Chemokine expression profile of an innate granuloma
- Department of Integrative Immunobiology, Duke University School of Medicine, United States
- Department of Ophthalmology, Duke University School of Medicine, United States
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, United States
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, United States
- Department of Pathology, Duke University School of Medicine, United States
- Department of Cell Biology, Duke University School of Medicine, United States
Figures
Figure 1 with 1 supplement
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. Characterization of predominant cell types and/or location of each cluster (initial characterization performed in Harvest et al., 2023); 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). (C) Temporal prevalence of CD45+ clusters, calculated as proportion of spots represented by each cluster within each timepoint. (D) SpatialDimPlot at 10 DPI as in (A), showing cluster overlay and annotated with cluster identity. (E) SpatialFeaturePlot at 10 DPI, showing log-normalized expression of Pf4 (murine homolog of CXCL4). Source code 1. Streamlined code for analysis using RStudio.
Figure 1—figure supplement 1
Sequencing depth of samples and spatial expression of CXCR3 ligands.
(A) SpatialFeaturePlot displaying raw counts (nCount) per spot at various days post-infection (DPI). Scale set at 0–60,000 reads. (B) Violin plot displaying raw counts (nCount) per cluster across all timepoints. (C) SpatialFeaturePlots displaying normalized gene expression data of CXCR3 ligands (i.e. Cxcl4, Cxcl9, and Cxcl10) at various DPI. Scale set at 0–3.0 expression.
Figure 2
Chemokines involved in neutrophil recruitment are upregulated during infection.
SpatialFeaturePlots displaying normalized 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.
Figure 3
Chemokines involved in monocyte recruitment are upregulated during infection.
SpatialFeaturePlots displaying normalized 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.
Figure 4 with 6 supplements
Qualitative heatmaps of chemokine and receptor expression during infection.
Normalized expression in SpatialFeaturePlots was visually ranked as absent (gray), 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.
Figure 4—figure supplement 1
Spatial expression of Cxcl12, Cxcl13, Cxcl14, and Cxcl16.
SpatialFeaturePlots displaying normalized gene expression data of selected Cxcl family members at various days post-infection (DPI). Scale set at 0–3.0 expression.
Figure 4—figure supplement 2
Spatial expression of Ccl3, Ccl4, Ccl5, Ccl6, and Ccl8.
SpatialFeaturePlots displaying normalized gene expression data of selected Ccl family members at various days post-infection (DPI). Scale set at 0–3.0 expression.
Figure 4—figure supplement 3
Spatial expression of Ccl9, Ccl11, Ccl19, Ccl20, and Ccl21a.
SpatialFeaturePlots displaying normalized gene expression data of selected Ccl family members at various days post-infection (DPI). Scale set at 0–3.0 expression.
Figure 4—figure supplement 4
Spatial expression of Ccl22, Ccl24, Ccl25, and Ccl27a.
SpatialFeaturePlots displaying normalized gene expression data of selected Ccl family members at various days post-infection (DPI). Scale set at 0–3.0 expression.
Figure 4—figure supplement 5
Spatial expression of Cxcr family members.
SpatialFeaturePlots displaying normalized gene expression data of selected Cxcr family members at various days post-infection (DPI). Scale set at 0–2.0 expression.
Figure 4—figure supplement 6
Spatial expression of Ccr family members.
SpatialFeaturePlots displaying normalized gene expression data of selected Ccr family members at various days post-infection (DPI). Scale set at 0–2.0 expression.
Figure 5 with 1 supplement
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 normalized expression level of each gene. Maximum expression level set to 1.5; annotated with cluster identity; 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). (C, D) Violin plots of 16 unique clusters showing normalized expression level of each gene across all timepoints. (E, F) Violin plots of various days post-infection (DPI) showing normalized expression level of each gene within all clusters.
Figure 5—figure supplement 1
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. This panels was created using BioRender.com. (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. This panels was created using BioRender.com. (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 at 1 DPI. 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, 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.
