Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling

Abstract

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Macrophage and DC populations show differential association with mammary ducts.
Fig. 2: Ductal MØs have a distinct gene expression signature.
Fig. 3: DMs are tissue-resident and occupy an intra-epithelial niche.
Fig. 4: DMs frequently contact all epithelial cells by dendrite movement.
Fig. 5: DMs proliferate during pregnancy and dominate the lactation immune landscape.
Fig. 6: DMs are essential for phagocytosis and remodelling during involution.
Fig. 7: Mammary tumour-associated MØs resemble DMs.

Data availability

The RNA-seq data that support the findings of this study have been deposited in the GEO under accession code GSE119869. Previously published microarray data that were re-analysed here are available under accession code GSE56755 (ref. 48). All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

FIJI macros are available from the authors on request.

References

  1. 1.

    Lavin, Y. & Merad, M. Macrophages: gatekeepers of tissue integrity. Cancer Immunol. Res. 1, 201–209 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Varol, C., Mildner, A. & Jung, S. Macrophages: development and tissue specialization. Ann. Rev. Immunol. 33, 643–675 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Wynn, T. A., Chawla, A. & Pollard, J. W. Macrophage biology in development, homeostasis and disease. Nature 496, 445–455 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Gautier, E. L. et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nature 13, 1118–1128 (2012).

    CAS  Google Scholar 

  5. 5.

    Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312–1326 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Gordon, S. & Plüddemann, A. Tissue macrophages: heterogeneity and functions. BMC Biol. 15, 53 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  7. 7.

    Hoeffel, G. & Ginhoux, F. Ontogeny of tissue-resident macrophages. Front. Immunol. 6, 486 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Ginhoux, F. & Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 44, 439–449 (2016).

  9. 9.

    Guilliams, M. & Scott, C. L. Does niche competition determine the origin of tissue-resident macrophages? Nat. Rev. Immunol. 17, 451–460 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Chua, A., Hodson, L., Moldenhauer, L., Robertson, S. & Ingman, W. Dual roles for macrophages in ovarian cycle-associated development and remodelling of the mammary gland epithelium. Development 137, 4229–4238 (2010).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Sun, X., Robertson, S. & Ingman, W. Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland. Cytokine 61, 377–388 (2013).

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Gouon-Evans, V., Rothenberg, M. E. & Pollard, J. W. Postnatal mammary gland development requires macrophages and eosinophils. Development 127, 2269–2282 (2000).

    CAS  PubMed  Google Scholar 

  13. 13.

    O’Brien, J. et al. Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species. Am. J. Pathol. 176, 1241–1255 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  14. 14.

    Jäppinen, N. et al. Fetal-derived macrophages dominate in adult mammary glands. Nat. Commun. 10, 281 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  15. 15.

    Stewart, T. A., Hughes, K., Hume, D. A. & Davis, F. M. Developmental stage-specific distribution of macrophages in mouse mammary gland. Front. Cell Dev. Biol 7, 250 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Nguyen, A. & Pollard, J. Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth. Dev. Biol. 247, 11–25 (2002).

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Pollard, J. W. & Hennighausen, L. Colony stimulating factor 1 is required for mammary gland development during pregnancy. Proc. Natl Acad. Sci. USA 91, 9312–9316 (1994).

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Ingman, W. V., Wyckoff, J., Gouon-Evans, V., Condeelis, J. & Pollard, J. W. Macrophages promote collagen fibrillogenesis around terminal end buds of the developing mammary gland. Dev. Dynam. 235, 3222–3229 (2006).

    CAS  Article  Google Scholar 

  19. 19.

    O’Brien, J., Martinson, H., Durand-Rougely, C. & Schedin, P. Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution. Development 139, 269–275 (2012).

