Kupffer cell and recruited macrophage heterogeneity orchestrate granuloma maturation and hepatic immunity in visceral leishmaniasis

Abstract

In murine models of visceral leishmaniasis (VL), parasitization of resident Kupffer cells (resKCs) is responsible for early growth of Leishmania infantum in the liver, which leads to granuloma formation and eventual parasite control. We employed the chronic VL model, and revealed an open niche established by KCs death and their migration outside of the sinusoids, resulting in their gradual replacement by monocyte-derived KCs (moKCs). While early granulomas were composed of resKCs, late granulomas were found outside of the sinusoids and contained resKC-derived macrophages, and monocyte-derived macrophages (momacs). ResKCs and moKCs within the sinusoids were identified as homeostatic/regulatory cells, while resKC-derived macrophages and momacs within late granulomas were pro-inflammatory. Despite the infection being largely confined to the resKC-derived macrophages, in the absence of monocyte recruitment, parasite control was strongly compromised. Macrophage heterogeneity, involving migration and reprogramming of resKCs, along with recruitment of monocyte-derived cells, is a hallmark of granuloma maturation and hepatic immunity in VL.

Keywords: Kupffer cells; cell death; chronic infection; ferroptosis; granuloma; liver; monocyte-derived macrophages; parasitic diseases; visceral leishmaniasis.

Extended Data Fig.1:. Myeloid cells gating strategy and macrophage heterogeneity at 42 days post-infection.

a, Dot plots showing the gating strategy used to identify resKCs, (F4/80^hiCD11bi^ntCD64⁺CLEC4F⁺TIM-4⁺), neutrophils (CD11b⁺Ly6G⁺SiglecF⁻), eosinophils (CD11b⁺Ly6G⁻SiglecF⁺) and monocytes (CD11b⁺Ly6G⁻SiglecF⁻Ly6C⁺CD64⁺) in live, single cells isolated from naïve WT livers. b, Representative FACS plots showing CLEC4F and TIM-4 expression gated on F4/80^hiCD11b^intCD64⁺ macrophages in naïve and 42 day-infected mice, and fluorescence minus one (FMO) staining for CLEC4F and TIM-4. FMO controls contain mixed naïve and infected samples. c, Frequency of macrophage subsets in naïve and 42-day infected mice by flow cytometry. Numbers indicate mean ± SD percentage of cells in the gate. Data pooled from 5 independent experiments (n=20). Values in c represent the mean ± SD. **, p < 0.01, ****, p < 0.0001.

Extended Data Fig.2:. WT and Ccr2−/− infected livers.

a, Original representative immunofluorescence images of rendered images from Fig.2i showing WT and Ccr2^−/− 42 d.p.i. livers, and F4/80 (cyan), CLEC4F (white), TIM-4 (red), and Hoechst (blue). Scale bars, 200 μm.

Extended Data Fig.3:. Cell death pathways in macrophages during VL.

a, Frequency of cells in F4/80^hiCD11b^intCD64⁺ subsets in 42 d.p.i. WT and Mlkl^−/− mice, by flow cytometry. Data pooled from 2 independent experiments (n=8 for WT and n=9 for Mlkl^−/−). b, Frequency of F4/80⁺ subsets in 42 d.p.i. WT and Casp1^−/− mice, quantified from immunofluorescence images (n=8). c, Number of cleaved caspase 3⁺ cells in each F4/80⁺ subset and in F4/80⁻ cells quantified from immunofluorescence images in naïve and 42-day infected WT mice. d, Frequency of each subset of F4/80⁺ and F4/80⁻ cells, gated on cleaved caspase 3⁺ cells, in naïve and 42-day infected WT mice. e, Representative immunofluorescence images showing apoptotic CLEC4F⁺TIM-4⁻ moKC (thin arrow), apoptotic CLEC4F⁻TIM-4⁺ resKC-derived macrophage (arrow), and apoptotic CLEC4F⁺TIM-4⁺ resKC (arrowhead) in 42-day infected mice. Scale bars, 8 and 10 μm, respectively. Data pooled from 2 independent experiments (n=6 for naïve and n=7 for 42 d.p.i.). Values in a-d represent mean ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Extended Data Fig.4:. Ferroptosis contributes to reduction in resKC numbers during VL.