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Figure 5—figure supplement 1—source data 1
Bacterial burden data for Figure 5—figure supplement 1B, D.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig5-figsupp1-data1-v1.xlsx
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Figure 5—figure supplement 1—source data 2
Flow cytometry data for Figure 5—figure supplement 1F–I.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig5-figsupp1-data2-v1.xlsx
Figure 6 with 1 supplement
CCR2 and monocyte recruitment are 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. Frequency of CD68+ macrophages from single-cell gate in the (F) liver, (H) spleen, and (J) blood. Frequency of Ly6G+ neutrophils from single-cell gate in the (G) liver, (I) spleen, and (K) blood. (F–K) Three experiments combined using only female mice. Each dot represents one mouse, with 10,000 events collected per sample. Two-way ANOVA (for multiple comparisons to assess genotype and infection); key comparisons and p-values shown. Line represents mean ± standard deviation.
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Figure 6—source data 1
Survival curve data for Figure 6A.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig6-data1-v1.xlsx
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Figure 6—source data 2
Bacterial burden data for Figure 6B.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig6-data2-v1.xlsx
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Figure 6—source data 3
Bacterial burden data for Figure 6C.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig6-data3-v1.xlsx
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Figure 6—source data 4
Flow cytometry data for Figure 6F–K.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig6-data4-v1.xlsx
Figure 6—figure supplement 1
CCR2 and monocyte recruitment are 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–E) Analysis of neutrophil (Ly6G+) and macrophage (CD68+) numbers via flow cytometry. Same samples as in Figure 6, but showing cell counts instead of percent; cell counts from single-cell gate, with 10,000 events collected per sample, or total cell counts from the whole liver or whole spleen calculated using hemocytometer values following tissue processing; three experiments combined using only female mice, each dot represents one mouse. Two-way ANOVA (for multiple comparisons to assess genotype and infection); key comparisons and p-values shown. Line represents mean ± standard deviation.
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Figure 6—figure supplement 1—source data 1
Flow cytometry data for Figure 6—figure supplement 1A–E.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig6-figsupp1-data1-v1.xlsx
Figure 7 with 4 supplements
Loss of CCR2-dependent monocyte trafficking results in abnormal granuloma architecture and failure of bacterial containment.
Wildtype (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 immunohistochemistry (IHC) markers for (A–D) WT female and (E–H) Ccr2–/– male. Necrotic core (NC), coagulative necrosis zone (NC), macrophage zone (M). For 10×, scale bar is 100 µm. For 20× and 40×, scale bar is 50 µm. Representative of two experiments with 2–4 mice per group, and multiple granulomas per section.
Figure 7—figure supplement 1
Loss of CCR2-dependent monocyte trafficking results in abnormal granuloma architecture and failure of bacterial containment.
Wildtype (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 immunohistochemistry (IHC) markers for (A–D) WT female and (E–H) Ccr2–/– female. For 10×, scale bar is 100 µm. For 20× and 40×, scale bar is 50 µm.
Figure 7—figure supplement 2
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 immunohistochemistry (IHC) markers. For 10×, scale bar is 100 µm.
Figure 7—figure supplement 3
Loss of CCR2-dependent monocyte trafficking results in abnormal granuloma architecture and failure of bacterial containment.
Wildtype (WT) and Ccr2–/– mice were infected intraperitoneally (IP) with 1 × 104 CFU C. violaceum and livers harvested 5 days post-infection (DPI) for immunofluorescent staining. Tissue sections were stained for nuclei (DAPI, blue), neutrophils (Ly6G, red), macrophages (CD68, green), and C. violaceum (white). (A) WT female and (B, C) Ccr2–/– females. For 2×, scale bar is 500 µm. For 10×, scale bar is 100 µm.
Figure 7—figure supplement 4
Ccr2–/– mice have increased CCL2 in the liver and serum.
Wildtype (WT) and Ccr2–/– mice were infected intraperitoneally (IP) with 1 × 104 CFU C. violaceum. (A–C) Livers harvested 5 days post-infection (DPI) for immunofluorescent staining. Tissue sections were stained for nuclei (DAPI, blue), neutrophils (Ly6G, red), and CCL2 (white). Serial sections from the same tissues in Figure 7—figure supplement 3. Quantification of CCL2 via ELISA in the liver (D) and serum (E) of WT and Ccr2–/– mice at 3 DPI; two experiments combined using male and female mice, each dot represents one mouse. Line represents mean ± standard deviation.
