Research ArticleMACROPHAGES

CD169+ macrophages orchestrate innate immune responses by regulating bacterial localization in the spleen

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Science Immunology  06 Oct 2017:
Vol. 2, Issue 16, eaah5520
DOI: 10.1126/sciimmunol.aah5520
  • Fig. 1 Splenic CD169+ macrophages trap and prevent early Lm replication and spread.

    (A) Confocal image (left) and isosurfaced image (right) of intracellular Lm located within a CD169+ macrophage in the MZ. Scale bars, 10 μm. 3D, three-dimensional. (B) Quantification of intracellular Lm within CD169+ macrophages in the spleens of WT mice. Each symbol represents a distinct region of the spleen. Data are shown as means ± SEM. (C) Quantification of viable Lm from sorted WT CD169+ macrophages at 1 hpi. Data are shown as means ± SEM. Representative example of gating strategy used to sort CD169+ macrophages from the spleen. CFU, colony-forming units; MZMs, MZ macrophages; UV, ultraviolet; FSC-A, forward scatter–area; FSC-H, forward scatter–height; SSC-A, side scatter–area; PE, phycoerythrin. (D) Confocal images of spleen sections from WT or CD169-DTR mice. Lm are rendered with spots. Scale bars, 50 μm. (E) Survival of WT or CD169-DTR mice (***P < 0.0005, Mantel-Cox log-rank test). (F) Quantification of Lm numbers from the spleens of WT or CD169-DTR mice at 10 hpi. Each symbol represents an individual mouse, shown as means ± SEM (*P < 0.05, Mann-Whitney test). (G) Quantification of Lm in the blood of WT or CD169-DTR mice. Each symbol represents an individual mouse, shown as mean ± SEM (*P < 0.05, Mann-Whitney test). All data are representative of two to three separate experiments with three to six mice per group.

  • Fig. 2 Splenic CD169+ macrophages express a unique gene profile.

    (A) PCA of 17,336 genes using each sample’s log2[fragments per kilobase of transcript per million mapped reads (FPKM) + 1]. (B) Heat map using z scores for 709 differentially expressed genes between naïve CD169+ macrophages and red pulp macrophages (F4/80+). (C) Transcript expression (FPKM) of paired-end sample sequencing represented as normalized values based on transcript length and depth of coverage. Genes represent transcripts that were previously identified to distinguish red pulp macrophages from other subsets of tissue macrophages. Data are shown as means ± SEM (*P < 0.05 and **P < 0.005, Student’s t test). ns, not significant. (D) Heat map using z scores for the identified 795 differentially expressed genes comparing CD169+ macrophages from naïve and Lm-infected mice. (E) Transcript expression (FPKM) of genes exclusively up-regulated in CD169+ macrophages 12 hours after Lm infection. Data are shown as means ± SEM (*P < 0.05 and **P < 0.005, Student’s t test).

  • Fig. 3 CD169+ macrophages mediate Lm translocation to the splenic T cell zones.

    (A) Confocal images of spleen sections from WT and CD169-DTR mice at 12 hpi. Lm are rendered with spots in green, B cell zones (B), T cell zones (T), and red pulp (RP). White and red arrowheads indicate Lm inside and outside the white pulp, respectively. Scale bars, 50 μm. (B) Frequency of Lm located in splenic T cell zones of WT or CD169-DTR mice at 12 hpi. Each symbol represents a distinct region of splenic tissue. Data are shown as means ± SEM (***P < 0.0005, Student’s t test). (C) Confocal image of spleen section from WT or CD169-DTR mice at 24 hpi. Lm are rendered with spots in green, B cell zones, T cell zones, and red pulp. White and red arrowheads indicate Lm inside and outside the white pulp, respectively. Scale bars, 150 μm. (D) Frequency of Lm found in splenic T cell zones of WT or CD169-DTR mice at 24 hpi. Each symbol represents a distinct region of splenic tissue. Data are shown as means ± SEM (***P < 0.0005, Student’s t test). (E) Confocal image of spleen section from WT and CD169-DTR mice at 24 hpi stained for F4/80 (light blue), B220 (dark blue), and CD8α+ DCs (red). Scale bars, 150 μm. (F) FACS quantification of F4/80+ macrophages in the spleens of WT or CD169-DTR mice at 48 hpi. Data are shown as means ± SEM. All data are representative of three separate experiments with two to four mice per group.

  • Fig. 4 CD169+ macrophages play a critical role in mediating innate immune cell reorganization.

