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.	cDCs, Macs, monocytes, and monocyte-derived cells; 42,922 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI.(K) Distribution of cells in UMAP originating from SD- (purple) or WD- (yellow) fed mice.(L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow).(M and N) Flow cytometry analysis of indicated cell populations in SD and WD-fed mice (24 weeks).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Representative gating strategies (M) and absolute number of indicated populations (N).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	p < 0.05, p < 0.01 Student’s t test.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Data are from 2 independent experiments with n = 5–6 per diet.(O and P) Top DEGs (O) and DEPs (P) for cell types from Figure 4H.(Q) Top 25 Murine KC genes as expressed by the human myeloid cell clusters.(R) Mapping of KC signature onto Visium trajectory for healthy (purple) and steatotic (orange) livers.(S) Expression of VSIG4 mRNA within human myeloid cells.(T) Expression of VSIG4 (red) and CD163 (gray, top) or CD169 (gray, bottom) by MICS analysis in healthy human liver.(U) Representative images showing KC location (red) as assessed by MICS analysis in the healthy (left) and steatotic (right) human liver.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	PV, portal vein; CV, central Vein, dashed line indicates zones of steatosis.(V) Representative image of CD68 and CD163 staining in 10–15-year-old human liver paraffin sections.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Image is representative of 6 different patients.(W) In silico gating strategy to isolate distinct myeloid cell populations identified from CITE-seq data.(X) Expression of VSIG4 and FOLR2 by live CD45 cells also expressing CD14 in indicated human liver biopsies by flow cytometry.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Data are representative of 21 biopsy samples analyzed.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Identification 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. (
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) UMAP of Visium data from 4 patient biopsy samples. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	D) Split of Visium spots based on % steatosis. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	E) Healthy and steatotic Visium liver tissue with clusters overlaid and H+E staining to identify steatotic zones. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	F) Zonation of Visium data (top) with zonation pattern mapped onto liver tissue (bottom). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	G) Indicated cell signatures from sc/snRNA-seq mapped onto Visium zonation trajectory, healthy (top), steatotic (bottom). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	H) Myeloid cells (40,821 cells) were isolated from Figure 4B and re-clustered with TotalVI. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	I) Expression of VSIG4 protein (top) and CD5L mRNA (bottom). (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	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. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	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.
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 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. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	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. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	D) Proportion of each cell type per patient profiled. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	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. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	F) Mapping of Visium UMAP zonation patterns onto tissue sections from patient H35 and H37. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	G) Expression of indicated zonation genes in patients H35–H38 assessed by Molecular Cartography. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	H and I) Expression of indicated proteins by MICS 100-plex protein analysis in the healthy (H) and steatotic (I) human liver. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	J) Murine myeloid cells (cDC1s, cDC2s, Mig.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	cDCs, Macs, monocytes, and monocyte-derived cells; 42,922 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.	K) Distribution of cells in UMAP originating from SD- (purple) or WD- (yellow) fed mice. (
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 and N) Flow cytometry analysis of indicated cell populations in SD and WD-fed mice (24 weeks).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Representative gating strategies (M) and absolute number of indicated populations (N).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	p < 0.05, p < 0.01 Student’s t test.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Data are from 2 independent experiments with n = 5–6 per diet. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	O and P) Top DEGs (O) and DEPs (P) for cell types from Figure 4H. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Q) Top 25 Murine KC genes as expressed by the human myeloid cell clusters. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	R) Mapping of KC signature onto Visium trajectory for healthy (purple) and steatotic (orange) livers. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	S) Expression of VSIG4 mRNA within human myeloid cells. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	T) Expression of VSIG4 (red) and CD163 (gray, top) or CD169 (gray, bottom) by MICS analysis in healthy human liver. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	U) Representative images showing KC location (red) as assessed by MICS analysis in the healthy (left) and steatotic (right) human liver.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	PV, portal vein; CV, central Vein, dashed line indicates zones of steatosis. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	V) Representative image of CD68 and CD163 staining in 10–15-year-old human liver paraffin sections.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Image is representative of 6 different patients. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	W) In silico gating strategy to isolate distinct myeloid cell populations identified from CITE-seq data. (
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	X) Expression of VSIG4 and FOLR2 by live CD45 cells also expressing CD14 in indicated human liver biopsies by flow cytometry.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Data are representative of 21 biopsy samples analyzed.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To date, no validated markers of bona fide human KCs have been described.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Explaining the difficulty to accurately define human KCs, we found monocytes and macs formed a single continuum in the human sc/snRNA-seq data, preventing a simple definition of human KCs (Figure 4B).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Notably, a similar continuum from monocyte to KCs was also observed in the NASH murine liver (Figures S5J and S6J–S6N, Table S8).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Consistent with our previous report (Remmerie et al., 2020), we observed both long-term resident Timd4-expressing KCs and recently recruited Timd4 monocyte-derived KCs (moKCs; Figures S6J–S6N).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	As the presence of such a continuum in the human liver suggests that there may also be monocyte contribution to the KC pool in the healthy human liver, we next zoomed in on myeloid cells to examine this, identifying 10 clusters (Figures 4H, S6O, and S6P; Table S2).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To define the KCs, we examined expression of the top 25 murine KC genes by these clusters, which identified cluster10 to be the genuine human KCs (Figure S6Q).