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Figure 7—figure supplement 4—source data 1
ELISA data for Figure 7—figure supplement 4D, E.
- https://cdn.elifesciences.org/articles/96425/elife-96425-fig7-figsupp4-data1-v1.xlsx
Tables
Table 1
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 and Kubes, 2019; Hughes and Nibbs, 2018; Sokol and Luster, 2015; Zlotnik and Yoshie, 2000; Zlotnik and Yoshie, 2012. Lymph node (LN); natural killer cell (NK); NK T cell (NKT); innate lymphoid cell (ILC); dendritic cell (DC).
| Ligand | Max expression | Alias and main functions |
|---|---|---|
| Cxcl1 | High | (NAP-3) Neutrophil migration |
| Cxcl2 | High | (MIP-2) (MIP2-α) Neutrophil migration; 90% identical to Cxcl1; involved in wound healing |
| Cxcl3 | High | (MIP2-β) Neutrophil migration; migration and adhesion of monocytes |
| Cxcl4 | High | (Pf4) Neutrophil and monocyte migration; released by platelets; wound repair and coagulation; angiogenesis |
| Cxcl5 | High | (LIX) Neutrophil migration; connective tissue remodeling |
| Cxcl9 | High | Th1, CD8, NK, monocyte migration; closely related to CXCL10 and CXCL11 |
| Cxcl10 | High | Th1, CD8, NK, monocyte migration |
| Cxcl11 | Absent | Th1, CD8, NK, monocyte migration |
| Cxcl12 | High | (SDF-1) Lymphocyte migration; bone marrow homing |
| Cxcl13 | Low | B cell migration within follicles of lymphoid tissues; highly expressed in liver, spleen, LN |
| Cxcl14 | Low | Monocyte migration to skin; potent activator of DC |
| Cxcl15 | Absent | Neutrophil migration during inflammation of lungs |
| Cxcl16 | Med | NKT and ILC migration and survival; found in red pulp of the spleen |
| Cxcl17 | Absent | Monocyte and DC migration in the lung |
| Ccl1 | Absent | (TCA3) T cell trafficking |
| Ccl2 | High | (MCP1) Monocyte trafficking |
| Ccl3 | High | (MIP-1α) Macrophage and NK cell migration |
| Ccl4 | High | (MIP-1β) Macrophage and NK cell migration |
| Ccl5 | High | (RANTES) Macrophage and NK cell migration; also chemotactic for T cells, eosinophils, basophils |
| Ccl6 | High | (C10) Myeloid cell differentiation; monocyte, T cell, and eosinophil chemotaxis |
| Ccl7 | Med | (MCP3) (MARC) Monocyte mobilization |
| Ccl8 | Med | (MCP2) Th2 response; skin homing |
| Ccl9 | High | (MIP-1γ) (MRP-2) DC migration |
| Ccl11 | Low | (Eotaxin) Eosinophil and basophil migration; selectively recruits eosinophils |
| Ccl12 | Low | (MCP5) Inflammatory monocyte trafficking |
| Ccl17 | Absent | (ABCD2) (TARC) T cell chemotaxis; lung and skin homing |
| Ccl19 | Med | (MIP-3β) T cell and DC migration to LN |
| Ccl20 | Low | (MIP-3α) Th17 responses; B cell and DC homing to gut-associated lymphoid tissue |
| Ccl21a | Med | (TCA4) T cell and DC migration to LN |
| Ccl21b | Absent | Very similar to Ccl21a |
| Ccl21c | Absent | Identical to Ccl21b |
| Ccl22 | Low | (ABCD1) Th2 response and migration; monocyte, DC, NK migration; produced by monocytes and DC |
| Ccl24 | Med | (MPIF-2) (Eotaxin-2) Eosinophil and basophil migration |
| Ccl25 | Low | (TECK) T cell homing to gut; T cell development; thymocyte, macrophage, and DC migration |
| Ccl26 | Absent | (Eotaxin-3) Eosinophil and basophil migration |
| Ccl27a | Low | T cell migration to skin |
| Ccl27b | Absent | T cell migration to skin |
| Ccl28 | Absent | (MEC) T and B cell migration to mucosal tissues |
| Cx3cl1 | Low | (Fractalkine) NK, monocyte, and T cell migration |
| Xcl1 | Low | (Lymphotactin) Cross-presentation by CD8+ DCs |
Table 2
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 and Kubes, 2019; Hughes and Nibbs, 2018; Sokol and Luster, 2015; Zlotnik and Yoshie, 2000; Zlotnik and Yoshie, 2012. Natural killer cell (NK); innate lymphoid cell (ILC); dendritic cell (DC); plasmacytoid DC (pDC); lymph node (LN); red blood cell (RBC).