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Walker, N., Bennett, R. & Kerr, J. Cell death by apoptosis during involution of the lactating breast in mice and rats. Am. J. Anat. 185, 19–32 (1989).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Lilla, J. N. & Werb, Z. Mast cells contribute to the stromal microenvironment in mammary gland branching morphogenesis. Dev. Biol. 337, 124–133 (2010).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Plaks, V. et al. Adaptive immune regulation of mammary postnatal organogenesis. Dev. Cell 34, 493–504 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Rios, A. C. et al. Intraclonal plasticity in mammary tumors revealed through large-scale single-cell resolution 3D imaging. Cancer Cell 35, 618–632 (2019).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Yu, Y.-R. A. R. et al. A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues. PLoS ONE 11, e0150606 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. 25.

    Ginhoux, F. et al. The origin and development of nonlymphoid tissue CD103+ DCs. J. Exp. Med. 206, 3115–3130 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Chakarov, S. et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science 363, eaau0964 (2019).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Schiavoni, G. et al. ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8α+ dendritic cells. J. Exp. Med. 196, 1415–1425 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Wang, H. et al. A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation. J. Immunol. 193, 1766–1777 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Sasmono, R. et al. A macrophage colony-stimulating factor receptor–green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101, 1155–1163 (2003).

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Feng, J. et al. IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools. J. Immunol. 186, 1458–1466 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. 31.

    Gouon-Evans, V., Lin, E. Y. & Pollard, J. W. Requirement of macrophages and eosinophils and their cytokines/chemokines for mammary gland development. Breast Cancer Res. 4, 155–164 (2002).

    PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Miller, J. C. et al. Deciphering the transcriptional network of the dendritic cell lineage. Nat. Immunol. 13, 888–899 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Ohtsuka, T. et al. Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol. Cell. Neurosci. 31, 109–122 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Gyorki, D. E., Asselin-Labat, M.-L., Rooijen, Nvan, Lindeman, G. J. & Visvader, J. E. Resident macrophages influence stem cell activity in the mammary gland. Breast Cancer Res. 11, R62 (2009).

    PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Chakrabarti, R. et al. Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche. Science 360, eaan4153 (2018).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  36. 36.

    Liu, Z. et al. Fate mapping via Ms4a3 expression history traces monocyte-derived cells. Cell 178, 1509–1525 (2019).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Jaitin, D. et al. Lipid-associated macrophages control metabolic homeostasis in a Trem2-dependent manner. Cell 178, 686–698 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Ewald, A. J., Werb, Z. & Egeblad, M. Monitoring of vital signs for long-term survival of mice under anesthesia. Cold Spring Harb. Protoc. 2011, 174–177 (2011).

    Google Scholar 

  39. 39.

    Ewald, A. J., Werb, Z. & Egeblad, M. Preparation of mice for long-term intravital imaging of the mammary gland. Cold Spring Harb. Protoc. 2011, 168–173 (2011).

    Google Scholar 

  40. 40.

    Hor, J. L. et al. Spatiotemporally distinct interactions with dendritic cell subsets facilitates CD4+ and CD8+ T cell activation to localized viral infection. Immunity 43, 554–565 (2015).

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Kedrin, D. et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat. Methods 5, 1019–1021 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Rios, A. C., Fu, N. Y., Lindeman, G. J. & Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Uderhardt, S., Martins, A., Tsang, J., Lämmermann, T. & Germain, R. Resident macrophages cloak tissue microlesions to prevent neutrophil-driven inflammatory damage. Cell 177, 541–555 (2019).

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Betts, C. B. et al. Mucosal immunity in the female murine mammary gland. J. Immunol. 201, 734–746 (2018).

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Rios, A. C. et al. Essential role for a novel population of binucleated mammary epithelial cells in lactation. Nat. Commun. 7, 11400 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Qian, B.-Z. & Pollard, J. W. Macrophage diversity enhances tumor progression and metastasis. Cell 141, 39–51 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Lohela, M. et al. Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations. Proc. Natl Acad. Sci. USA 111, E5086–E5095 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48.