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Unlike in mice, these were preferentially located in the mid zone (Figure S6R).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Cluster9 also expressed many of these genes but lacked TIMD4 (Figure S6Q), suggesting that these cells may be recently recruited moKCs.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The presence of moKCs in the liver is consistent with reports that host-derived macs are identified in transplanted donor livers (Bittmann et al., 2003; Pallett et al., 2020) and suggests that the KC population may be a mix of embryonic and monocyte-derived cells.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Although not the case at mRNA level, VSIG4 was found to be the best human KC protein marker in the CITE-seq data, while FOLR2, CD163, and CD169 were also identified as useful markers of these cells for flow cytometry and confocal microscopy on frozen and paraffin sections (Figures 4I and S6S–S6X).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Co-staining human livers for VSIG4 protein and KC-specific CD5L mRNA and MICS 100-plex protein analysis also confirmed the mid-zonal localization of KCs (Figures 4J and S6U).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To assess if KC identity was further conserved in evolution, we profiled macaque, pig, hamster, chicken, and zebrafish livers (Figure 4K).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	We identified the KCs in an unbiased manner by mapping the conserved human-mouse KC signature onto the datasets (Figures 4K and S7A–S7C).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	We then examined the main features of each KC population identified (Figures S7D–S7H; Table S9).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	A strong overlap in transcriptomes across species was observed likely due to the conserved expression of core KC transcription factors (Figure S7I).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, each species also harbored a number of unique KC genes (Figure S7J; Table S9).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	VSIG4 protein expression was also conserved in pig and macaque KCs (Figures S7K–S7M).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Similarly, we were also able to identify most of the other hepatic cells across species on the basis of conserved genes (Figures S7N and S7O).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	cDC2s were the main exception to this, as specific cDC2 marker genes were not conserved across all species (Figure S7O).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Alongside KCs, we also identified distinct clusters of macs in the human myeloid cells (Figure 4H).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	To better understand the nature of these clusters we performed confocal microscopy to examine the specific locations of CD68VSIG4 macs in the human liver (Figure S8A).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This identified CD68VSIG4 macs in the liver capsule, in close proximity to central and PVs as well as at bile ducts (BDs) (Figures 5A–5C and S8A).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Similar populations were also observed at the PVs and CVs and at the BDs in the healthy macaque liver (Figure S7M).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Examination of the scRNA-seq data and comparison with murine signatures identified immature and mature LAMs, with immature LAMs expressing some monocyte genes (Figures 4H, 5D, S8B, and S8C).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Although recently suggested to be specific to fibrotic human livers (Ramachandran et al., 2019), we identified LAMs in all patients profiled with scRNA-seq, but there was a trend toward increased proportions of LAMs in the livers with >10% steatosis (Figure S8D) consistent with the increased population of LAMs in murine NAFLD (Figures S6J–S6N).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	As in the healthy mouse, Visium identified human LAMs in portal zones of non-steatotic livers.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, in steatotic human livers, LAMs were primarily located peri-centrally, in zones with steatosis (Figure 5E), suggesting that monocytes are recruited to distinct locations in the healthy and obese liver where they then differentiate into LAMs.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This altered location of LAMs was further validated by confocal microscopy and Molecular Cartography (Figures 5F–5H).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	However, this analysis did not identify any capsule macs, suggesting that these cells may be absent from our UMAP, likely as a result of the small amount of capsule tissue on a biopsy.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	The Mac1 population expressing IGSF21 was present in very low numbers throughput the tissue (Figures 5G and 5H).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	This coupled with their similar transcriptomic profile to moKCs could suggest that these are moKC precursors as observed in the mouse, but this requires further study.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Focusing on the LAMs, the change in their location in the steatotic human liver was also observed in the murine NAFLD model.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Here, LAMs were found across portal, periportal, and mid zones (Figures 5I, S8E, and S8F), fitting with the presence of steatosis in these regions and consistent with our previous report (Remmerie et al., 2020).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Comparison of DEGs between LAMs in standard diet (SD) and WD-fed mice identified that LAMs had a more mature phenotype in WD-fed mice, downregulating their expression of some monocyte genes and increasing their expression of prototypical mac markers, consistent with the presence of both immature and mature LAMs in the human liver (Figure 5J).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Fitting with a more mature phenotype, WD-derived LAMs also expressed lower levels of Il1b, Tnf, and Il10 compared with SD LAMs (Figure 5K).
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	While further studies are required to assess the precise functions of these cells in NAFLD, this could further suggest a protective rather than a pathogenic role for LAMs (Daemen et al., 2021).Figure S7Conserved and unique features of KCs across species, related to Figure 4(A and B) Expression of human-murine KC signature genes across cell types in mouse (A) and human (B).(C) Unbiased identification of KCs in mouse and human using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019).(D–H) Annotated UMAPs from indicated species and expression of top KC-specific genes compared with other cells per species.(I) Expression of previously identified core murine transcription factors (Bonnardel et al., 2019) by KCs across species.(J) Venn diagram showing convergence and divergence of expression of top 50 KC genes per species across species, see Table S9 for genes lists per species.(K) Top DEPs (identified with cross reactive human antibodies) in the pig CITE-seq data.(L) Expression of VSIG4 in the porcine liver by confocal microscopy.(M) Expression of VSIG4, CD68 (protein), and CD5L (mRNA) in macaque liver.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	PV, portal vein; HA, hepatic artery; BD, bile duct.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	All images are representative of 2 livers.(N and O) Conserved expression of indicated genes across CD45 (N) and CD45 (O) cell types and species.
PMC8809252	Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.	Figure 5LAMs are found at the bile duct in the healthy liver, but at zones of steatosis in the obese liver(A) Expression of indicated markers at the capsule of the healthy human liver (H14) by confocal microscopy.
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.