| Receptor | Max expression | Alias, cellular expression, and main functions |
|---|---|---|
| Cxcr1 | Absent | (IL8R-α) Neutrophil, monocyte, NKs, mast cell, basophil, CD8 T cells; neutrophil migration and activation |
| Cxcr2 | Med | (IL8R-β) Neutrophil, monocyte, NKs, mast cell, basophil, CD8 T cells; B cell and neutrophil migration; neutrophil egress from BM |
| Cxcr3 | Med | Various T cells, NKs, pDCs, B cells; effector T cell migration and activation |
| Cxcr4 | Med | Most leukocytes; bone marrow homing and retention |
| Cxcr5 | Absent | B cells, T cells; T and B cell migration within LN to B cell zones |
| Cxcr6 | Med | Various T cells, ILCs, NKs, plasma cells; T cell and ILC function |
| Ccr1 | High | Monocyte, macrophage, neutrophil, Th1, basophil, DC |
| Ccr2 | High | Monocyte, macrophage, Th1, DC, basophil, NK; monocyte migration, Th1 immunity |
| Ccr3 | Absent | Highly expressed on eosinophils and basophils; allergic airway; eosinophil trafficking |
| Ccr4 | Absent | Various T cells, monocytes, B cells, DCs; T cell homing to skin and lung |
| Ccr5 | High | Monocytes, macrophages, various T cells, NK, DC, neutrophils, eosinophils; adaptive immunity |
| Ccr6 | Absent | Various T cells, DCs, NKs; DC and B cell maturation and migration; adaptive immunity |
| Ccr7 | Med | Various T cells, DCs, B cells; migration of adaptive lymphocytes and DCs to lymphoid tissues |
| Ccr8 | Absent | Various T cells, monocytes, macrophages; surveillance in skin; expressed in the thymus |
| Ccr9 | Absent | T cells, thymocytes, B cells, DCs, pDCs; T cell migration to gut; key regulator of thymocyte migration and maturation |
| Ccr10 | Absent | T cells, melanocytes, plasma cells; immunity at mucosal sites, especially skin |
| Xcr1 | Low | DCs; antigen cross-presentation |
| Cx3cr1 | Low | Monocytes, macrophages, microglia, DCs, T cells; migration and adhesion of leukocytes; marker of anti-inflammatory monocytes; thought to promote a patrolling phenotype and pro-survival signals |
| Atypical receptors | ||
| Ackr1 | Low | (DARC) RBCs, endothelial cells, neurons; chemokine scavenging, neutrophil transmigration; chemokine transcytosis on lymphatic endothelium and RBCs |
| Ackr2 | Low | Endothelial cells, DCs, B cells, macrophages; chemokine scavenging |
| Ackr3 | Low | (Cxcr7) Stromal cells, B cells, T cells, neurons, mesenchymal cells; pro-survival, adhesion, shaping CXCR4 gradients; involved in CXCR4 gradients |
| Ackr4 | Low | (Ccrl1) Epithelial cells, leukocytes, astrocytes, microglia; chemokine scavenging and transcytosis; chemokine scavenging in thymus |
| Ccrl2 | High | Chemokine receptor-like protein; binds chemerin; related to CCR1; expressed on neutrophils and monocytes |
Table 3
Expression level of selected proteins and 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 Bui et al., 2020; David and 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).