    Franklin, R. A. et al. The cellular and molecular origin of tumor-associated macrophages. Science 344, 921–925 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Tymoszuk, P. et al. In situ proliferation contributes to accumulation of tumor‐associated macrophages in spontaneous mammary tumors. Eur. J. Immunol. 44, 2247–2262 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  50. 50.

    Broz, M. L. et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 26, 638–652 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Engelhardt, J. J. et al. Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells. Cancer Cell 21, 402–417 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Linde, N. et al. Macrophages orchestrate breast cancer early dissemination and metastasis. Nat. Commun. 9, 21 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  53. 53.

    Martinson, H., Jindal, S., Durand-Rougely, C., Borges, V. & Schedin, P. Wound healing‐like immune program facilitates postpartum mammary gland involution and tumor progression. Int. J. Cancer 136, 1803–1813 (2015).

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Monks, J. et al. Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release. Cell Death Differ. 12, 107–114 (2005).

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Monks, J., Smith-Steinhart, C., Kruk, E., Fadok, V. & Henson, P. Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland. Biol. Reprod. 78, 586–594 (2008).

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Han, H. et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14, 627–645 (2002).

    Article  Google Scholar 

  57. 57.

    Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84 (2006).

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676 (2012).

    CAS  Google Scholar 

  59. 59.

    Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  60. 60.

    Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. 61.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  62. 62.

    Phipson, B., Lee, S., Majewski, I. J., Alexander, W. S. & Smyth, G. K. Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann. Appl. Stat. 10, 946–963 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  63. 63.

    Liberzon, A. et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics 27, 1739–1740 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. 64.

    Wu, D. & Smyth, G. K. Camera: a competitive gene set test accounting for inter-gene correlation. Nucleic Acids Res. 40, e133 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. 65.

    Sheridan, J. M. et al. A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1. BMC Cancer 15, 221 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank W. Alexander, M. Kauppi and A. Stock for their assistance with the chimaera experiments; F. Jackling for animal management; M. Chopin and S. Nutt for providing mice; Y. Hu for assistance with bioinformatics; J. Whittle, S. Naik, G. Belz, S. Heinzel, J. Schreuder and D. Lin for discussions; and C. Nowell at the MIPS imaging facility. We are grateful to the WEHI Centre for Dynamic Imaging, flow cytometry and animal facilities. This work was supported by the Australian National Health and Medical Research Council (NHMRC) grant nos 1016701, 1054618, 1100807 and 1113133; NHMRC IRIISS; the Victorian State Government through VCA funding and Operational Infrastructure Support and the Australian Cancer Research Foundation. C.A.D. was supported by an Australian Government Research Training Program Scholarship. A.C.R. was supported by a National Breast Cancer Foundation (NBCF)/Cure Cancer Australia Fellowship. S.N.M., G.K.S., G.J.L. and J.E.V. were supported by NHMRC Fellowships (grant nos 1136550, 1058892, 1078730, and 1037230 and 1102742, respectively).

Author information

Affiliations

Authors

Contributions

C.A.D. designed and performed experiments, analysed data and wrote the manuscript. B.P. performed the RNA-seq experiments. F.V. performed the gland clearing and implantation. L.C.G. and G.K.S. analysed the RNA-seq data. Z.L., C.B. and F.G. provided mice and assisted with experiments. G.J.L. provided general guidance. S.N.M. designed experiments. A.C.R. designed experiments and provided general guidance. J.E.V. designed experiments, provided general guidance and co-wrote the manuscript.

