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Infant mice NK cells do not significantly expand during B. pertussis infection. P7 infant and adult mice were inoculated with B. pertussis or PBS sham inoculum. At 7 dpi, lungs and spleens were harvested for flow cytometry. Top, the numbers of (A) NK cells, (B) NKT-like cells, or (C) T cells are based on the total number of Live <t>CD45</t> cells in the lungs. Bottom, the numbers of (D) NK cells, (E) NKT-like cells, or (F) T cells are based on the total number of live lymphocytes in the spleen. Symbols represent individual mice (n = 3–7 mice/group), and bars indicate mean ± SEM. Statistical significance was analyzed using Ordinary One-way ANOVA with Tukey’s multiple comparisons test. *p≤0.05, ***p≤0.001, ****p≤0.0001.

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1) Product Images from "Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice"

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

Journal: Journal of Leukocyte Biology

doi: 10.1093/jleuko/qiae020

Figure Legend Snippet: Infant mice NK cells do not significantly expand during B. pertussis infection. P7 infant and adult mice were inoculated with B. pertussis or PBS sham inoculum. At 7 dpi, lungs and spleens were harvested for flow cytometry. Top, the numbers of (A) NK cells, (B) NKT-like cells, or (C) T cells are based on the total number of Live CD45 cells in the lungs. Bottom, the numbers of (D) NK cells, (E) NKT-like cells, or (F) T cells are based on the total number of live lymphocytes in the spleen. Symbols represent individual mice (n = 3–7 mice/group), and bars indicate mean ± SEM. Statistical significance was analyzed using Ordinary One-way ANOVA with Tukey’s multiple comparisons test. *p≤0.05, ***p≤0.001, ****p≤0.0001.

Techniques Used: Infection, Flow Cytometry

Infant mice NK cells produce less IFN-γ than adult NK cells during B. pertussis infection and have an immature phenotype. (A) Schematic outline of the experimental design. P7 infant and adult GREAT (IFN-γ-YFP reporter) mice were inoculated with B. pertussis or PBS, and lungs were harvested for flow cytometry and other assays at 7 dpi. (B) Flow cytometry analysis showing the percentage of IFN-γ-expressing cells among live CD45+ NK, NKT-like, CD4+, and CD8+ T cells in adult mice (upper panels) and infant mice (lower panels). Plots are from single mice and are representative of 4–5 mice per group. (C) Graphed data corresponding to results in (B) from all mice (n = 4–5 mice per group). (D) Histogram showing expression of CD27 on lung NK cells from B. pertussis-infected infant and adult mice. (E) Mean fluorescence intensity of CD27 expression on lung NK cells from B. pertussis-infected infant and adult mice. (F) Production of lung IL-12p70 in B. pertussis-infected and PBS sham-inoculated infant and adult mice. (C, E, and F) Symbols represent individual mice (n = 4–5 mice/group), and bars indicate mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Figure Legend Snippet: Infant mice NK cells produce less IFN-γ than adult NK cells during B. pertussis infection and have an immature phenotype. (A) Schematic outline of the experimental design. P7 infant and adult GREAT (IFN-γ-YFP reporter) mice were inoculated with B. pertussis or PBS, and lungs were harvested for flow cytometry and other assays at 7 dpi. (B) Flow cytometry analysis showing the percentage of IFN-γ-expressing cells among live CD45+ NK, NKT-like, CD4+, and CD8+ T cells in adult mice (upper panels) and infant mice (lower panels). Plots are from single mice and are representative of 4–5 mice per group. (C) Graphed data corresponding to results in (B) from all mice (n = 4–5 mice per group). (D) Histogram showing expression of CD27 on lung NK cells from B. pertussis-infected infant and adult mice. (E) Mean fluorescence intensity of CD27 expression on lung NK cells from B. pertussis-infected infant and adult mice. (F) Production of lung IL-12p70 in B. pertussis-infected and PBS sham-inoculated infant and adult mice. (C, E, and F) Symbols represent individual mice (n = 4–5 mice/group), and bars indicate mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Techniques Used: Infection, Flow Cytometry, Expressing, Fluorescence

