Spatial transcriptomics dataset reveals 16 unique clusters during infection with C. violaceum.

(A) SpatialDimPlots showing hematoxylin and eosin (H&E) and cluster overlay of spatial transcriptomics data corresponding to various days post-infection (DPI). Each circle is an individual barcoded spot that is 55 µm in diameter. (B) UMAP plot of 16 unique clusters identified based on differentially expressed genes during the course of infection. (C) Temporal expression of each cluster, and characterization of predominant cell types present and/or location of each cluster (initial characterization performed in Harvest et al., 2023); spot size is arbitrary. Macrophage zone (M), hepatocyte (HEP), representative HEP (rep HEP), necrotic core center (NC-C), NC-periphery (NC-P), coagulative necrosis (CN), CN-macrophage (CN-M), endothelial cell (EC), outside granuloma (OG). (D) SpatialDimPlot at 10 DPI as in (A), showing cluster overlay and annotated with cluster identity. (E) SpatialFeaturePlot at 10 DPI, showing expression of Pf4 (murine homolog of CXCL4).

Expression level of chemokine ligands during infection with C. violaceum.

Expression was visually ranked as absent, low, medium, or high based on SpatialFeaturePlots. Maximum expression rank recorded here. Table generated from: (David & Kubes, 2019; Hughes & Nibbs, 2018; Sokol & Luster, 2015; Zlotnik & Yoshie, 2000, 2012). Lymph node (LN); Natural killer cell (NK); NK T cell (NKT); innate lymphoid cell (ILC); dendritic cell (DC).

Expression level of chemokine receptors during infection with C. violaceum.

Expression was visually ranked as absent, low, medium, or high based on SpatialFeaturePlots. Maximum expression rank recorded here. Table generated from: (David & Kubes, 2019; Hughes & Nibbs, 2018; Sokol & Luster, 2015; Zlotnik & Yoshie, 2000, 2012). Natural killer cell (NK); innate lymphoid cell (ILC); dendritic cell (DC); plasmacytoid DC (pDC); lymph node (LN); red blood cell (RBC).

Expression level of selected chemoattractive molecules during infection with C. violaceum.

Expression was visually ranked as absent, low, medium, or high based on SpatialFeaturePlots. Maximum expression rank recorded here. Table generated from: (Bui et al., 2020; David & Kubes, 2019; Parks et al., 2004; Wang et al., 2018). Dendritic cell (DC); plasmacytoid DC (pDC); Kupffer cell (KC); natural killer cell (NK); syndecan 1 (SDC1).

Chemokines involved in neutrophil recruitment are upregulated during infection.

SpatialFeaturePlots displaying gene expression data of CXCR2 ligands (i.e. Cxcl1, Cxcl2, Cxcl3, and Cxcl5) at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Chemokines involved in monocyte recruitment are upregulated during infection.

SpatialFeaturePlots displaying gene expression data of CCR2 ligands (i.e. Ccl2, Ccl7, and Ccl12) at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Qualitative heatmaps of chemokine and receptor expression during infection.

Expression in SpatialFeaturePlots was visually ranked as absent (grey), low (blue), medium (yellow), or high (red) for (A) CXCL family chemokines, (B) CCL family chemokines, (C) CXC chemokine receptors, and (D) CC chemokine receptors. Visual rankings were based on both the intensity of expression and the relative number of spots that expressed the gene. (A-B) Scale set at 0 – 3.0 expression; (C-D) Scale set at 0 – 2.0 expression. Arrows indicate Ligand – Receptor interactions. Ligands are color-coded based on the maximum expression level reached at any time during the course of infection.

Chemokines involved in monocyte recruitment peak after chemokines involved in neutrophil recruitment.

Comparative analysis of Cxcl1 (A, C, and E) and Ccl2 (B, D, and F). (A-B) UMAP plots of 16 unique clusters showing expression level of each gene. Maximum expression level set to 1.5; annotated with cluster identity. (C-D) Violin plots of 16 unique clusters showing expression level of each gene. (E-F) Violin plots of various days post-infection (DPI) showing expression level of each gene.

CCR2 and monocyte recruitment is essential for a successful granuloma response to C. violaceum.

Wildtype (WT) and Ccr2−/− mice were infected intraperitoneally (IP) with 1×104 CFU C. violaceum. (A) Survival analysis of WT (N = 10) and Ccr2−/− (N = 9) mice. Two experiments combined. Mantel-Cox test, ****p<0.0001. (B-K) Livers and spleens were harvested 5 days post-infection (DPI). Bacterial burdens in the (B) liver and (C) spleen of WT and Ccr2−/− mice. Two experiments combined. Each dot represents one mouse. (B) Two-tailed t test (normally distributed data); ***p=0.0002. (C) Mann-Whitney (abnormally distributed data); **p=0.0012. Dotted line, limit of detection. Solid line, median. (D) Gross images of WT and Ccr2−/− livers 5 DPI. (E) Gating strategy for analysis of neutrophil (Ly6G+) and macrophage (CD68+) numbers via flow cytometry. Liver samples from infected mice shown. Macrophage numbers in (F) liver, (H) spleen, and (J) blood. Neutrophil numbers in (G) liver, (I) spleen, and (K) blood. (F-K) Two experiments combined using only female mice. Each dot represents one mouse, with 10,000 events collected per sample. Line represents mean ± standard deviation.