a, Lipid peroxidation measured by bodipy median fluorescence intensity in TIM-4^+/− cells (gated on F4/80^hiCD11b^intCD64⁺ macrophages) and in CD11b⁺ cells (excluding F4/80^hi macrophages), in WT naïve and 42 d.p.i. by flow cytometry. Data pooled from 4 independent experiments (n=16). b, Intracellular GSH levels measured in whole tissue homogenates from naïve and 42 d.p.i. WT mice. Data pooled from 2–3 independent experiments (n=12 for naïve and n=8 for 42 d.p.i.). c, Lipid peroxidation in TIM-4⁺ and TIM-4⁻ cells from WT and Bach1^−/− mice at 42 d.p.i. Data representative of 2 independent experiments (n=4 each). d, Representative dot plots from immunofluorescence images showing the frequencies of macrophages based on CLEC4F and TIM-4 expression in WT and Bach1^−/− at 42 d.p.i. Numbers indicate mean ± SD percentage of cells in the gate. e-f Frequency (e) and number (f) of F4/80⁺ subsets in 42 d.p.i WT and Bach1^−/− mice, quantified from immunofluorescence images. g, Number of F4/80⁺ macrophages in WT and Bach1^−/− mice, at 42 d.p.i., quantified from immunofluorescence images. h, Representative rendered images of WT and Bach1^−/− 42 d.p.i. livers showing CLEC4F⁺TIM-4⁺resKCs (yellow), CLEC4F⁻TIM-4⁺resKC-derived macrophages (red), CLEC4F⁺TIM-4⁻moKCs (magenta) and CLEC4F⁻TIM-4⁻momacs (cyan), and original immunofluorescence images showing F4/80 (cyan), CLEC4F (white), TIM-4 (red) and Hoechst (blue). Scale bars, 200 μm. Data pooled from 3 independent experiments (n=11). Values from a-c and e-g represent mean ± standard deviation. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Extended Data Fig.5:. Clusters and differentially expressed genes identified by single-cell RNA sequencing.

a, UMAP plot of scRNA-seq data of sorted live, single, CD45.2⁺F4/80⁺CD11b^intCD64⁺ cells from uninfected and 42 d.p.i. mice, showing 3 clusters in naïve and 6 clusters in infected mice, as defined by Remmerie et al.. b, Heatmap showing the average expression of the top 10 differentially expressed genes defining the clusters from sorted live, single, CD45.2⁺F4/80⁺CD11b^intCD64⁺ cells of uninfected and 42 d.p.i. mice. c, Average gene expression and corresponding cell percent of KC-associated transcription factors within each of the clusters. Data from 1,200 naïve cells and 6,152 cells from 42 d.p.i. mice, after QC.

Extended Data Fig.6:. Activation and infection status of macrophage subsets.

a, Individual images from Fig.5d showing F4/80 (cyan), CLEC4F (white), TIM-4 (red), and iNOS (green) in WT 19-, 42-, and 72-day infected livers. Scale bars, 30μm. b, Frequency of infected cells identified outside F4/80⁺ granulomas at 19 and 42 d.p.i., obtained from immunofluorescence images. Data pooled from 2 independent experiments for each time point (n=8 for 19- and 42 d.p.i., n=7 for 72 d.p.i.), obtained from images of 2–3 regions from each liver containing granulomas (ROIs=24 for 19 d.p.i., ROIs=23 for 42 d.p.i., ROIs=16 for 72 d.p.i.). c, Individual images from merged images in Fig.5n showing F4/80 (cyan), CLEC4F (white), TIM-4 (red), and L. infantum (yellow) in WT and Ccr2^−/− 42-day infected livers. Scale bars, 20μm. d, Representative and rendered images of F4/80⁺ granulomas and iNOS expression in WT and Ccr2^−/− 42-day infected mice, showing F4/80 (cyan), CLEC4F (white), TIM-4 (red), and iNOS (green) staining, and rendered CLEC4F⁺TIM-4⁺ (yellow), CLEC4F⁻TIM-4⁺ (red), CLEC4F⁺TIM-4⁻ (magenta), and CLEC4F⁻TIM-4⁻ (cyan) F4/80⁺ cells. Scale bars, 30μm. e, iNOS expression by different F4/80⁺ subsets in WT and Ccr2^−/− 42 d.p.i. mice, quantified from immunofluorescence images. f, IFN-γ, TNF and IL-1β levels measured by Luminex in liver homogenates from WT and Ccr2^−/− mice at 42 d.p.i. Data pooled from 2 independent experiments (n=9 for WT and n=8 for Ccr2^−/−). Values in b, e-f represent the mean ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Figure 1:. Heterogeneity of F4/80⁺ macrophages during VL defined by the differential expression of CLEC4F and TIM-4.

