Fig 2: AM1_Pro-infl subset gene signatures and Mtb transcriptional profile. (a) Pathway enrichment analysis results for the transcriptional profile of the AM_Pro-Infl population. The reactome database has been used as the main data source. (b) Umap plot showing gene expression levels (in log-normalized counts) for the Marco PRR. Higher expression values are displayed in yellow/green, while low expression values are shown in blue. (c) Heatmap showing relative expression levels for the iron gene signature in Mtb. (d) Heatmap showing relative expression levels for the suf operon in Mtb. (e) Violin plots showing expression levels (in log-normalized counts) for the genes part of the ergothioneine biosynthesis pathway in Mtb. (f) Flow cytometry gating strategy for the sorting of CD11clow and CD11chigh macrophage populations (MerTK+CD64+). Fluorescence Minus One (FMO) controls for both CD11c and mCherry signals have been used as gating controls. n = 5 for the infected population, n = 2 for the uninfected population, n = 3 for the bacterial transcriptome. The statistical significance for the genes part of the bacterial transcriptome has been calculated using the Wald test as implemented in the DESeq2 package (FDR < 0.05; see Materials and methods; Love et al., 2014). For pathway enrichment analysis, only pathways with FDR < 0.05 are shown (g:SCS method for multiple testing correction; see Materials and methods) Unless otherwise specified, the statistical significance is provided when appropriate for each plot (*, p-adj. < 0.05; **, p-adj. < 0.01).

Fig 3: Pathway enrichment analysis and assessment of genes associated with an iron signature for the IM_2 and IM_3 subsets. (a) Flow cytometry analysis of the two ontologically distinct macrophage populations from infected (at 3 w.p.i.) and uninfected mice lungs. Macrophages (MerTK+ CD64+) have been gated from other immune cells, and expression of the surface marker SiglecF has been used to separate the two main populations (AMs and IMs). As evidenced in the text, recruited populations of macrophages are present in lower numbers in uninfected mice compared with infected tissues. FSC-A, forward scatter area. (b) Umap plot showing expression levels (in log-normalized counts) for the Ly6a/Sca-1 marker gene. IM_2 and IM_3 subpopulations both express this marker, although only IM_3 shows a pro-inflammatory phenotype. Higher expression values are displayed in yellow/green, while low expression values are shown in blue. (c) Pathway enrichment analysis results for the transcriptional profile of IM_3 and IM_2 infected macrophages. The reactome database has been used as the main data source. (d) Umap plots showing gene expression levels (in log-normalized counts) for some of the genes (Hp, Slc48a1, Sod2, and Slc11a1) involved in the heme-iron response in IMs. Higher expression values are displayed in yellow/green, while low expression values are shown in blue. (e) Umap plots (split-view) showing gene expression levels (in log-normalized counts) for the ferritin genes (Ftl1 and Fth1) across different infection conditions. Higher expression values are shown in blue, while low expression values are displayed in gray. n = 5 for the infected population, n = 2 for the uninfected population. All the genes highlighted in the different figures show statistically significant greater expression in their clusters compared with cells in different clusters (FDR < 0.05; Wilcoxon rank-sum test; see Materials and methods). For pathway enrichment analysis, only pathways with FDR < 0.05 are shown (g:SCS method for multiple testing correction; See Materials and methods).

Fig 4: Localization and Heterogeneity of Macrophages in MAFLD(A) Confocal microscopy showing cells expressing CLEC4F (red), F4/80 (green), TIM4 (blue), and CD31 (gray) in the livers of SD- or WD-fed mice at the indicated time points. Smaller images show results for individual channels; the larger image is merged from all channels. Scale bar, 50 µm. Images are representative of 5–6 mice per time point and are extracted from 4 × 4 tiled images. White arrows point to CLEC4F+TIM4- moKCs. Dashed line highlights the zones enriched for CLEC4F- macs.(B) Tile scans (4x4) of livers from SD- and WD-fed mice (36 weeks) showing annotation of identified macs per subset in indicated colors. Indicated regions (dashed lines) identify the areas from the WD-fed mouse used in (A). Shaded gray boxes identify large vessels (portal or central veins) that were excluded from quantification analysis in (C) and (E). Images are representative of 6 mice.(C) Quantification of indicated populations shown in (B) as a % of total F4/80+ macs. **p < 0.01, ****p < 0.0001, Student’s t test compared with SD control. Error bars indicate ±SEM.(D) Tile scan of liver from a WD-fed mouse (36 weeks; same mouse as from A and B) with identified Desminhi regions demarcated in blue. The rest of the tissue is classified as Desminlo, excluding the larger vessels, for quantification in (E).(E) Quantification of indicated populations in Desminhi and Desminlo zones (from D) as a % of each mac subset. *p < 0.05, Student’s t test compared with Desminlo area. Quantification data in (C) and (E) are pooled from 2 independent experiments with n = 6. Error bars indicate ±SEM.(F–I) Monocyte- and mac-containing clusters (based on expression of Mafb, Ly6c2, Ccr2, Fcgr1, Adgre1) were isolated from the CITE-Seq data (18,241 cells) and re-clustered.(F) UMAP showing annotated monocyte and macrophage clusters.(G) Distribution of cells on SD or WD at indicated time points, with SD data coming from cells pooled after 12, 24, and 36 weeks.(H and I) Expression of indicated genes by the different clusters (SD + WD pooled). See also Figure S5.

Fig 5: ALK1-BMP9/10 axis regulates KC development(A) Mouse BM monocytes were cultured in the presence of CSF1 and indicated concentrations of human ac-LDL, prior to analyzed for expression of indicated genes by qPCR. Data are pooled from 2 experiments. One-way ANOVA with Bonferroni post-test compared with 0 ng/mL.(B) NicheNet circos plot highlighting conserved ligand-receptor pairs and induced target genes between KCs and indicated niche cells in human and mouse.(C) Feature plots showing expression of ALK1 (Acvrl1) in human myeloid cells (left) and GDF2/BMP10 in CD45- cells (right).(D) Livers were harvested from Fcgr1-CrexAcvrl1fl/fl mice or Acvrl1fl/fl or Acvrl1+/+ controls and KCs examined (left) and quantified (right) using VSIG4 expression.(E) Expression of indicated KC markers by mac populations in Fcgr1-CrexAcvrl1fl/fl or Acvrlfl/fl control mice. Data are pooled from 3 independent experiments with n = 9 per group. Student’s t test.(F) Expression of indicated markers in livers of Fcgr1-CrexAcvrl1fl/fl or Acvrl1fl/fl or Acvrl1+/+ control mice by confocal microscopy. Scale bars, 50 µm. Images are representative of 2 mice per group.(G) Schematic of chimera experiment setup.(H) % chimerism normalized to levels in blood Ly6Chi monocytes in Clec4f-Dtr mice 7 or 13 days after DT administration following partial irradiation and receiving either Acvrlfl/fl or Fcgr1-CrexAcvrlfl/fl BM 4 weeks earlier. Data are pooled from 2 independent experiments with n = 10–12 mice per group.(I) Schematic of Fc trap experiment setup.(J) Representative FACS plots showing VSIG4 and CLEC2 expression by total macs. Numbers represent % of total mac population in the indicated gate (left) and % of VSIG4+ and VSIG4- macs among total CD45+ cells in the different treatment conditions. One-way ANOVA with Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S8.