cDCs, Macs, monocytes, and monocyte-derived cells; 42,922 cells) from mice fed the SD or WD for 24 or 36 weeks were isolated from Figure S5J and re-clustered with TotalVI.(K) Distribution of cells in UMAP originating from SD- (purple) or WD- (yellow) fed mice.(L) Proportion of indicated cell types arising from mice fed the SD (purple) or WD (yellow).(M and N) Flow cytometry analysis of indicated cell populations in SD and WD-fed mice (24 weeks).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Representative gating strategies (M) and absolute number of indicated populations (N).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

p < 0.05, p < 0.01 Student’s t test.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Data are from 2 independent experiments with n = 5–6 per diet.(O and P) Top DEGs (O) and DEPs (P) for cell types from Figure 4H.(Q) Top 25 Murine KC genes as expressed by the human myeloid cell clusters.(R) Mapping of KC signature onto Visium trajectory for healthy (purple) and steatotic (orange) livers.(S) Expression of VSIG4 mRNA within human myeloid cells.(T) Expression of VSIG4 (red) and CD163 (gray, top) or CD169 (gray, bottom) by MICS analysis in healthy human liver.(U) Representative images showing KC location (red) as assessed by MICS analysis in the healthy (left) and steatotic (right) human liver.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

PV, portal vein; CV, central Vein, dashed line indicates zones of steatosis.(V) Representative image of CD68 and CD163 staining in 10–15-year-old human liver paraffin sections.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Image is representative of 6 different patients.(W) In silico gating strategy to isolate distinct myeloid cell populations identified from CITE-seq data.(X) Expression of VSIG4 and FOLR2 by live CD45 cells also expressing CD14 in indicated human liver biopsies by flow cytometry.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Data are representative of 21 biopsy samples analyzed.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Identification 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. (

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) UMAP of Visium data from 4 patient biopsy samples. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

D) Split of Visium spots based on % steatosis. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

E) Healthy and steatotic Visium liver tissue with clusters overlaid and H+E staining to identify steatotic zones. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

F) Zonation of Visium data (top) with zonation pattern mapped onto liver tissue (bottom). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

G) Indicated cell signatures from sc/snRNA-seq mapped onto Visium zonation trajectory, healthy (top), steatotic (bottom). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

H) Myeloid cells (40,821 cells) were isolated from Figure 4B and re-clustered with TotalVI. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

I) Expression of VSIG4 protein (top) and CD5L mRNA (bottom). (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

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. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

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.

