or in vivo (12 samples) enzymatic digestion.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Alternatively, livers were snap frozen and nuclei isolated by tissue homogenization (14 samples).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Live cells/intact nuclei were purified using FACS.

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.

10 samples 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 121,980 cells/nuclei were clustered using TotalVI. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

K) Murine lymphoid cells (B cells, T cells, NK cells, ILC1s, pDCs; 21,322 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

M) Top DEGs between CTLs isolated from mice fed the SD (purple) or WD (yellow).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To determine the degree of conservation between the mac subsets and their different microenvironmental niches between the mouse and the human liver, we next generated a proteogenomic atlas of the human liver using sc/snRNA-seq and CITE-seq on 19 liver biopsies (Figures 4A, 4B, S6A, and S6B; Tables S1 and S5).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Of these, most were histologically healthy with only 5 patients showing >10% hepatic steatosis in the absence of any significant fibrosis (Table S6).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Cellular proportions varied according to the isolation technique used, and while there was some variability between patients, this was not linked to the surgery (Figures S6C–S6E).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Further confirming the lack of fibrosis in the steatotic livers, we did not detect any increase in CTLs (Figures S5E–S5I; Table S5), which have been shown to correlate with non-alcoholic steatohepatitis (NASH) (Haas et al., 2019).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A significant increase in CTLs was detected in the setting of murine NASH induced by feeding a western diet (WD) for up to 36 weeks, demonstrating that this is not due a limitation in detecting these differences using CITE-seq (Figures S5J–S5M; Table S7).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

As Visium reliably located murine hepatic cells, we used this to locate the cells of the human liver in 4 biopsies (Figure 4C).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, the Visium spots from patients with >10% steatosis were found to cluster separately from the healthy samples (<10% steatosis; Figures 4D and 4E).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

We therefore used the healthy samples to calculate a baseline zonation and then transferred this trajectory onto the steatotic samples (Figures 4F and S6F).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This identified the steatosis to be predominantly present in regions expressing peri-central zonation genes like CYP2E1.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This zonation pattern was further validated using Molecular Cartography (Figure S6G).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This fits with previous clinical studies demonstrating peri-central steatosis to be most common in non-alcoholic fatty liver disease (NAFLD) patients, especially in early disease (Chalasani et al., 2008; Kleiner and Makhlouf, 2016).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, as these peri-central regions are larger than in the healthy controls, it could also imply that the presence of steatosis alters expression of the zonated hepatocyte genes, but this remains to be tested.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Notably, the overall cellular distribution was not impacted by the presence of steatosis, although neutrophils and monocytes and monocyte-derived cells were preferentially localized peri-centrally in the steatotic patients correlating with the presence of steatosis (Figure 4G).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

MICS 100-plex protein analysis further validated the cellular distributions predicted by Visium and confirmed the increase in neutrophils in the steatotic livers (Figures S6H and S6I).Figure 4Identification of bona fide Kupffer cells across species(A) Cells/nuclei were isolated from liver biopsies (∼1–2 mm; 14 cells, 5 nuclei) from patients undergoing either liver resection, cholecystectomy or gastric bypass.

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.

Either total live, live CD45, and live CD45 or live CD45, CD3, and CD19 cells were sorted.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

7 cell samples were stained with a panel of 198 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, 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.

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.

Inset in bottom panels.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Scale bars, 20 μm.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Images are representative of 2–4 livers.(K) Livers (2/species) were isolated from healthy macaque, pig, chicken, hamster, and zebrafish.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

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).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Total live cells (hamster, chicken, and pig), DsRedGFP cells (zebrafish) or nuclei (macaque) were FACS-purified.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

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).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

KCs were identified using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019) (bottom).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

See also Figures S6 and S7 and Tables S1, S2,S3, S5, S6,S8, and S9.Figure S6Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables generation of a human liver atlas and identification of bona fide human KCs, related to Figure 4(A and B) Top DEGs (A) and DEPs (B) for the cell types from Figure 4B.(C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocol used; 152,535 cells from ex vivo digestions and 15,063 nuclei.(D) Proportion of each cell type per patient profiled.(E) Proportion of indicated cell types as a % of total CD45 cells calculated from ex vivo digested samples per surgery type.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Ch; cholecystectomy, Re; resection, GB; gastric bypass.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

p < 0.05; one-way ANOVA with Bonferroni post-test.(F) Mapping of Visium UMAP zonation patterns onto tissue sections from patient H35 and H37.(G) Expression of indicated zonation genes in patients H35–H38 assessed by Molecular Cartography.(H and I) Expression of indicated proteins by MICS 100-plex protein analysis in the healthy (H) and steatotic (I) human liver.(J) Murine myeloid cells (cDC1s, cDC2s, Mig.