Adoptive transfer of NK cells from adult mice to infant mice reduces bacterial dissemination during B. pertussis infection. (A) Schematic outline of the experimental design. Purified splenic NK cells or whole splenocytes from adult CD45.2 mice (or PBS control) were adoptively transferred to infant CD45.1 mice at P6. At P7, mice were inoculated with B. pertussis and euthanized at 7 dpi, and tissue was harvested for flow cytometry and bacterial CFU assessment. (B) Flow cytometry analysis of the percentage of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice. (C) Number of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice (n = 3–5 per group). (D, E) Bacterial burden in the lungs (D) and blood (E) of B. pertussis-infected infant mice receiving no cells, NK cells, or whole splenocytes from adult mice at 7 dpi (n = 7–14 per group). (C, D, and E) Symbols represent individual mice, and bars indicate mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Figure Legend Snippet: Adoptive transfer of NK cells from adult mice to infant mice reduces bacterial dissemination during B. pertussis infection. (A) Schematic outline of the experimental design. Purified splenic NK cells or whole splenocytes from adult CD45.2 mice (or PBS control) were adoptively transferred to infant CD45.1 mice at P6. At P7, mice were inoculated with B. pertussis and euthanized at 7 dpi, and tissue was harvested for flow cytometry and bacterial CFU assessment. (B) Flow cytometry analysis of the percentage of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice. (C) Number of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice (n = 3–5 per group). (D, E) Bacterial burden in the lungs (D) and blood (E) of B. pertussis-infected infant mice receiving no cells, NK cells, or whole splenocytes from adult mice at 7 dpi (n = 7–14 per group). (C, D, and E) Symbols represent individual mice, and bars indicate mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Techniques Used: Adoptive Transfer Assay, Infection, Purification, Flow Cytometry

Image Search Results

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Infant mice NK cells do not significantly expand during B. pertussis infection. P7 infant and adult mice were inoculated with B. pertussis or PBS sham inoculum. At 7 dpi, lungs and spleens were harvested for flow cytometry. Top, the numbers of (A) NK cells, (B) NKT-like cells, or (C) T cells are based on the total number of Live CD45 cells in the lungs. Bottom, the numbers of (D) NK cells, (E) NKT-like cells, or (F) T cells are based on the total number of live lymphocytes in the spleen. Symbols represent individual mice (n = 3–7 mice/group), and bars indicate mean ± SEM. Statistical significance was analyzed using Ordinary One-way ANOVA with Tukey’s multiple comparisons test. *p≤0.05, ***p≤0.001, ****p≤0.0001.

Article Snippet: To confirm NK cell purity, 250,000 cells from either unfractionated splenocytes or NK cell enrichment were stained with viability dye 7-AAD (Biolegend), PE-CD45.2 (clone A2,), FITC-, PerCP-cy5.5-, or PE-NK-1.1 (clone PK 136), APC-eF780-CD3e (clone 145–2C11) and PE-Cy7-CD69 and purity was confirmed by flow cytometry.

Techniques: Infection, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Infant mice NK cells produce less IFN-γ than adult NK cells during B. pertussis infection and have an immature phenotype. (A) Schematic outline of the experimental design. P7 infant and adult GREAT (IFN-γ-YFP reporter) mice were inoculated with B. pertussis or PBS, and lungs were harvested for flow cytometry and other assays at 7 dpi. (B) Flow cytometry analysis showing the percentage of IFN-γ-expressing cells among live CD45+ NK, NKT-like, CD4+, and CD8+ T cells in adult mice (upper panels) and infant mice (lower panels). Plots are from single mice and are representative of 4–5 mice per group. (C) Graphed data corresponding to results in (B) from all mice (n = 4–5 mice per group). (D) Histogram showing expression of CD27 on lung NK cells from B. pertussis-infected infant and adult mice. (E) Mean fluorescence intensity of CD27 expression on lung NK cells from B. pertussis-infected infant and adult mice. (F) Production of lung IL-12p70 in B. pertussis-infected and PBS sham-inoculated infant and adult mice. (C, E, and F) Symbols represent individual mice (n = 4–5 mice/group), and bars indicate mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Techniques: Infection, Flow Cytometry, Expressing, Fluorescence