Corresponding author

Correspondence to Jane E. Visvader.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Macrophage and dendritic cell populations in the mammary gland (supporting information for Fig. 1).

a, Analysis of a whole-mount 3D confocal image of mammary tissue from a ten-week-old CD11c-GFP mouse immunostained for GFP and keratin 5 (K5). K5 surface (magenta), GFP signal spots (cyan) (left). Image representative of 2 mice. Scale bar, 200 µm. Middle panel: distance from spot centre to K5 surface. x-axis: centre of 10 µm bins. Right panel, box blot: minimum, maximum and quartiles. n=663 cells (>5 µm) and 591 cells (<5 µm) pooled from 2 mice. Normalised CD11b mean intensity within GFP spots (diameter 10 µm). b, Overlay of MØ1/2, MØ3 and CD45+ cells by FACS (gated as in Fig. 1c). c, FACS quantification of nine-week-old mice treated with 400 µg anti-Csf1r or isotype by i.p. injection 7, 4 and 2 days before collection (n=3 mice). DC2, CD11b+ DCs; DC1, CD11bCD24hi DCs. Scale bars, s.e.m. Two-way ANOVA with Sidak correction. *P=0.0231 (MØ1), *P=0.0159 (MØ2), *P=0.0101 (MØ3), ns P=0.6122 (DC1), ns P=0.8956 (DC2). d, FACS plots from mice at 4, 6 and 20 weeks of age (n=3 mice, 4, 6 weeks; n=4 mice, 20 weeks). Same samples shown in Fig. 3g. e, FACS plots from mice at 9 weeks (n=2 mice). f, FACS plots from mice at 9 weeks and Lyve1-FITC mean MFI normalised to autofluorescence (n=2 mice). g, YFP MFI in Irf8-YFP mice normalised to autofluorescence. DC1, CD11b DCs; DC2, CD11b+ DCs (n=2 mice). h, 3D image of Irf8-YFP tissue at 16 weeks immunostained for K5 (magenta) and YFP (cyan). Image representative of 2 mice. Inset: View along duct axis. Arrows indicate Irf8hi cells on outer basal surface. Scale bars, 100 µm (overview) and 30 µm (enlargement). i, Analysis of (h): distance from YFP+ spot centre to K5 surface (n=2 mice). j, Analysis from a 3D image of Csf1r-GFP tissue immunostained for GFP and K5 (Fig. 1d). Histogram of distance from GFP+ spot centre to K5 surface (n=2 mice). Source data

Extended Data Fig. 2 Immune regulation of duct growth during puberty.

a, Quantification of MØs and DCs by FACS in six-week-old CD11c-CreT/+Irf8fl/fl and CD11c-Cre+/+Irf8fl/fl mice. Fold-change % CD45+ cells was calculated relative to control (n=3 mice, control; n=4, knockout). Mean values are displayed. Error bars, s.e.m. Two-way ANOVA with Sidak correction. *P=0.0142, ns: P=0.6517 (MØ3), P=0.9552 (MØ1/2), P=0.8773 (DC2). b, Carmine-stained whole-mounts from six-week-old CD11c-CreT/+Irf8fl/fl and CD11c-Cre+/+Irf8fl/fl mice and branch quantification (n=6 mice, control; n=11, knockout). Error bars, s.e.m. Two-tailed Student’s t-test. Scale bars, 2 mm. c, FACS analysis one day after DT treatment of CD11b-DTR mice (n=3 mice, control; n=4 mice, depletion). Eos: eosinophil, Neut: neutrophil, Mono: monocyte. Means are stated. Error bars, s.e.m. Two-way ANOVA with Sidak correction ****P<0.0001. d, Whole-mounts from CD11b-DTR or WT mice treated with DT (5 weeks) and collected at 6 weeks (n=4 mice, control; n=5 mice, depletion). Error bars, s.e.m. Two-tailed Student’s t-test **P=0.0018. Scale bars, 2 mm. e, FACS analysis of CD11c-CreT/+RBPJfl/fl (RBPJ cKO) and CD11c-Cre+/+RBPJfl/fl (control) mice at 6 weeks, frequency relative to epithelial cells (CD45CD24+) (Two-way ANOVA with Sidak correction, ns: all P>0.9999) and MFI relative to control mean. Two-way ANOVA with Sidak correction ****P<0.0001, ns: P=0.5882 (MHCII), n=0.1659 (CD11c), n=0.9271 (Ly6C) (n=5 mice, controls; n=3 mice, cKO). Error bars, s.e.m. f, Whole-mounts from control and RBPJ cKO mice and quantification of ductal area and branch frequency (n=5 mice, control; n=3 mice, cKO). Error bars, s.e.m. Two-sided Student’s t-tests, P=0.6548 (area), P=0.0538 (branches). Scale bars, 2 mm. g, 3D images from control and RBPJ cKO mice immunostained for MHCII (yellow) and K5 (magenta). Opaque signal rendering as in Fig. 3c. Image representative of 5 mice (control) and 3 mice (cKO). Scale bars, 50 µm. Source data