| Other | Max expression | Alias, cellular expression, and main functions |
|---|---|---|
| Fpr1 | High | (Formyl peptide receptor 1) Expressed on myeloid cells and lymphocytes; widely expressed by neutrophils, eosinophils, basophils, monocytes, and platelets (among others); involved in leukocyte chemotaxis and activation |
| Fpr2 | Med | (Formyl peptide receptor 2) Expressed on neutrophils, eosinophils, monocytes, macrophages, T cells; involved in leukocyte chemotaxis and activation |
| C5ar1 | Med | (Complement C5a receptor 1) Expressed on basophils, DCs, mast cells, non-immune cells; involved in leukocyte chemotaxis and activation |
| Ltb4r1 | Low | (Leukotriene B4 receptor) Expressed on neutrophils, macrophages, T cells; involved in leukocyte chemotaxis and activation |
| Cmklr1 | Low | (Chemerin chemokine-like receptor 1) Expressed mainly on myeloid cells; present in thymus, bone marrow, spleen, fetal liver, and lymphoid organs; involved in migration of macrophages, DCs, and pDCs |
| Mmp2 | High | (Gelatinase A) Inactivates CXCL12, CCL7; degrades S100A9 |
| Mmp8 | Med | (Neutrophil collagenase) Stored in secondary granules; cleaves and enhances CXCL5; inactivates CXCL-9 and CXCL-10 |
| Mmp9 | High | (Gelatinase B) Mainly expressed by neutrophils; cleaves and enhances CXCL5; cleaves SDC1 to promote neutrophil infiltration; inactivates CXCL4 and CXCL1; inactivates CXCL-9 and CXCL-10; upregulated during respiratory epithelial healing; also expressed by KCs |
| Mmp12 | High | (Macrophage elastase) Activates TNF release from macrophages |
| Mmp13 | Med | (Collagenase 3) Inactivates CXCL-12; inactivates CCL2, CCL8, CCL13 |
| Itgam | Med | (CR3A) (Cd11b) Regulates adhesion and migration of monocytes, granulocytes, macrophages, NKs; involved in complement system |
| Mif | High | (Macrophage migration inhibitory factor) Binds to CXCR2 and CXCR4 to promote chemotaxis of leukocytes |
| Icam1 | High | (Intracellular adhesion molecule 1) Promotes leukocyte migration from circulation to sites of inflammation |
| S100a8 | High | Heterodimerizes with S100a9; involved in leukocyte recruitment and inflammation |
| S100a9 | High | Heterodimerizes with S100a8; involved in leukocyte recruitment and inflammation |
Table 4
Top 20 differentially expressed genes per cluster.
The FindAllMarkers function was used to identify the top differentially expressed genes for each cluster across all timepoints. Genes were sorted from highest to lowest average log2 fold change (avg_log2FC) values within each cluster. Genes of interest shown in red. Full dataset found in Table 4—source data 1.