    (A) Splenic Lm quantification in WT or CD169-DTR mice at 24 and 48 hpi. Each symbol represents an individual mouse. Data are shown as means ± SEM (*P < 0.05 and **P < 0.005, Mann-Whitney test). (B) Confocal images of WT or CD169-DTR mice at 24 hpi. Spleen sections stained for Lm (green) and Ly6G (red). Lm are rendered using spots. White arrowheads indicate Lm foci devoid of neutrophil clusters. Scale bars, 100 μm. (C) Confocal images of spleen sections from WT or CD169-DTR mice at 24 hpi showing organized clustering versus disrupted hierarchical clustering of innate immune cells. Lm are rendered with spots in green. CD169+ macrophages are shown in blue, NK cells are shown in red, and CD11b+ cells are shown in turquoise. Scale bars, 100 μm. (D) Representative images of spleen sections showing IFN-γ (yellow), NK1.1 (red), and Lm (white). Lm are rendered with spots. Scale bars, 80 μm. (E and F) Lm numbers quantified from (E) lungs and (F) brains of WT or CD169-DTR mice. ND, not detected. Each symbol represents an individual mouse. Data are shown as means ± SEM (*P < 0.05 and **P < 0.005, Mann-Whitney test). All data are representative of two to four separate experiments with two to four mice per group.

  • Fig. 5 CD169+ macrophages mediate the transport of bacteria to T cell zones by trans-infecting CD8α+ DCs.

    (A) Confocal images of spleen sections from WT or CD169-DTR mice at 6 hpi. Colocalization of CD11c and CD8α is shown in white. Lm are rendered with spots in green. White arrowheads indicate Lm in the MZ. Scale bars, 70 to 80 μm. (B) Confocal images of spleen sections from WT or CD169-DTR mice at 9 hpi. Spleen sections stained for Lm (green), CD169 (blue), and CD11c (red). Colocalization of CD11c and CD8α is shown in white. Lm are rendered with spots. White arrows indicate areas of Lm. Scale bars, 30 μm. (C) High-magnification confocal image showing Lm-infected CD169+ macrophages and CD8α+ DCs in close contact at 3 hpi. Spleen sections stained for Lm (green), CD169 (blue), CD11c (red), and CD8α (light blue). Scale bars, 20 μm. (D and E) Quantification of viable Lm recovered from FACS-sorted myeloid cells from pooled WT or CD169-DTR mice at (D) 1 hpi and (E) 3 hpi (n = 2 to 4). (F) General gating strategy used for CD169+ macrophages and DC subset identification from the spleen. (G) Representative confocal image of WT mice at 3 hpi. Spleen sections stained for Lm (white), CD169 (blue), and CD11c (red). Lm are rendered with spots. White and green arrowheads indicate Lm in CD169+ cells and CD169+CD11c+ cells, respectively. Scale bars, 20 μm. All data are representative of three to four separate experiments with two to four mice per group.

  • Fig. 6 CD8α+ DC-deficient mice are highly susceptible to Lm infection in the absence of CD169+ macrophages.

    (A and B) Splenic Lm quantification of WT, Batf3−/−, CD169-DTR, or CD169-DTR–Batf3−/− mice at (A) 12 hpi and (B) 24 hpi. Each symbol represents individual mouse. Data are shown as means ± SEM (*P < 0.05, Mann-Whitney test). (C) Confocal images of spleen sections from WT, Batf3−/−, CD169-DTR, or CD169-DTR–Batf3−/− mice at 24 hpi. Spleen sections stained for Lm (red), F4/80 (blue), and CD8 (yellow). Lm are rendered with spots. Scale bars, 100 μm. (D) Frequency of Lm found in splenic T cell zones at 24 hpi in WT, CD169-DTR, or CD169-DTR–Batf3−/− mice. Data are shown as means ± SEM. (E) Lm quantification in the spleens of WT, Batf3−/−, CD169-DTR, or CD169-DTR–Batf3−/− mice at 1 hpi. Each symbol represents an individual mouse. Data are shown as means ± SEM (**P < 0.005, Mann-Whitney test). (F) Frequency of intracellular Lm located within CD169+ macrophages in WT or Batf3−/− mice at 3 hpi. All data are representative of two to four separate experiments.

  • Fig. 7 Cytosolic entry of Lm required for CD8α+ DC recruitment.