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 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. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

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. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

D) Proportion of each cell type per patient profiled. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

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. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

F) Mapping of Visium UMAP zonation patterns onto tissue sections from patient H35 and H37. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

G) Expression of indicated zonation genes in patients H35–H38 assessed by Molecular Cartography. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

H and I) Expression of indicated proteins by MICS 100-plex protein analysis in the healthy (H) and steatotic (I) human liver. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

J) Murine myeloid cells (cDC1s, cDC2s, Mig.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

cDCs, Macs, monocytes, and monocyte-derived cells; 42,922 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.

K) Distribution of cells in UMAP originating from SD- (purple) or WD- (yellow) fed mice. (

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 and N) Flow cytometry analysis of indicated cell populations in SD and WD-fed mice (24 weeks).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Representative gating strategies (M) and absolute number of indicated populations (N).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

p < 0.05, p < 0.01 Student’s t test.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Data are from 2 independent experiments with n = 5–6 per diet. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

O and P) Top DEGs (O) and DEPs (P) for cell types from Figure 4H. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Q) Top 25 Murine KC genes as expressed by the human myeloid cell clusters. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

R) Mapping of KC signature onto Visium trajectory for healthy (purple) and steatotic (orange) livers. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

S) Expression of VSIG4 mRNA within human myeloid cells. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

T) Expression of VSIG4 (red) and CD163 (gray, top) or CD169 (gray, bottom) by MICS analysis in healthy human liver. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

U) Representative images showing KC location (red) as assessed by MICS analysis in the healthy (left) and steatotic (right) human liver.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

PV, portal vein; CV, central Vein, dashed line indicates zones of steatosis. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

V) Representative image of CD68 and CD163 staining in 10–15-year-old human liver paraffin sections.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Image is representative of 6 different patients. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

W) In silico gating strategy to isolate distinct myeloid cell populations identified from CITE-seq data. (

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

X) Expression of VSIG4 and FOLR2 by live CD45 cells also expressing CD14 in indicated human liver biopsies by flow cytometry.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Data are representative of 21 biopsy samples analyzed.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To date, no validated markers of bona fide human KCs have been described.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Explaining the difficulty to accurately define human KCs, we found monocytes and macs formed a single continuum in the human sc/snRNA-seq data, preventing a simple definition of human KCs (Figure 4B).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Notably, a similar continuum from monocyte to KCs was also observed in the NASH murine liver (Figures S5J and S6J–S6N, Table S8).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Consistent with our previous report (Remmerie et al., 2020), we observed both long-term resident Timd4-expressing KCs and recently recruited Timd4 monocyte-derived KCs (moKCs; Figures S6J–S6N).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

As the presence of such a continuum in the human liver suggests that there may also be monocyte contribution to the KC pool in the healthy human liver, we next zoomed in on myeloid cells to examine this, identifying 10 clusters (Figures 4H, S6O, and S6P; Table S2).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To define the KCs, we examined expression of the top 25 murine KC genes by these clusters, which identified cluster10 to be the genuine human KCs (Figure S6Q).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Unlike in mice, these were preferentially located in the mid zone (Figure S6R).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Cluster9 also expressed many of these genes but lacked TIMD4 (Figure S6Q), suggesting that these cells may be recently recruited moKCs.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The presence of moKCs in the liver is consistent with reports that host-derived macs are identified in transplanted donor livers (Bittmann et al., 2003; Pallett et al., 2020) and suggests that the KC population may be a mix of embryonic and monocyte-derived cells.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Although not the case at mRNA level, VSIG4 was found to be the best human KC protein marker in the CITE-seq data, while FOLR2, CD163, and CD169 were also identified as useful markers of these cells for flow cytometry and confocal microscopy on frozen and paraffin sections (Figures 4I and S6S–S6X).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Co-staining human livers for VSIG4 protein and KC-specific CD5L mRNA and MICS 100-plex protein analysis also confirmed the mid-zonal localization of KCs (Figures 4J and S6U).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To assess if KC identity was further conserved in evolution, we profiled macaque, pig, hamster, chicken, and zebrafish livers (Figure 4K).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

We identified the KCs in an unbiased manner by mapping the conserved human-mouse KC signature onto the datasets (Figures 4K and S7A–S7C).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

We then examined the main features of each KC population identified (Figures S7D–S7H; Table S9).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

A strong overlap in transcriptomes across species was observed likely due to the conserved expression of core KC transcription factors (Figure S7I).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, each species also harbored a number of unique KC genes (Figure S7J; Table S9).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