| M | HEP1 | HEP0 | HEP4 | HEP3 | NC-C | CN-M2 | HEP5 | CN-M1 | CN | EC2 | NC-P | HEP2 | EC1 | rep HEP | OG |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| Mmp2 | Spink1 | Mup11 | Acot3 | Mup21 | Ewsr1 | Col11a1 | Gm31583 | Ptgs2 | F13a1 | Hbb-bt | Hcar2 | Elovl3 | Derl3 | Ly6d | Ccl8 |
| Aebp1 | Gstm3 | Mup17 | Cyp4a14 | Elovl3 | Parp10 | Ptprn | Mpo | Il11 | Cxcl3 | Hba-a1 | Cxcl3 | Cyp4a12b | 3930402G23Rik | Moxd1 | Gm32468 |
| Olfml3 | Ifi27l2b | Cyp2b13 | Cyp2c69 | Serpina1e | Fth1 | Ccl11 | Gdf10 | Cxcl10 | Pf4 | Hba-a2 | Ptges | Hsd3b5 | Hyou1 | BC049987 | Kdelr3 |
| Cd74 | Klk1b4 | Mup12 | Sult2a1 | Cib3 | Ptprc | Prnd | Cd207 | Cxcl9 | Mmp9 | Hbb-bs | Tnf | Gm32468 | Sult3a1 | Esco2 | Hbb-bt |
| Pacs2 | Vnn3 | Mup16 | Cyp2a4 | Sds | Csf3r | Cthrc1 | Gck | Il6 | Ptges | mt-Atp8 | Ccl4 | Lhpp | Sdf2l1 | Gsta1 | Cyp1b1 |
| Ngp | Cib3 | Mup7 | Cyp4a10 | Mfsd2a | Pacs2 | Gpnmb | Cyp8b1 | Serpine1 | Cstdc4 | mt-Nd4l | Cxcl2 | Cyp4a12a | Apcs | Cdkn3 | Lgals1 |
| Ewsr1 | Cdh1 | Mup1 | Sult2a2 | Acmsd | Lyn | Actg2 | Abcd2 | Hspa1a | Gpr84 | Malat1 | Il1f9 | Fitm1 | Pdia4 | Chrna4 | Vwf |
| Clu | Frzb | Mup3 | Fmo3 | Slc22a7 | Osbpl9 | Fbln2 | 1700001C19Rik | Adm | Itgam | mt-Nd3 | Fth1 | Oat | Dnajb9 | Nat8 | Cthrc1 |
| Cdk11b | Spon2 | Cyp2b9 | Slc16a5 | Etnppl | Hectd1 | Col12a1 | Defb1 | Gm15056 | Fpr2 | mt-Nd5 | Ccl3 | Slc1a2 | A1bg | Nat8f5 | Cpe |
| Parp8 | Snta1 | Cyp7b1 | Cyp2b9 | Slc10a2 | Iqgap1 | Sulf1 | Prox1os | Nos2 | Adam8 | mt-Nd2 | Slfn4 | Cyp2a5 | Prg4 | Mup1 | Pcdh17 |
| Nisch | Wfdc2 | Mup20 | A1bg | Selenbp2 | Clk1 | Mmp13 | Socs2 | Gbp5 | Lyz2 | mt-Co2 | Asprv1 | Tuba8 | Gm26917 | Thrsp | Rasl11a |
| Cpxm1 | Gstm2 | Gm13775 | Cyp2c40 | Mmd2 | Lilr4b | Sfrp1 | Bik | Olr1 | Clec4d | Elane | Slc7a11 | Cyp2c55 | Mt2 | Gm32468 | Ccdc80 |
| Poglut1 | Spic | mt-Atp8 | Slc22a27 | G6pc | Thrap3 | Fkbp10 | Afmid | Rnd1 | Cav1 | Gm26917 | Acod1 | Rhbg | Cyp17a1 | Cdca3 | Mrc2 |
| Col6a2 | Tmem268 | Mup9 | Cyp2c37 | Arl4d | Stip1 | Lox | Rad51b | Retnlg | Mmp8 | mt-Atp6 | Slpi | Slc13a3 | Creld2 | Hebp2 | Hbb-bs |
| Loxl1 | Tstd1 | Serpina3m | Cyp2c38 | Kcnk5 | Fbxl5 | Acta2 | 1810059H22Rik | Il1a | Il1f9 | mt-Nd1 | Ccrl2 | Cyp7a1 | Vnn1 | Ect2 | Ccbe1 |
| Gpx3 | Prelp | Itih4 | Acot1 | Lpin1 | Zfp207 | Col15a1 | Tmem25 | F3 | Fpr1 | mt-Nd4 | Il1rn | Glul | Hist1h4h | Pbk | mt-Nd1 |
| Col1a1 | Slc39a4 | Slco1a1 | Etnppl | Tat | Klf2 | Nbl1 | Angptl6 | Cxcl2 | Capg | Gm29966 | Slc25a37 | Slc1a4 | Rcan2 | Cdc20 | Plxdc2 |
| Igha | Mki67 | Cyp2b10 | Gstt3 | Upp2 | Hck | Col5a2 | Fam89a | Procr | Stfa2l1 | mt-Co3 | Mmp12 | Rdh16 | Hspa5 | Gpam | Nat8f5 |
| Ikbkb | Cdk1 | Car3 | Gm13775 | Pck1 | Rhob | Col5a1 | Mug1 | AA467197 | Pqlc3 | Gm42418 | Clec4e | Serpina7 | mt-Atp6 | Nek2 | Chrna4 |
| Rpl4 | Mcm5 | Fbxo31 | Ptgds | Fam47e | Lilrb4a | Tnc | Ccl27a | Plaur | Pdpn | mt-Co1 | Il1b | Cyp1a2 | mt-Co2 | Aurka | Snhg18 |
-
Table 4—source data 1
Top differentially expressed genes for each cluster across all timepoints.