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Adoptive transfer of NK cells from adult mice to infant mice reduces bacterial dissemination during B. pertussis infection. (A) Schematic outline of the experimental design. Purified splenic NK cells or whole splenocytes from adult CD45.2 mice (or PBS control) were adoptively transferred to infant CD45.1 mice at P6. At P7, mice were inoculated with B. pertussis and euthanized at 7 dpi, and tissue was harvested for flow cytometry and bacterial CFU assessment. (B) Flow cytometry analysis of the percentage of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice. (C) Number of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice (n = 3–5 per group). (D, E) Bacterial burden in the lungs (D) and blood (E) of B. pertussis-infected infant mice receiving no cells, NK cells, or whole splenocytes from adult mice at 7 dpi (n = 7–14 per group). (C, D, and E) Symbols represent individual mice, and bars indicate mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Techniques: Adoptive Transfer Assay, Infection, Purification, Flow Cytometry

A . Left , Representative immunostaining of macrophages in the splenic marginal zone expressing CD169 (red) and Tim4 (green); DAPI nuclear staining (blue). Right , FACS contour plots for CD169 and Tim4 expression in splenic CD45 + CD11b low F4/80 low cells and quantitation of Tim4 expression in CD169 + MMMs; n=4. B . Top Left , FACS pseudocolor plots of circulating Ly6C monocytes with histograms identifying CD169 + and CD169 − populations within Ly6C low monocytes, and corresponding cell quantitation; NTM, normalized to mode. Top Right , principal component analysis of top 500 differentially expressed genes (DEGs) from bulk RNAseq of sorted Ly6C low CD169 − and Ly6C low CD169 + cells, and heat maps with dendrograms for 334 DEGs (q<0.05) between the same sub-populations. Bottom , FACS pseudocolor plots of circulating Ly6C low CD169 + Tim4 + macrophages in naïve and spx mice blood with flow histograms identifying CD64 and MHCII surface expression (in black), together with quantitation of frequency or absolute number of the populations shown; n=5-7, statistics: unpaired t test. C. Top, Representative blood FACS dot plots and quantitation of tdTomato (Tdt) expression in blood Ly6C + monocytes from CX3CR1 CreERT ; Rosa26 tdTomato mice after a tamoxifen (TAM) pulse to induce Cre recombination. Shown are data from several time points after TAM and 14 d after splenectomy (Spx) performed at 26 d post-TAM; *p<0.05, n=3, statistics: unpaired t-test. Bottom , representative FACS plots showing CD169 expression in Tdt+ cells 26 d post-TAM pulse and overlay of the CD169 + Tdt + cells (green) on blood Ly6C + monocytes. D . Top , UMAP plots derived from single cell RNA sequencing of blood leukocytes from 3 naïve mice (∼400K total cells, 11 identified cell clusters), heat map demonstrating CD169 ( Siglec1 ) expression primarily in the monocyte cluster, and quantitative expression of select macrophage genes in cells with and without CD169 expression (adjusted FDR p values are shown). E . Left, FACS plots identifying CD169 + Tim4 + cardiac macrophages in intact and Spx mice, overlay of these macrophages on contour plots of CCR2 and LYVE1 expression (in intact mice), and quantitation of overall LYVE1 and CCR2 expression (n=6). Right , quantitation of frequency and number of cardiac CD169 + Tim4 + LYVE1 low macrophages naïve and Spx C57BL/6 mice; n=5-7/group, statistics: unpaired t test.

