1. Developmental Biology
  2. Immunology and Inflammation
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Tissue-resident macrophages promote extracellular matrix homeostasis in the mammary gland stroma of nulliparous mice

  1. Ying Wang
  2. Thomas S Chaffee
  3. Rebecca S LaRue
  4. Danielle N Huggins
  5. Patrice M Witschen
  6. Ayman M Ibrahim
  7. Andrew C Nelson
  8. Heather L Machado
  9. Kathryn L Schwertfeger  Is a corresponding author
  1. Department of Laboratory Medicine and Pathology, University of Minnesota, United States
  2. University of Minnesota Supercomputing Institute, University of Minnesota, United States
  3. Comparative and Molecular Biosciences Graduate Program, University of Minnesota, United States
  4. Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane School of Medicine, United States
  5. Department of Zoology, Faculty of Science, Cairo University, Egypt
  6. Masonic Cancer Center, University of Minnesota, United States
  7. Center for Immunology, University of Minnesota, United States
Research Article
Cite this article as: eLife 2020;9:e57438 doi: 10.7554/eLife.57438
6 figures, 1 table and 5 additional files

Figures

Figure 1 with 2 supplements
Identification of Lyve-1+ macrophages in the mammary gland.

(A) Mammary glands from 10-week-old mice (n = 4) were assessed for CD45+ and CD45 Lyve-1+ cells by flow cytometry. (B) Mammary glands from 10-week-old mice (n = 4) were assessed for CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ cells by flow cytometry. (C) Mammary glands were harvested from 6-week-old mice, immunostaining was performed for F4/80 and Lyve-1, and the localization of single- and double-positive cells associated with TEBs was examined (n = 5, three images/localization). Representative images of F4/80+Lyve-1 (i, arrowhead) and F4/80+Lyve-1+ (ii, arrowhead) cells are shown. Yellow lines show the margin of the mammary gland. Insets show higher magnification. (D, E) Representative images of F4/80+Lyve-1+ cells in the adipose stroma and fibrous capsule of the mammary gland. Representative images show F4/80 (iii) and co-staining (iv). Inserts show higher magnification. (F) Human mammary glands obtained from reduction mammoplasty samples demonstrate the presence of CD68+Lyve-1+ macrophages in the interlobular stroma (n = 5, three images/sample). Representative images show F4/80 (v) and co-staining (vi).

Figure 1—figure supplement 1
Identification of Lyve-1+ macrophages in the mammary gland.

(A) Gating of flow cytometry for CD45+ and CD45 Lyve-1+ cells. (B) Gating of flow cytometry for CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ cells.

Figure 1—figure supplement 2
Localization of Lyve-1+ macrophages in the mammary gland.

Image of a mammary gland from a 6-week-old female mouse (n = 3) stained for F4/80 (red), Lyve-1 (green) and DAPI (blue). Insets show examples of the mammary gland capsule (a–d) and lymph nodes (e–h).

Figure 2 with 1 supplement
Identification of a distinct Lyve-1+ macrophage subpopulation by transcriptional profiling.

(A) Heat map of RNA-seq analysis of CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ cells isolated from 6-week-old (lane 1) and 10-week-old (lanes 2–3) mice. Genes shown have an adjusted p-value <0.01 and a fold-change of >1 and <10 (abs values). For each lane, n = 4 pooled mice. (B) UMAP of scRNA-seq analysis of CD45+ cells isolated from 10-week-old mice as generated by Seurat. (C) Heat map of the top 20 differentially regulated genes in each cluster. (D) Feature plots and violin plots of selected macrophage genes. (E) Feature plots and violin plots of genes associated with cluster four that were also found in the bulk RNA-seq analysis. (F) GSEA demonstrating that the single-cell populations (clusters) are enriched for in the data from the bulk RNA-seq analysis.

Figure 2—figure supplement 1
Identification of a distinct Lyve-1+ macrophage subpopulation by transcriptional profiling.

(A) Post-sort analysis of CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ cells that were submitted for bulk RNA-seq analysis. (B) scRNA-seq data showing genes associated with macrophages expressed in clusters 0 and 4. (C) Violin plots showing expression levels of Itgam and Itgax across clusters. (D) Graph showing Lyve-1 expression in F4/80+/CD11b macrophages by flow cytometry.

Figure 3 with 1 supplement
F4/80+Lyve-1+ cells are enriched for CD206 expression.