- https://cdn.elifesciences.org/articles/96425/elife-96425-table4-data1-v1.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Wildtype C57BL/6 mice (WT) | Jackson Laboratory (West Grove, PA) | Ref# 000664 | |
| Strain, strain background (Mus musculus) | Ccr2RFP (Ccr2–/–) | Jackson Laboratory | Ref# 017586 | |
| Strain, strain background (Bacteria) | Chromobacterium violaceum (C. violaceum) | ATCC (Manassas, VA) | Ref# 12472 | |
| Antibody | Rat anti-mouse Ly6G monoclonal (IA8) in BV421 | BD Biosciences (Franklin Lakes, NJ) | Ref# 562737 | 1:300 (FC) |
| Antibody | Rat anti-mouse monoclonal (FA-11) CD68 in FITC | BioLegend (San Diego, CA) | Ref# 137005 | 1:300 (FC) |
| Antibody | Rabbit anti-C. violaceum polyclonal | Cocalico Biologicals (Denver, PA) | Custom polyclonal antibody | 1:2000 (IHC, IF) |
| Antibody | Rat anti-mouse Ly6G monoclonal (IA8) | BioLegend | Ref# 127601 | 1:300 (IHC) |
| Antibody | Rabbit anti-mouse CD68 polyclonal | Abcam (Waltham, MA) | Ref# ab125212 | 1:200 (IHC) |
| Antibody | Rat anti-mouse CD68 monoclonal (FA-11) in Alexa Fluor 488 | Abcam | Ref# ab201844 | 1:100 (IF) |
| Antibody | Rat anti-mouse Ly6G monoclonal (IA8) in Alexa Fluor 647 | BioLegend | Ref# 127610 | 1:100 (IF) |
| Antibody | Rabbit anti-mouse MCP1 (CCL2) polyclonal | Abcam | Ref# ab315478 | 1:100 (IF) |
| Antibody | Goat anti-rabbit secondary polyclonal in Alexa Fluor 594 | Invitrogen (Waltham, MA) | Ref# A32740 | 1:1000 (IF) |
| Commercial assay or kit | Avidin/Biotin Blocking Kit | Vector Laboratories (Newark, CA) | Ref# SP-2001 | |
| Commercial assay or kit | SignalStain Boost IHC Detection Reagent (HRP, Anti-Rabbit) | Cell Signaling (Danvers, MA) | Ref# 8114 | |
| Commercial assay or kit | ImmPRESS HRP Goat Anti-Rat Detection Kit | Vector Laboratories | Ref# MP-7404 | |
| Commercial assay or kit | DAB Substrate Kit, HRP | Vector Laboratories | Ref# SK-4100 | |
| Commercial assay or kit | H&E Stain Kit (Modified Mayer’s Hematoxylin and Bluing Reagent) | Abcam | Ref# ab245880 | |
| Commercial assay or kit | MCP-1/CCL2 Mouse Uncoated ELISA Kit | Thermo Scientific (Waltham, MA) | Ref# 88-7391-22 | |
| Chemical compound, drug | Reparixin | MedChemExpress (Monmouth Junction, NJ) | Ref# HY-15251 | |
| Software, algorithm | RStudio | Posit PBC (Boston, MA) | ||
| Software, algorithm | FlowJo | BD Biosciences | ||
| Software, algorithm | Prism 9 | GraphPad (Boston, MA) | ||
| Software, algorithm | Fiji | ImageJ (Burleson, TX) | ||
| Other | Collagenase Type IV | Gibco | Ref# 17104019 | Tissue dissociation media |
| Other | 1× DMEM, +4.5 g/l D-Glucose, +L-Glutamine, +110 mg/l Sodium Pyruvate | Gibco | Ref# 11995-065 | Cell culture media |