VSIG4 protein expression was also conserved in pig and macaque KCs (Figures S7K–S7M).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Similarly, we were also able to identify most of the other hepatic cells across species on the basis of conserved genes (Figures S7N and S7O).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

cDC2s were the main exception to this, as specific cDC2 marker genes were not conserved across all species (Figure S7O).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Alongside KCs, we also identified distinct clusters of macs in the human myeloid cells (Figure 4H).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

To better understand the nature of these clusters we performed confocal microscopy to examine the specific locations of CD68VSIG4 macs in the human liver (Figure S8A).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This identified CD68VSIG4 macs in the liver capsule, in close proximity to central and PVs as well as at bile ducts (BDs) (Figures 5A–5C and S8A).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Similar populations were also observed at the PVs and CVs and at the BDs in the healthy macaque liver (Figure S7M).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Examination of the scRNA-seq data and comparison with murine signatures identified immature and mature LAMs, with immature LAMs expressing some monocyte genes (Figures 4H, 5D, S8B, and S8C).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Although recently suggested to be specific to fibrotic human livers (Ramachandran et al., 2019), we identified LAMs in all patients profiled with scRNA-seq, but there was a trend toward increased proportions of LAMs in the livers with >10% steatosis (Figure S8D) consistent with the increased population of LAMs in murine NAFLD (Figures S6J–S6N).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

As in the healthy mouse, Visium identified human LAMs in portal zones of non-steatotic livers.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, in steatotic human livers, LAMs were primarily located peri-centrally, in zones with steatosis (Figure 5E), suggesting that monocytes are recruited to distinct locations in the healthy and obese liver where they then differentiate into LAMs.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This altered location of LAMs was further validated by confocal microscopy and Molecular Cartography (Figures 5F–5H).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

However, this analysis did not identify any capsule macs, suggesting that these cells may be absent from our UMAP, likely as a result of the small amount of capsule tissue on a biopsy.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The Mac1 population expressing IGSF21 was present in very low numbers throughput the tissue (Figures 5G and 5H).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

This coupled with their similar transcriptomic profile to moKCs could suggest that these are moKC precursors as observed in the mouse, but this requires further study.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Focusing on the LAMs, the change in their location in the steatotic human liver was also observed in the murine NAFLD model.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Here, LAMs were found across portal, periportal, and mid zones (Figures 5I, S8E, and S8F), fitting with the presence of steatosis in these regions and consistent with our previous report (Remmerie et al., 2020).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Comparison of DEGs between LAMs in standard diet (SD) and WD-fed mice identified that LAMs had a more mature phenotype in WD-fed mice, downregulating their expression of some monocyte genes and increasing their expression of prototypical mac markers, consistent with the presence of both immature and mature LAMs in the human liver (Figure 5J).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Fitting with a more mature phenotype, WD-derived LAMs also expressed lower levels of Il1b, Tnf, and Il10 compared with SD LAMs (Figure 5K).

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

While further studies are required to assess the precise functions of these cells in NAFLD, this could further suggest a protective rather than a pathogenic role for LAMs (Daemen et al., 2021).Figure S7Conserved and unique features of KCs across species, related to Figure 4(A and B) Expression of human-murine KC signature genes across cell types in mouse (A) and human (B).(C) Unbiased identification of KCs in mouse and human using the human-murine KC signature and the signature finder algorithm (Pont et al., 2019).(D–H) Annotated UMAPs from indicated species and expression of top KC-specific genes compared with other cells per species.(I) Expression of previously identified core murine transcription factors (Bonnardel et al., 2019) by KCs across species.(J) Venn diagram showing convergence and divergence of expression of top 50 KC genes per species across species, see Table S9 for genes lists per species.(K) Top DEPs (identified with cross reactive human antibodies) in the pig CITE-seq data.(L) Expression of VSIG4 in the porcine liver by confocal microscopy.(M) Expression of VSIG4, CD68 (protein), and CD5L (mRNA) in macaque liver.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

PV, portal vein; HA, hepatic artery; BD, bile duct.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

All images are representative of 2 livers.(N and O) Conserved expression of indicated genes across CD45 (N) and CD45 (O) cell types and species.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Figure 5LAMs are found at the bile duct in the healthy liver, but at zones of steatosis in the obese liver(A) Expression of indicated markers at the capsule of the healthy human liver (H14) by confocal microscopy.

PMC8809252

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

Scale bars, 20 μm.