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Left , Representative immunostaining of macrophages in the splenic marginal zone expressing CD169 (red) and Tim4 (green); DAPI nuclear staining (blue). Right , FACS contour plots for CD169 and Tim4 expression in splenic CD45 + CD11b low F4/80 low cells and quantitation of Tim4 expression in CD169 + MMMs; n=4. B . Top Left , FACS pseudocolor plots of circulating Ly6C monocytes with histograms identifying CD169 + and CD169 − populations within Ly6C low monocytes, and corresponding cell quantitation; NTM, normalized to mode. Top Right , principal component analysis of top 500 differentially expressed genes (DEGs) from bulk RNAseq of sorted Ly6C low CD169 − and Ly6C low CD169 + cells, and heat maps with dendrograms for 334 DEGs (q<0.05) between the same sub-populations. Bottom , FACS pseudocolor plots of circulating Ly6C low CD169 + Tim4 + macrophages in naïve and spx mice blood with flow histograms identifying CD64 and MHCII surface expression (in black), together with quantitation of frequency or absolute number of the populations shown; n=5-7, statistics: unpaired t test. C. Top, Representative blood FACS dot plots and quantitation of tdTomato (Tdt) expression in blood Ly6C + monocytes from CX3CR1 CreERT ; Rosa26 tdTomato mice after a tamoxifen (TAM) pulse to induce Cre recombination. Shown are data from several time points after TAM and 14 d after splenectomy (Spx) performed at 26 d post-TAM; *p<0.05, n=3, statistics: unpaired t-test. Bottom , representative FACS plots showing CD169 expression in Tdt+ cells 26 d post-TAM pulse and overlay of the CD169 + Tdt + cells (green) on blood Ly6C + monocytes. D . Top , UMAP plots derived from single cell RNA sequencing of blood leukocytes from 3 naïve mice (∼400K total cells, 11 identified cell clusters), heat map demonstrating CD169 ( Siglec1 ) expression primarily in the monocyte cluster, and quantitative expression of select macrophage genes in cells with and without CD169 expression (adjusted FDR p values are shown). E . Left, FACS plots identifying CD169 + Tim4 + cardiac macrophages in intact and Spx mice, overlay of these macrophages on contour plots of CCR2 and LYVE1 expression (in intact mice), and quantitation of overall LYVE1 and CCR2 expression (n=6). Right , quantitation of frequency and number of cardiac CD169 + Tim4 + LYVE1 low macrophages naïve and Spx C57BL/6 mice; n=5-7/group, statistics: unpaired t test.

Article Snippet: Cell suspensions were incubated with anti-mouse CD16/32 (clone 93, BioLegend) for 10 min at 4°C to block Fcγ receptors, and then stained for 60 minutes in staining buffer with anti-mouse fluorochrome-conjugated antibodies in panel appropriate combinations for specific experiments as follows: Ly6C-PE-Cy7 (HK1.4, eBioscience), Ly6C-PE Vio770 (1G7.G10, Miltenyi Biotec), CD45.1-FITC (A20, Miltenyi Biotec), CD45.2-PE (104, BD Biosciences), CD169-PE (REA197, Miltenyi Biotec; and 3D6.112, BioLegend), CD11b-Alexa Fluor 700 (M1/70, eBioscience), Tim4-Alexa Fluor 647 (RMT4-54, BioLegend), Gr1/Ly6G-eFluor 450 (RB6-8C5, eBioscience), F4/80-PerCP-Cy5.5 (BM8, eBioscience), MARCO-FITC (ED31, Novus Biologicals), CD45-600 Super Bright (30-F11, eBioscience), CD45-PE-Cy7 (30-F11, BD Biosciences), LYVE1 Alexa Fluor 488 (ALY7, eBioscience), CD45-605 NC (30-F11, eBioscience), CD54(ICAM-1)-PE (eBioKat-1, eBioscience), MHCII (I-A/I-E)-APC-eFluor780 (M5/114.15.2, eBioscience), TGFβ1-APC (TW7-16B4, BioLegend), CD54-FITC (YN1/1.7.4, BioLegend), CD169-PerCP/Cy5.5 (3D6.112, BioLegend), CD169-PE (3D6.112, BioLegend), CD64-Brilliant Violet 605 (X54-5/7.1, BioLegend), CD206-FITC (MR5D3, AbD Serotech), CD45-eVolve 605 (30-F11, eBioscience), CD206-Alexa Fluor 647 (MR5D3, Bio-Rad), IL17A-Alexa Fluor 700 (TC11-18H10.1, BioLegend), CD34-Alexa Fluor 647 (SA376A4, BioLegend), CD117(c-kit)-PE/Cy7 (ACK2, BioLegend), NOS2-TRITC (N-20, Santa Cruz Biotechnology), and CD16/32-PE (93, BioLegend, without pre-Fcγ receptor blocking).