a, Representative confocal microscopy images showing WT naïve, 19-, 42-, and 72-day infected livers stained with anti-F4/80 (cyan), anti-CLEC4F (white), and anti-TIM-4 (red), and surface rendered F4/80⁺ cells classified according to their CLEC4F and TIM-4 expression: CLEC4F⁺TIM-4⁺ (yellow), CLEC4F⁻TIM-4⁺ (red), CLEC4F⁺TIM-4⁻ (magenta), CLEC4F⁻TIM-4⁻ (cyan). Scale bars, 200μm. b, Frequency and number of F4/80⁺ cells classified according to their CLEC4F and TIM-4 expression, in naïve and infected WT mice at different days post-infection. Data pooled from 2 independent experiments (n=6 for naïve and n=7 for infected mice). c, Inset shows granulomas identified in (a) and defined as clusters of F4/80⁺ cells with volumes above 1.03×10⁴ μm³. Scale bars, 40μm. d, Proportion of CLEC4F⁺ and/or TIM-4⁺ cells in F4/80⁺ granulomas at different times post-infection. Data pooled from 2–4 independent experiments (n=7 for 19- and 72 d.p.i. and n=15 for 42 d.p.i.). Values in b and d represent the mean ± SD.

Figure 2:. TIM-4⁻ macrophages are monocyte-derived at 42 d.p.i.

a, Representative contour plots showing the percentages of chimerism in F4/80⁺CD11b^intCD64⁺ cells of naïve and 42 days infected CD45.1⁺ parabiont. b, Percentages of chimerism in F4/80⁺CD11b^intCD64⁺ cells of uninfected and infected CD45.1⁺ parabiotic partner at 42 d.p.i. c, Frequency of CD45.2⁺ cells in infected CD45.1⁺ parabiont and gated on F4/80⁺CD11b^intCD64⁺CLEC4F^+/−TIM-4^+/− macrophages or on Ly6C⁺CD11b⁺Ly6G⁻SiglecF⁻ monocytes. Data pooled from two independent experiments (n=3 for uninfected and n=4 for infected pairs). d-e, Number of monocytes in the liver of naïve (d) and 19- and 42-day infected WT and Ccr2^−/− mice (e), obtained by flow cytometry. Data pooled from 5 independent experiments (naïve, n=14 for WT and n=17 for Ccr2^−/−; 19 d.p.i., n=8 for WT and n=9 for Ccr2^−/−; 42 d.p.i., n=12 for WT and n=14 for Ccr2^−/−). f, Number of F4/80⁺ cells in WT and Ccr2^−/− at 42 d.p.i., quantified from confocal microscopy images. g, Frequency of macrophage subsets based on CLEC4F and TIM-4 expression in WT and Ccr2^−/− mice at 42 d.p.i., quantified from confocal microscopy images. h, Representative dot plots from confocal microscopy images showing the frequency of macrophage subsets in infected WT and Ccr2^−/− mice at 42 d.p.i. Numbers indicate mean ± SD percentage of cells in the gate. i, Representative rendered confocal microscopy images of WT and Ccr2^−/− 42 d.p.i. livers showing CLEC4F⁺TIM-4⁺ (yellow) and CLEC4F⁻TIM-4⁺ (red) macrophages, CLEC4F⁺TIM-4⁻ (magenta) moKCs and CLEC4F⁻TIM-4⁻ (cyan) momacs. Scale bars, 200μm. Data pooled from 2 independent experiments (n=8 for WT and n=9 for Ccr2^−/−). j- k, Representative live images of naïve (j) and 42-day infected (k) Clec4f-Cre-ZsGreen mice showing Clec4f active expression (red), Clec4f previous expression (green), and sinusoids (magenta). Scale bars, 100μm and 30μm (insets). l, Frequency of tdTomato⁺ cells identified inside and outside granulomas, at 42 d.p.i. Data pooled from 2 independent experiments (n=4). Values in b-g and l show mean ± SD. *, p < 0.05, ***, p < 0.001, ****, p < 0.0001.