(A) Violin plot of Mrc1 expression in the single-cell RNA-seq dataset shown in Figure 2. (B) Mammary glands from 10-week-old mice (n = 4) were assessed for CD206 expression in CD45+CD11b+F4/80+Lyve-1- and CD45+CD11b+F4/80+Lyve-1+ cells by flow cytometry. (C) Mammary glands were harvested from 6-week-old mice (n = 5, three images/localization), immunostaining was performed for F4/80 and CD206, and the localization of single- and double-positive cells associated with TEBs was examined. Representative images of F4/80+CD206 (i, arrowheads) and F4/80+CD206+ (ii, arrowheads) cells are shown. (D, E) Representative images of F4/80+CD206+ cells in the adipose stroma and fibrous capsule of the mammary gland. Representative images show F4/80 (iii) and co-staining (iv). The yellow line in panel (E) shows the margin of the mammary gland. Insets show higher magnification. (F) Flow cytometry analysis showing F4/80hi and F4/80int cells in the mammary gland and CD206 expression in each population. (G) Flow cytometry analysis of F4/80 expression on Lyve-1 and Lyve-1+ cells. n = 4 mice analyzed.

Figure 3—figure supplement 1
F4/80+Lyve-1+ cells are enriched for CD206 expression.

(A) Gating of flow cytometry for Lyve-1 expression in CD45+CD11b+F4/80+CD206 and CD45+CD11b+F4/80+CD206+ cells. (B) Gating of flow cytometry for CD206 expression in CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ cells.

Lyve-1+ macrophages exhibit low turnover and self-renewal in the mammary gland.

(A) Schematic of shielded bone marrow chimera experiment. (B) Representative flow cytometry plots showing the gating and identification of host (CD45.2) and donor (CD45.1) cells in the blood and mammary gland. Quantification of flow cytometry demonstrates that although most of the monocytes in the blood are donor-derived, the majority of macrophages in the mammary gland are host-derived. Cells were first gated on Live and pan-CD45 expression. In the quantification graphs, n = 23 mice from three separate experiments. (C) Quantification of flow cytometry demonstrates that the majority of Lyve-1+ macrophages remain host-derived 6 weeks after bone marrow transplant. (D) Mammary glands were isolated from mice after a 2 hr BrdU pulse and immunostained for BrdU and Lyve-1 (n = 3, three images/localization). Examples of BrdU+Lyve-1+ cells are shown, insets show higher magnification.

Figure 5 with 1 supplement
Lyve-1+ cells localize to HA-enriched ECM in the mammary gland.

(A) Mammary glands from 6-week-old mice were immunostained for HA using HA binding protein (HABP, green) and smooth muscle actin (SMA, red) (n = 3, three images/sample). (B) Mammary glands from 6-week-old mice were immunostained for HA using HA binding protein (HABP, red) and Lyve-1 (green) showing staining of the fibrous capsule and fibrous septae in the adipose stroma. (C) Quantification of Lyve-1+ cells associated with the capsule and adipose stromal regions in the mammary gland. N = 6 mice, three images analyzed per mammary gland. ****p<0.0001. (D) Co-staining of HABP and Lyve-1 in human mammary gland demonstrating the presence of HA-associated Lyve-1+ cells (n = 3, five images/sample). Arrowheads show Lyve-1+ cells and inserts show higher magnification. (E) Mammary tumor sections from 4T1 tumors were immunostained for F4/80 (red) and Lyve-1 (green) (n = 4, three images/localization). Representative images are shown of the peri-tumoral stroma and the tumor parenchyma. Inserts show higher magnification. (F) Mammary tumor sections from 4T1 tumors were immunostained for Lyve-1 (red) and HABP (green). Insets show higher magnification.

Figure 5—figure supplement 1
Lyve-1+ cells localize to HA-enriched ECM in the mammary gland.

Mammary glands were stained for F4/80, Lyve-1 and HABP to confirm that Lyve-1+ cells associated with HA are also F4/80+. Insets show higher magnification.

Figure 6 with 1 supplement
Macrophage depletion impacts ECM in the stroma.