| Other | 1× RPMI Medium 1640, +L-Glutamine | Gibco | Ref# 11875-093 | Cell culture media |
| Other | PenStrep +10,000 units/ml Penicillin, +10,000 µg/ml Streptomycin | Gibco | Ref# 15140-122 | Antibiotics |
| Other | HyClone Characterized Fetal Bovine Serum | Cytiva (Marlborough, MA) | Ref# SH30396.03 | Cell culture media |
| Other | 1× DPBS, -Calcium Chloride, -Magnesium Chloride | Gibco | Ref# 14190-144 | Cell culture media |
| Other | 70 µm Cell Strainers | Genesee Scientific (El Cajon, CA) | Ref# 25-376 | Tissue dissociation reagent |
| Other | 40 µm Cell Strainers | Genesee Scientific | Ref# 25-375 | Tissue dissociation reagent |
| Other | Percoll | GE Healthcare (Chicago, IL) | Ref# 17-0891-01 | Tissue dissociation reagent |
| Other | 1× RBC Lysis Buffer | eBioscience | Ref# 00-4333-57 | Flow cytometry reagent |
| Other | Falcon Round-Bottom Polystyrene Test Tubes | Thermo Scientific | Ref# 14-959-1A | Flow cytometry tubes |
| Other | Mouse BD Fc Block | BD Biosciences | Ref# 553142 | Blocking reagent; used at 1 µg (FC), 2% (IF) |
| Other | Intracellular Fixation & Permeabilization Buffer | eBioscience | Ref# 88-8824-00 | Flow cytometry reagent |
| Other | 10% Neutral Buffered Formalin | VWR (Radnor, PA) | Ref# 16004–128 | Histology reagent |
| Other | 16% Paraformaldehyde | VWR | Ref# 15710S | Immunofluorescence reagent |
| Other | Sucrose | Sigma-Aldrich | Ref# S1888 | Immunofluorescence reagent |
| Other | Tissue-Tek O.C.T. Compound | Sakura (Torrance, CA) | Ref# 4583 | Immunofluorescence reagent |
| Other | Epredia Xylene | Fisher Chemical | Ref# 99-905-01 | Immunohistochemistry reagent |
| Other | ImmEdge Pen | Vector Laboratories | Ref# H-4000 | Immunohistochemistry reagent |
| Other | Normal Goat Serum Blocking Solution, 2.5% | Vector Laboratories | Ref# S-1012 | Immunohistochemistry reagent |
| Other | SignalStain Antibody Diluent | Cell Signaling | Ref# 8112 | Immunohistochemistry reagent |
| Other | Permount | Fisher Chemical | Ref# SP15-100 | Immunohistochemistry reagent |
| Other | T-PER Tissue Protein Extraction Reagent | Thermo Scientific | Ref# 78510 | Tissue dissociation reagent |
| Other | Sulfuric Acid | Ricca Chemical (Arlington, TX) | Ref# 8310-32 | ELISA Stop Buffer |
| Other | Fluoroshield with DAPI | Sigma-Aldrich | Ref# F6057 | Immunofluorescence reagent |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/96425/elife-96425-mdarchecklist1-v1.docx
-
Source code 1
Streamlined code for analysis using RStudio.
- https://cdn.elifesciences.org/articles/96425/elife-96425-code1-v1.zip
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Chemokine expression profile of an innate granuloma
eLife 13:RP96425.
https://doi.org/10.7554/eLife.96425.3