Figure 3:. Late granulomas form outside of the sinusoids and contain polyclonal clusters of macrophages derived from res-KCs.

a, Representative live images from a 42-day infected Clec4f-Cre-ZsGreen mouse showing Clec4f active expression (red), Clec4f previous expression (green), F4/80 (cyan), and sinusoids (magenta). Arrow shows a F4/80⁺tdTomato⁻ZsGreen⁺ cell, and arrowheads show F4/80⁺tdTomato⁻ZsGreen⁻ cells. Scale bars, 30μm. b, Frequency of F4/80⁺ cells inside the sinusoids according to tdTomato and ZsGreen expression, in 42 d.p.i. mice. c, Representative images of Clec4f-Cre-ZsGreen mice at 42 d.p.i., showing Clec4f active expression (red), Clec4f previous expression (green), F4/80 (cyan), and sinusoids (magenta). Scale bars, 200μm. Inset #1, dotted line shows clustered tdTomato⁻ZsGreen⁺ cells in granulomas outside the sinusoids. Arrowheads show one F4/80⁺ and one F4/80⁻ tdTomato⁺ZsGreen⁺ cell outside the sinusoids but in close contact with LSECs. Arrow points to 3 F4/80⁺tdTomato⁺ZsGreen⁺ cells outside the sinusoids. Scale bars, 30 μm. Inset #2, dotted lines show a tdTomato⁻ZsGreen⁺ cluster with 3 tdTomato⁺ZsGreen⁺ closer to the LSECs. Arrow points to 2 tdTomato⁺ZsGreen⁺ partially inside the sinusoids and partially stained with F4/80. Scale bars, 15 μm. Data pooled from 2 independent experiments (n=4) d, Representative rendered images showing tdTomato (red) and ZsGreen (green) expression inside and outside the sinusoids (magenta), in 42-day infected Clec4f-Cre-ZsGreen mouse. Scale bars, 80 μm. e, Representative immunofluorescence image of a 42-day infected WT liver showing the spatial distribution of CLEC4F⁺TIM-4⁺ resKCs (yellow), CLEC4F⁻TIM-4⁺ resKC-derived macrophages (red), CLEC4F⁺TIM-4⁻ moKCs (magenta), CLEC4F⁻TIM-4⁻ momacs (cyan), and LSECs (green). Scale bars, 30 μm. f, Bar graphs showing the localization of each macrophage subset according to their interaction with LSECs, in 42 d.p.i. and naïve mice. Data pooled from 2 independent experiments (n=4 for naïve and n=8 for 42 d.p.i.). (Quantification of 4 regions of interest (ROIs) from each infected mouse, n=32 ROIs, and 2–3 ROIs from each uninfected mouse, n=11 ROIs). g, Scatter plots from immunofluorescence images showing the frequency of each F4/80⁺ population based on their distribution outside or inside granulomas at 42 d.p.i. Data pooled from 3 independent experiments (n=11). h, Intravital microscopy images representing a 42-day infected liver from Clec4f-Cre-Confetti mouse stained with anti-CD31 (magenta) and showing tdTomato (red-nucleus), RFP (red-cytoplasm), YFP (yellow-cytoplasm), GFP (green-nucleus), and CFP (blue-membrane) KCs. Scale bars, 50 μm. i, Composition of macrophage granulomas based on KC and KC-derived macrophages colors. Data pooled from live imaging carried out in 2 independent experiments (n=4 and quantification of 284 granulomas). Values from b, f-g and i represent mean ± standard deviation. *, p < 0.05, **, p < 0.01, ****, p < 0.0001.