Techniques: Immunostaining, Expressing, Staining, Quantitation Assay, Derivative Assay, RNA Sequencing Assay

A Left , Parabiosis schema joining CD45 isotype-mismatched host (spleen-intact or after splenectomy [Spx]) and donor parabiont mice, with MI induced in the host. Middle and Right , FACS plots and quantitation of donor chimerism in host mouse blood (total CD45 + leukocytes) and heart (Ly6C low CD169 + macrophages) in spleen-intact and Spx host mice 48 h after MI. n=4-7/group, statistics: unpaired t-test. B Top Left , Parabiosis schema joining CD169 DTR host and donor parabiont MaFIA mice, with host mice given either vehicle or diphtheria toxin (DT) at the time of MI. Right , Representative FACS dot plots of donor GFP + CD169 + macrophages in 48 h post MI hearts from host mice and flow histograms of CD169 expression in GFP + CD64 + MHCII + Tim4 + cells delineated as NTM or cell counts. Bottom Left , quantitation of GFP + frequency in host cardiac CD169 + Tim4 + macrophages and total CD169 + Tim4 + cells as a percentage of all autofluorescent(Auto) + macrophages in vehicle and DT treated host MI mice; n=3-4/group, statistics: unpaired t test. C. Left , Parabiosis schema joining CD169 DTR host mice and either spleen-intact or Spx MaFIA donor parabionts, with host mice given DT at the time of MI to deplete CD169 + macrophages. Right , Example FACS dot plots and quantitation of donor CD45 + Auto + CD64 + MHCII + GFP + CD169 + Tim4 + macrophages in the host MI heart 48 h post MI; n=3-4/group, statistics: unpaired t test.

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A Left , Parabiosis schema joining CD45 isotype-mismatched host (spleen-intact or after splenectomy [Spx]) and donor parabiont mice, with MI induced in the host. Middle and Right , FACS plots and quantitation of donor chimerism in host mouse blood (total CD45 + leukocytes) and heart (Ly6C low CD169 + macrophages) in spleen-intact and Spx host mice 48 h after MI. n=4-7/group, statistics: unpaired t-test. B Top Left , Parabiosis schema joining CD169 DTR host and donor parabiont MaFIA mice, with host mice given either vehicle or diphtheria toxin (DT) at the time of MI. Right , Representative FACS dot plots of donor GFP + CD169 + macrophages in 48 h post MI hearts from host mice and flow histograms of CD169 expression in GFP + CD64 + MHCII + Tim4 + cells delineated as NTM or cell counts. Bottom Left , quantitation of GFP + frequency in host cardiac CD169 + Tim4 + macrophages and total CD169 + Tim4 + cells as a percentage of all autofluorescent(Auto) + macrophages in vehicle and DT treated host MI mice; n=3-4/group, statistics: unpaired t test. C. Left , Parabiosis schema joining CD169 DTR host mice and either spleen-intact or Spx MaFIA donor parabionts, with host mice given DT at the time of MI to deplete CD169 + macrophages. Right , Example FACS dot plots and quantitation of donor CD45 + Auto + CD64 + MHCII + GFP + CD169 + Tim4 + macrophages in the host MI heart 48 h post MI; n=3-4/group, statistics: unpaired t test.