(A) 5-week-old female mice were treated with pexidartinib for 2 weeks and mammary glands were harvested for analysis of macrophages for CD45+CD11b+F4/80+ by flow cytometry. Flow cytometry demonstrated depletion of CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ cells in the mammary gland following pexidartinib treatment. Quantification of total cell counts and fold-change depletion are shown. Total Macrophages Number DMSO vs pexidartinib, p=0.0007; Lyve1 Macrophages Number DMSO vs pexidartinib, p=0.0015; Lyve1+ Macrophages Number DMSO vs pexidartinib, p=0.0095; Lyve1 Fold Change DMSO vs pexidartinib, p=0.037; Lyve1+ Fold Change DMSO vs pexidartinib, p=0.0052. These data demonstrate one representative experiment, experiments were repeated three times with similar results, n = 4 mice per treatment group. (B) Mammary glands from mice treated with either pexidartinib or solvent control were stained for HA (HABP, green) or with trichrome to visualize collagen-containing ECM (collagen, blue). (C) Quantification of HABP and collagen staining. Between 3 and 6 images/sample were analyzed for HABP and collagen staining, n = 5 per treatment group. Three replicates were used for HABP analysis and one replicate was done for collagen analysis. HA deposition (% of area) DMSO vs pexidartinib, p<0.0001 (pooled) and collagen deposition (% of area) DMSO vs pexidartinib, p<0.0001. (D) Quantification of HA in mammary glands isolated from mice treated with pexidartinib or solvent control normalized to gland weight, p=0.0015, n = 4 mice for control group and n = 5 mice for pexidartinib group. (E) GSEA analysis of the C5 database for gene sets enriched in cluster 4 from the scRNA-seq analysis showing the top nine enriched gene sets by NES. (F) GSEA plots of representative lists identified in panel (E). *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.0001; using Welch’s t-test.

Figure 6—figure supplement 1
Macrophage depletion does not impact ductal elongation of branching.