Loss of CCR2-dependent monocyte trafficking results in abnormal granuloma architecture and failure of bacterial containment.

WT and Ccr2−/− mice were infected intraperitoneally (IP) with 1×104 CFU C. violaceum and livers were harvested 5 days post-infection (DPI). Serial sections of livers stained by hematoxylin and eosin (H&E) or various IHC markers for (A-D) WT male and (E-H) Ccr2−/− male. Necrotic core (NC), coagulative necrosis zone (NC), macrophage zone (M). For 10X, scale bar is 100 µm. For 20X and 40X, scale bar is 50 µm. Representative of two experiments with 2 – 4 mice per group, and multiple granulomas per section.

Spatial expression of CXCR3 ligands.

SpatialFeaturePlots displaying gene expression data of CXCR3 ligands (i.e. Cxcl4, Cxcl9, and Cxcl10) at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Spatial expression of Cxcl12, Cxcl13, Cxcl14, and Cxcl16.

SpatialFeaturePlots displaying gene expression data of selected Cxcl family members at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Spatial expression of Ccl3, Ccl4, Ccl5, Ccl6, and Ccl8.

SpatialFeaturePlots displaying gene expression data of selected Ccl family members at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Spatial expression of Ccl9, Ccl11, Ccl19, Ccl20, and Ccl21a.

SpatialFeaturePlots displaying gene expression data of selected Ccl family members at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Spatial expression of Ccl22, Ccl24, Ccl25, and Ccl27a.

SpatialFeaturePlots displaying gene expression data of selected Ccl family members at various days post-infection (DPI). Scale set at 0 – 3.0 expression.

Spatial expression of Cxcr family members.

SpatialFeaturePlots displaying gene expression data of selected Cxcr family members at various days post-infection (DPI). Scale set at 0 – 2.0 expression.

Spatial expression of Ccr family members.

SpatialFeaturePlots displaying gene expression data of selected Ccr family members at various days post-infection (DPI). Scale set at 0 – 2.0 expression.

Reparixin does not inhibit neutrophil chemotaxis into the liver of infected mice.

(A) Schematic of the experimental procedure. Mice were injected subcutaneously (SQ) with 20 mg/kg of reparixin, or with PBS. The following day, mice were infected intraperitoneally (IP) with 1×104 CFU of C. violaceum and treated again with reparixin or PBS. Mice were treated daily thereafter until harvesting on day 3 post-infection. (B) Bacterial burdens in the liver and spleen of PBS- or reparixin-treated mice at 3 days post-infection (DPI). (C) Schematic of the experimental procedure as in A, except mice were harvested on day 1 post-infection. (D) Bacterial burdens in the liver and spleen of PBS- or reparixin-treated mice at 1 DPI. (E) Gating analysis of neutrophil (Ly6G+) and macrophage (CD68+) numbers via flow cytometry. Neutrophil numbers in the (F) liver and (G) spleen. Macrophage numbers in the (H) liver and (I) spleen. Each dot represents one mouse, with 10,000 events collected per sample. Line at median. (B and D) Dotted line, limit of detection. Solid line, median. Mann-Whitney (abnormally distributed data) for all except liver CFU at 1 DPI, which was analyzed using a two-tailed t test (normally distributed data). Not significant (ns). (B) Liver, p=0.6286; spleen, p=0.4286. (D) Liver, p=0.0641; spleen, p=0.8485. (B) One experiment. (D) Two experiments combined. (F-I) Two experiments combined.

Loss of CCR2-dependent monocyte trafficking results in abnormal granuloma architecture and failure of bacterial containment.

WT and Ccr2−/− mice were infected intraperitoneally (IP) with 1×104 CFU C. violaceum and livers harvested 5 days post-infection (DPI). Serial sections of livers stained by hematoxylin and eosin (H&E) or various IHC markers for (A-D) WT female and (E-H) Ccr2−/− female. For 10X, scale bar is 100 µm. For 20X and 40X, scale bar is 50 µm.

Ccr2−/− mice have increased necrosis and clotting.

Ccr2−/− mice were infected intraperitoneally (IP) with 1×104 CFU C. violaceum. (A) Liver section from Ccr2−/− male mouse 5 days post-infection (DPI), stained for C. violaceum; zoom showing individual puncta of C. violaceum. (B-D) A Ccr2−/− female mouse from survival curve in Figure 6A that was sacrificed at 7 DPI according to euthanasia criteria. (B) Gross pathology. (C) Liver section stained by hematoxylin and eosin (H&E) showing clotting. (D) Serial sections of liver stained with H&E or various IHC markers. For 10X, scale bar is 100 µm.