Techniques: Quantitation Assay, Expressing

FACS plots and quantitation of Ly6C low CD169 + Tim4 + macrophages and total Ly6C low cells in blood ( A ) and in heart ( B ) 1 d post-MI in wild-type (WT) and splenectomized (Spx) WT mice, and CD169 DTR mice given diphtheria toxin (CD169 DTR /DT) at the time of MI; n=5-7/group, statistics: one-way ANOVA, Bonferroni post-test. Isotype antibody is shown in gray. C . FACS plots and group data for blood CD45 + CD11b + Ly6G + neutrophils and ICAM-1/CD54 + neutrophils 1 d post MI in WT, Spx, and CD169 DTR /DT mice; n=6-8/group; statistics: one-way ANOVA, Bonferroni post-test. D . Left, Representative confocal images of immunofluorescent Ly6G staining in WT, Spx, and CD169 DTR /DT hearts 1 d post-MI demonstrating Ly6G + neutrophil (red) infiltration (arrows); nuclear staining with DAPI (blue). Scale bar 20 μm. Right , FACS plots and corresponding quantitation of cardiac CD45 + CD11b + Ly6G + neutrophils (red) and annexin V + apoptotic neutrophils (blue) in the same groups 1 d post-MI; n=3-4/group, statistics: one-way ANOVA, Dunnett’sT3 post-test. E . FACS density plots for Lin − c-kit + CD34 + CD16/32 + granulocyte monocyte precursors (GMPs) in bone marrow from WT, Spx, and CD169 DTR /DT mice 1 d post MI, together with quantitation. Flow gates were based on isotype antibody control. n=5-7/group; statistics: one-way ANOVA, Tukey’s post-test. F . Left , Representative FACS dot plots identifying cardiac neutrophils as CD11b + Ly6G + cells in WT mice and as Ly6G + tdTomato + cells in Catchup mice at baseline and 1 d post-MI. Right Top , Representative histograms of Ly6G and tdTomato fluorescence intensity in heart mononuclear cells from the same groups. Right Bottom , FACS dot plots gated on cardiac CD169 + Tim4 + macrophages illustrating tdTomato expression in Catchup mice 1 d post-MI. Auto, autofluorescence. G . Representative FACS histograms of intracellular IL4 and IL10 staining in cardiac Ly6C low cells 1 d post MI in WT, Spx and CD169 DTR /DT mice, together with cell quantitation of the Ly6C low subsets. N=6-7/group; statistics: one-way ANOVA, Bonferroni post-test. H . Representative FACS pseudocolor plots for intracellular TGFβ and IL10 staining in cardiac CD169 + macrophages 1 d post MI in WT and Spx mice, with accompanying quantitation; n=4-5/group, statistics: unpaired t test.

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: FACS plots and quantitation of Ly6C low CD169 + Tim4 + macrophages and total Ly6C low cells in blood ( A ) and in heart ( B ) 1 d post-MI in wild-type (WT) and splenectomized (Spx) WT mice, and CD169 DTR mice given diphtheria toxin (CD169 DTR /DT) at the time of MI; n=5-7/group, statistics: one-way ANOVA, Bonferroni post-test. Isotype antibody is shown in gray. C . FACS plots and group data for blood CD45 + CD11b + Ly6G + neutrophils and ICAM-1/CD54 + neutrophils 1 d post MI in WT, Spx, and CD169 DTR /DT mice; n=6-8/group; statistics: one-way ANOVA, Bonferroni post-test. D . Left, Representative confocal images of immunofluorescent Ly6G staining in WT, Spx, and CD169 DTR /DT hearts 1 d post-MI demonstrating Ly6G + neutrophil (red) infiltration (arrows); nuclear staining with DAPI (blue). Scale bar 20 μm. Right , FACS plots and corresponding quantitation of cardiac CD45 + CD11b + Ly6G + neutrophils (red) and annexin V + apoptotic neutrophils (blue) in the same groups 1 d post-MI; n=3-4/group, statistics: one-way ANOVA, Dunnett’sT3 post-test. E . FACS density plots for Lin − c-kit + CD34 + CD16/32 + granulocyte monocyte precursors (GMPs) in bone marrow from WT, Spx, and CD169 DTR /DT mice 1 d post MI, together with quantitation. Flow gates were based on isotype antibody control. n=5-7/group; statistics: one-way ANOVA, Tukey’s post-test. F . Left , Representative FACS dot plots identifying cardiac neutrophils as CD11b + Ly6G + cells in WT mice and as Ly6G + tdTomato + cells in Catchup mice at baseline and 1 d post-MI. Right Top , Representative histograms of Ly6G and tdTomato fluorescence intensity in heart mononuclear cells from the same groups. Right Bottom , FACS dot plots gated on cardiac CD169 + Tim4 + macrophages illustrating tdTomato expression in Catchup mice 1 d post-MI. Auto, autofluorescence. G . Representative FACS histograms of intracellular IL4 and IL10 staining in cardiac Ly6C low cells 1 d post MI in WT, Spx and CD169 DTR /DT mice, together with cell quantitation of the Ly6C low subsets. N=6-7/group; statistics: one-way ANOVA, Bonferroni post-test. H . Representative FACS pseudocolor plots for intracellular TGFβ and IL10 staining in cardiac CD169 + macrophages 1 d post MI in WT and Spx mice, with accompanying quantitation; n=4-5/group, statistics: unpaired t test.