    (A) Confocal microscopy of spleen sections from WT mice at 12 hpi with WT Lm (top), ActA knockout (KO) Lm (middle), and LLO KO Lm (bottom). Lm are rendered with spots in green. CD169 is shown in blue, and colocalization of CD11c and CD8α is shown in red. Scale bars, 50 μm. (B) Quantification of intracellular Lm within CD169+ macrophages at 12 hpi. Each symbol represents a different confocal image quantified. All data are representative of two separate experiments with two to three mice per group.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/16/eaah5520/DC1

    Materials and Methods

    Fig. S1. Characterization of WT and CD169-DTR mice.

    Fig. S2. Splenic CD169+ macrophages are essential for protection against various blood-borne pathogens.

    Fig. S3. Sorting strategy and quality control for RNA sequencing.

    Fig. S4. CD169-DTR mice have elevated but displaced cytokine production.

    Fig. S5. CD169+ macrophages recruit CD8α+ DCs to early sites of infection.

    Fig. S6. Characterization of CD8α+ DCs in CD169-DTR mice.

    Fig. S7. Myeloid cell differentials comparing Batf3−/− knockout and CD169-DTR–Batf3−/− knockout mice.

    Fig. S8. Model depicting the role of CD169+ macrophages after Lm infection.

    Table S1. Top 15 GO enriched biological processes up-regulated in CD169+ macrophages versus red pulp macrophages at steady state.

    Table S2. Top 15 GO enriched biological processes up-regulated in CD169+ macrophages during infection versus steady state.

    Table S3. Top 15 GO enriched biological processes down-regulated in CD169+ macrophages during infection versus steady state.

    Table S4. List of identified TF candidates for up-regulated genes in CD169+ macrophages isolated from infected mice versus uninfected mice.

    Table S5. Enriched immune-related GO terms for corresponding TFs in CD169+ macrophages during infection versus steady state.

    Table S6. Antibodies used for fluorescence-activated cell sorting (FACS) and confocal microscopy.

    Movie S1. Listeria is captured by CD169+ macrophages early after infection.

    Movie S2. Listeria is captured by CD169+ macrophages early after infection.

    Movie S3. CD169+ macrophages transfer bacteria to CD8α+ DCs.

    Source data (Excel file)

    References (3638)

  • Supplementary Materials

    Supplementary Material for:

    CD169+ macrophages orchestrate innate immune responses by regulating bacterial localization in the spleen

    Oriana A. Perez, Stephen T. Yeung, Paola Vera-Licona, Pablo A. Romagnoli, Tasleem Samji, Basak B. Ural, Leigh Maher, Masato Tanaka, Kamal M. Khanna*

    *Corresponding author. Email: kamal.khanna{at}nyumc.org

    Published 6 October 2017, Sci. Immunol. 2, eaah5520 (2017)
    DOI: 10.1126/sciimmunol.aah5520

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of WT and CD169-DTR mice.
    • Fig. S2. Splenic CD169+ macrophages are essential for protection against various blood-borne pathogens.
    • Fig. S3. Sorting strategy and quality control for RNA sequencing.
    • Fig. S4. CD169-DTR mice have elevated but displaced cytokine production.
    • Fig. S5. CD169+ macrophages recruit CD8α+ DCs to early sites of infection.
    • Fig. S6. Characterization of CD8α+ DCs in CD169-DTR mice.
    • Fig. S7. Myeloid cell differentials comparing Batf3−/− knockout and CD169- DTR–Batf3−/− knockout mice.
    • Fig. S8. Model depicting the role of CD169+ macrophages after Lm infection.
    • Table S1. Top 15 GO enriched biological processes up-regulated in CD169+ macrophages versus red pulp macrophages at steady state.
    • Table S2. Top 15 GO enriched biological processes up-regulated in CD169+ macrophages during infection versus steady state.
    • Table S3. Top 15 GO enriched biological processes down-regulated in CD169+ macrophages during infection versus steady state.
    • Table S4. List of identified TF candidates for up-regulated genes in CD169+ macrophages isolated from infected mice versus uninfected mice.
    • Table S5. Enriched immune-related GO terms for corresponding TFs in CD169+ macrophages during infection versus steady state.
    • Table S6. Antibodies used for fluorescence-activated cell sorting (FACS) and confocal microscopy.
    • Legends for movies S1 to S3
    • References (36–38)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Listeria is captured by CD169+ macrophages early after infection.
    • Movie S2 (.mov format). Listeria is captured by CD169+ macrophages early after infection.
    • Movie S3 (.mov format). CD169+ macrophages transfer bacteria to CD8α+ DCs. Source data (Excel file)
    • Source data (Excel file).

    Files in this Data Supplement:

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