Quantification of ductal elongation and branching in mammary glands from mice treated with either solvent or pexidartinib for 2 weeks. Ductal Branching DMSO vs pexidartinib, p=0.1081; Ductal Length DMSO vs pexidartinib, p=0.1661. P values generated using Mann-Whitney U Test.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Mus musculus, female)FVB/NEnvigoFVB/NHsd
Strain, strain background (Mus musculus, female)Balb/c CD45.2 MiceJackson LaboratoriesStock No.: 000651Balb/cJ Mouse Strain
Strain, strain background (Mus musculus)Balb/c CD45.1 MiceJackson LaboratoriesStock No.: 006584CByJ.SJL(B6)-Ptprca/J Mouse Strain
AntibodyvioletFluor 450-Conjugated anti-CD45.1 (mouse monoclonal)Tonbo BiosciencesCat #: 75–0453 U025
RRID:AB_2621949
Flow cytometry: 1:200
AntibodyAlexa Fluor 700-Conjugated anti-CD45.2 (mouse monoclonal)BioLegendCat #: 109822
RRID:AB_493731
Flow cytometry: 1:200
AntibodyBUV395-conjugated anti-Ly6G (rat monoclonal)BioLegendCat #: 563978
RRID:AB_2716852
Flow cytometry: 1:400
AntibodyBrilliant Violet 421-Conjugated Anti-CD64 (mouse monoclonal)BioLegendCat #: 139309
RRID:AB_2562694
Flow cytometry: 1:100
AntibodyBrilliant violet 711-conjugated anti-CD11c (Armenian hamster monoclonal)BioLegendCat #: 117349
RRID:AB_2563905
Flow cytometry: 1:200
AntibodyPE/Cy7-conjugated anti-CD11b (rat monoclonal)BioLegendCat #: 101216
RRID:AB_312799
Flow cytometry: 1:200
AntibodyPE-conjugated anti-F4/80 (rat monoclonal)BioLegendCat #: 123109
RRID:AB_893498
Flow cytometry: 1:200
AntibodyBiotinylated anti-Lyve-1 (goat polyclonal)R and D SystemsCat #: BAF2125
RRID:AB_2138529
Flow cytometry:4 μg/mL
AntibodyAnti-CD16/CD32 (rat monoclonal)eBioscienceCat #: 14-0161-82
RRID:AB_467133
Flow cytometry: 1:200
AntibodyRat anti-mouse monoclonal F4/80 (clone: CI: A3-1)Bio-Rad laboratoriesCat# MCA497GA
RRID:AB_323806
IF 1:100
AntibodyGoat polyclonal anti-mouse LYVE-1 (Ala24-Thr234)R and D SystemsCat# AF2125
RRID:AB_2297188
IF 1:60
AntibodyAnti-mannose receptor
(rabbit polyclonal)
AbcamCat# ab64693
RRID:AB_1523910
IF 1:1000
AntibodyAnti-BrdU antibody [BU1/75 (ICR1)]AbcamCat# ab6326
RRID:AB_305426
IF 1:200
AntibodyHyaluronic acid bindingprotein, biotinylatedMillipore SigmaCat# 385911–50 UGIF 1:100
AntibodyActin-smooth muscle (rabbit polyclonal)Spring BioscienceCat# E2464
RRID:AB_95752
IF 1:50
AntibodyCD68 monoclonal antibody (514H12)Thermo- Fisher ScientificCat# MA1-80133
RRID:AB_929283
IF 1:100
AntibodyHuman LYVE-1 antibody (goat polyclonal)R and D SystemsCat# AF2089
RRID:AB_355144
IF 1:60
AntibodyAlexa 594 secondary antibody
(donkey anti-mouse)
Thermo- Fisher ScientificCat# A-32744
RRID:AB_2762826
IF 1:400
AntibodyAlexa 594 secondary antibody (donkey anti-rat)Thermo- Fisher ScientificCat# A-21209
RRID:AB_2535795
IF 1:400
AntibodyAlexa Fluor 488 conjugated streptavidin secondary antibodyThermo- Fisher ScientificCat# S11223IF 1:400
AntibodyAlexa 488 secondary antibody
(donkey anti-goat)
Thermo- Fisher ScientificCat# A-11055
RRID:AB_2534102
IF 1:400
AntibodyAlexa 488 secondary antibody
(goat anti-rabbit)
Thermo- Fisher ScientificCat# A-11008
RRID:AB_143165
IF 1:400
AntibodyAlexa 594 secondary antibody
(goat anti-rat)
Thermo- Fisher ScientificCat # A-11007
RRID:AB_10561522
IF 1:400
AntibodyAlexa 594 secondary antibody (goat anti-rabbit)Thermo- Fisher ScientificCat# A-11012
RRID:AB_2534079
IF 1:400
AntibodyAlexa Fluor 647 conjugated streptavidin secondary antibodyThermo- Fisher ScientificCat# S-32357IF 1:400
Commercial
assay or kit
Hyaluronan enzyme-linked immunosorbent assayEchelon BiosciencesCat #: K-1200
Chemical compound, drugBusulfanSigma-AldrichCat #: B2635-10G
Chemical compound, drugPexidartinibSelleck ChemCat #: S7818
Chemical compound, drugHeparinSigma-AldrichCat #: H3149-10KU
Chemical compound, drugCollagenase ASigma-AldrichCat #: 10103586001
Chemical compound, drugDNase ISigma-AldrichCat #: DN25-100MG
Software, algorithmGraphPad Prism 8.0GraphPad Prism 8.0RRID:SCR_002798http://www.graphpad.com/scientific-software/prism/
Software, algorithmImageJ 2.0.0ImageJ 2.0.0RRID:SCR_003070https://imagej.nih.gov/ij/download.html
Software, algorithmHisat2Kim et al., 2015RRID:SCR_015530version 2.0.2
Software, algorithmDESeq2 softwareLove et al., 2014RRID:SCR_015687version 1.20.0
Software, algorithmggplot2RRRID:SCR_014601v3.0.0
Software, algorithmpheatmapRRRID:SCR_0164181.0.12
OtherFlow Cytometry Perm Buffer (10X)Tonbo BiosciencesCat #: TNB-1213-L150
OtherFixable Viability Dye eFluor 780eBioscienceCat #: 65-0865-14Flow cytometry: 1:1000 dilution
OtherAPC-conjugated streptavidineBioscienceCat #: 17-4317-82Flow cytometry: 1:100 dilution
OtherProLong Gold Antifade Mountant
(DAPI)
Thermo- Fisher ScientificCat# P36931
OtherTrichrome stain kitAbcamCat# ab150686
OtherAntigen Retrieval Buffer (100X EDTA Buffer, pH 8.0)AbcamCat# ab936801x
OtherAntigen unmasking solution, PH 6.0Thermo- Fisher ScientificCat# NC94010671x

Additional files

Supplementary file 1

List of 155 differentially expressed genes in CD45+CD11b+F4/80+Lyve-1 and CD45+CD11b+F4/80+Lyve-1+ bulk RNA-seq analysis.

https://cdn.elifesciences.org/articles/57438/elife-57438-supp1-v3.xlsx
Supplementary file 2

List of the top 100 differentially regulated genes in each cluster from the scRNA-seq analysis.

https://cdn.elifesciences.org/articles/57438/elife-57438-supp2-v3.xlsx
Supplementary file 3

GSEA analysis using gene sets created from a differentially expressed ranked list from the bulk RNA-seq data (adjusted p-value <0.1 and a fold-change >0.25) to determine enrichment of cluster 4.

https://cdn.elifesciences.org/articles/57438/elife-57438-supp3-v3.xlsx
Supplementary file 4

List of top gene sets identified by GSEA analysis including core enriched genes.

https://cdn.elifesciences.org/articles/57438/elife-57438-supp4-v3.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/57438/elife-57438-transrepform-v3.docx

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