Techniques: Quantitation Assay, Staining, Control, Fluorescence, Expressing

$A . Kaplan-Meier survival curves over 10 d following MI or sham operation in WT, Spx, and CD169 DTR /DT mice, and after MI in CD45.2 Spx mice with adoptive transfer of naïve splenic CD169 + Tim4 + cells from syngeneic CD45.1 WT mice 24 h post-MI (Spx-MI+AT). Statistical comparisons: log-rank test. B . Gross images of post-MI cardiac rupture with hemothorax or hemopericardium, and Kaplan-Meier curves for freedom from rupture over 10 d post-MI in WT, Spx, CD169 DTR /DT, and Spx-MI+AT mice. Statistical comparisons: log-rank test. C . Left , Representative post-mortem whole hearts and end-diastolic long-axis 2-dimensional echocardiograms from WT-MI, Spx-MI, CD169 DTR /DT-MI and Spx-MI+AT mice (10 d post-MI). Right , Quantitation of LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) at 10 d post-MI; n=4-6/group, statistics: one-way ANOVA, Bonferroni post-test. D . Left , Representative confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the heart infarct border zone (BZ) from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. DAPI (blue) nuclear staining. iNOS + CD206 + cells appear yellow. Higher magnification is shown in the middle panel. Right , quantitation of iNOS + CD206 + and iNOS − CD206 + cells/mm 2 in the hearts. N=3/group, statistics: one-way ANOVA, Bonferroni post-test. NS, not significant. E . Top Left , Confocal images of MMP-9 immunostaining (red) in infarct BZ of hearts from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. Nuclear staining with DAPI (blue). Top Right , Quantitative group data for total MMP-9 mean fluorescence intensity per region of interest (ROI). AU, arbitrary units. N=4/group, statistics: one-way ANOVA, Bonferroni post-test. Bottom , Representative Masson’s trichrome stains of infarcted hearts (10 d post-MI) from the same groups demonstrating MI border zone (BZ) fibrosis, together with BZ fibrosis quantitation. n=4/group; statistics: one-way ANOVA, Bonferroni post-test. F . FACS quantitation of Ly6C hi blood monocytes and serum IL-10 levels in WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice at 10 d post-MI. N=4-7/group, statistics: one-way ANOVA, Bonferroni post-test.$