Figure 4:. Macrophage heterogeneity revealed by single-cell RNA sequencing.

a, UMAP plot of scRNA-seq data of sorted live, single, CD45.2⁺F4/80⁺CD11b^intCD64⁺ cells from livers of uninfected and 42 d.p.i. mice, showing 3 clusters for naïve and 7 clusters for infected mice. b, Annotated UMAP plot showing the single-cell expression of conserved KC signature genes described by Guilliams et al. c, Single-cell expression of Ccr2, Clec4f and Timd4 in CD45.2⁺F4/80⁺CD11b^intCD64⁺ cells from naïve and 42 d.p.i. mice d, Average gene expression and percent of cells in which they are expressed within each of the identified clusters in naïve and 42 day-infected CD45.2⁺F4/80⁺CD11b^intCD64⁺ cells. e, Frequency of the different subsets identified by scRNA-seq in naïve and 42 d.p.i. mice. f, Heatmap showing the differential expression of Mac2 specific genes identified by Remmerie et al.. Data from 1,200 naïve cells and 6,152 cells from 42 d.p.i. mice, after QC filters.

Figure 5:. Functional heterogeneity of macrophage subsets and the contribution of of monocyte-derived macrophages to L. infantum control.

a-b, Heatmaps showing the average expression levels of lipid and iron metabolism genes (a), and chemokines and cytokines (b) at 42 d.p.i. c, Number of iNOS⁺F4/80⁺ granulomas at different times post-infection in WT mice, quantified from immunofluorescence images. d, Representative images showing F4/80 (cyan), CLEC4F (white), TIM-4 (red), and iNOS (green) in WT 19-, 42-, and 72-day infected livers. Scale bars, 30μm. e, Number of F4/80⁺iNOS⁺ macrophages, based on CLEC4F and TIM-4 expression in infected WT livers at 42 d.p.i., and quantified from immunofluorescence images. Data pooled from 5 independent experiments (n=19). f, Representative images showing F4/80 (cyan), CLEC4F (white), TIM-4 (red), and iNOS (green) above, and only F4/80⁺iNOS⁺ granulomas below, in a WT liver at 42 d.p.i. g, UMAP plots showing the single-cell expression of Nos2, Cxcl10, Tnf, Ccl24, and Il10 in CD45.2⁺F4/80⁺CD11b^intCD64⁺ cells from 42-day infected mice. h, Frequency of infected cells found inside F4/80⁺ granulomas, at 19 and 42 d.p.i., and obtained from immunofluorescence images. i, Representative images of F4/80⁺ macrophages in WT 19- and 42-day infected livers, stained with anti-F4/80 (cyan), anti-CLEC4F (white), anti-TIM-4 (red), and anti-L. infantum (yellow). Scale bars, 30 μm. Data pooled from 2 independent experiments for each time point (n=8 for 19- and 42 d.p.i., n=7 for 72 d.p.i.), and from images of 2–3 regions from each liver containing granulomas (ROIs=24 for 19 d.p.i., ROIs=23 for 42 d.p.i., ROIs=16 for 72 d.p.i.). j, Number of F4/80⁺ granulomas quantified from immunofluorescence images. Data pooled from 2 independent experiments (n=7 for 19- and 42 d.p.i. and n=6 for 72 d.p.i.). k, Distribution of parasites inside and outside F4/80⁺ granulomas at 19 and 42 d.p.i., quantified from immunofluorescence images. Data pooled from 2 independent experiments (n=7). l, Parasite loads in WT and Ccr2^−/− mice at 42 d.p.i. Data pooled from 3 independent experiments (n=12 for WT and n=13 for Ccr2^−/−). m, Number of L. infantum amastigotes per field in 42 day-infected WT and Ccr2^−/− mice, quantified from confocal microscopy images. Data pooled from 2 independent experiments (n=8). n, Representative images showing F4/80 (cyan), CLEC4F (white), TIM-4 (red), and L. infantum (yellow) in WT and Ccr2^−/− 42-day infected livers. Scale bars, 20μm. o, IL-4 levels measured by Luminex in liver homogenates from WT and Ccr2^−/− mice at 42 d.p.i. Data pooled from 2 independent experiments (n=9 for WT and n=8 for Ccr2^−/−).Values in c,e, h, j-k, l-m and o represent the mean ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. Data shown in heatmaps a-b were downsampled to a maximum of 500 cells per cluster for a more even cell type representation.