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . Kaplan-Meier survival curves over 10 d following MI or sham operation in WT, Spx, and CD169 DTR /DT mice, and after MI in CD45.2 Spx mice with adoptive transfer of naïve splenic CD169 + Tim4 + cells from syngeneic CD45.1 WT mice 24 h post-MI (Spx-MI+AT). Statistical comparisons: log-rank test. B . Gross images of post-MI cardiac rupture with hemothorax or hemopericardium, and Kaplan-Meier curves for freedom from rupture over 10 d post-MI in WT, Spx, CD169 DTR /DT, and Spx-MI+AT mice. Statistical comparisons: log-rank test. C . Left , Representative post-mortem whole hearts and end-diastolic long-axis 2-dimensional echocardiograms from WT-MI, Spx-MI, CD169 DTR /DT-MI and Spx-MI+AT mice (10 d post-MI). Right , Quantitation of LV ejection fraction (EF) and end-diastolic and end-systolic volume (EDV and ESV) at 10 d post-MI; n=4-6/group, statistics: one-way ANOVA, Bonferroni post-test. D . Left , Representative confocal images of immunofluorescent staining for CD206 (green) and iNOS (red) in the heart infarct border zone (BZ) from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. DAPI (blue) nuclear staining. iNOS + CD206 + cells appear yellow. Higher magnification is shown in the middle panel. Right , quantitation of iNOS + CD206 + and iNOS − CD206 + cells/mm 2 in the hearts. N=3/group, statistics: one-way ANOVA, Bonferroni post-test. NS, not significant. E . Top Left , Confocal images of MMP-9 immunostaining (red) in infarct BZ of hearts from WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice 10 d post-MI. Nuclear staining with DAPI (blue). Top Right , Quantitative group data for total MMP-9 mean fluorescence intensity per region of interest (ROI). AU, arbitrary units. N=4/group, statistics: one-way ANOVA, Bonferroni post-test. Bottom , Representative Masson’s trichrome stains of infarcted hearts (10 d post-MI) from the same groups demonstrating MI border zone (BZ) fibrosis, together with BZ fibrosis quantitation. n=4/group; statistics: one-way ANOVA, Bonferroni post-test. F . FACS quantitation of Ly6C hi blood monocytes and serum IL-10 levels in WT-MI, Spx-MI, CD169 DTR /DT-MI, and Spx-MI+AT mice at 10 d post-MI. N=4-7/group, statistics: one-way ANOVA, Bonferroni post-test.

Techniques: Adoptive Transfer Assay, Quantitation Assay, Staining, Immunostaining, Fluorescence

A . FACS plots and characterization of human CD45 + CD14 + HLA- DR + CD64 + CD169 + Tim4 + circulating macrophages from a subject with acute STEMI. The accompanying overlaid contour plot illustrates CD14 + HLA-DR + (red) and CD64 + CD169 + Tim4 + (green) subsets superimposed on all CD45 + leukocytes in an SSC-A versus FSC-A gate. B . Top , FACS gating strategy for sorting CD45 + CD14 + HLA-DR + CD169 + cells from human peripheral blood for further characterization using ImageStream analysis. Bottom , ImageStream visualization of FACS-sorted CD45 + CD169 + blood cells from STEMI patients and control subjects undergoing elective percutaneous coronary intervention (PCI), and group data for size distribution of CD169 + cells; scale bar 10 μm. C . FACS contour plots and quantitation of circulating CD45 + CD14 + HLA-DR + CD64 + CD169 + Tim4 + macrophages in STEMI and control PCI subjects. n=11-14/group, statistics: unpaired t test.

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Figure Lengend Snippet: A . FACS plots and characterization of human CD45 + CD14 + HLA- DR + CD64 + CD169 + Tim4 + circulating macrophages from a subject with acute STEMI. The accompanying overlaid contour plot illustrates CD14 + HLA-DR + (red) and CD64 + CD169 + Tim4 + (green) subsets superimposed on all CD45 + leukocytes in an SSC-A versus FSC-A gate. B . Top , FACS gating strategy for sorting CD45 + CD14 + HLA-DR + CD169 + cells from human peripheral blood for further characterization using ImageStream analysis. Bottom , ImageStream visualization of FACS-sorted CD45 + CD169 + blood cells from STEMI patients and control subjects undergoing elective percutaneous coronary intervention (PCI), and group data for size distribution of CD169 + cells; scale bar 10 μm. C . FACS contour plots and quantitation of circulating CD45 + CD14 + HLA-DR + CD64 + CD169 + Tim4 + macrophages in STEMI and control PCI subjects. n=11-14/group, statistics: unpaired t test.

Techniques: Control, Quantitation Assay

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Techniques: Immunostaining, Expressing, Staining, Quantitation Assay, Derivative Assay, RNA Sequencing Assay

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Techniques: Quantitation Assay, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Techniques: Quantitation Assay, Staining, Control, Fluorescence, Expressing

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Techniques: Adoptive Transfer Assay, Quantitation Assay, Staining, Immunostaining, Fluorescence

Journal: medRxiv

Article Title: Splenic CD169 + Tim4 + Marginal Metallophilic Macrophages Are Essential for Wound Healing After Myocardial Infarction

doi: 10.1101/2024.08.09.24311769

Techniques: Control, Quantitation Assay

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