Extended Data Fig. 3 Gene expression analysis of adult virgin mammary DCs and MØs.

a, Top 50 positive SM signature genes. Heatmap shows mean-centred log2-expression with genes ranked by average FDR across pairwise comparisons (see Methods for a–i) (n=2 samples per population, each pooled from 12 mice, applies to a–f). b, Top 50 positive DC1 signature genes. c, Top 50 positive DC2 signature genes. d, DE genes between DMs versus SMs. Left heatmap shows most significant genes upregulated in DMs, right shows most significant down-regulated genes. e, Heat map showing relative expression of genes previously associated with MØs4 in the mammary DC/MØ populations. f, Heat map showing relative expression of genes previously associated with DCs32 in the mammary DC/MØ populations. g, GFP MFI from FACS analysis of Cx3cr1GFP/+ mice relative to wild-type (WT) controls (Fig. 2f) (n=2 mice). h, Barcode enrichment plots showing that genes associated with MHCIIloLyve1hi lung MØs26 are enriched in mammary MØ1/2 (roast p = 4e-5) whereas genes associated with MHCIIhiLyve1lo lung MØs26 are enriched in DMs (roast p = 5e-5). Genes are ordered in the plot right to left from most up- to most down-regulated in DMs versus SMs (Fig. 2). The x axis shows moderated t-statistics. Vertical bars designate MHCIIloLyve1hi or MHCIIhiLyve1lo lung MØ genes and the worms show relative enrichment. i, Epithelial and MØ expression of known receptor-ligand interactions for genes specifically associated with DMs or MØs. Epithelial gene expression data are from Sheridan et al., 201565. Lum, luminal; Prog, progenitor. Source data

Extended Data Fig. 4 Intra-epithelial macrophages are abundant within the mammary epithelium throughout postnatal development.

a, Optical sections of mammary ducts at 9 weeks of age, immunostained for K5 (magenta), MHCII (yellow) and GFP, YFP or CD11b (cyan) in WT or indicated reporter mice. Arrows indicate intra-epithelial MØs. Hollow arrowhead indicates a CD11b+ stromal cell. Images representative of 3 mice (Csf1r, CD11c and CD11b) and 6 mice (Cx3cr1). Scale bars, 20 μm. b, 3D image of a mammary gland at 2 weeks of age, immunostained for K5 (magenta) and MHCII (yellow). Enlargement: inner duct surface with opaque signal. Image representative of 3 mice. Scale bars, 200 µm (overview) and 20 µm (enlargement). c, 3D image of terminal end buds (TEBs) at 5 weeks and enlarged optical section, immunostained for GFP (cyan) and K5 (magenta) and labelled for F-actin (yellow). Image representative of 3 mice. Arrows indicate dendritic-shaped Cx3cr1hi cells within the TEB. Scale bars, 100 µm (overview) and 40 µm (enlargement). d, 3D images of ducts of the nipple, mid and distal regions of glands and enlarged optical sections, from a Cx3cr1GFP/+ mouse at 8 weeks, immunostained for K5 (magenta), GFP (cyan) and keratin 8 (K8, yellow). Images representative of 2 mice. Arrows indicate Cx3cr1hi DMs between the K8+ luminal and K5+ basal layers. Scale bars, 50 µm.

Extended Data Fig. 5 DMs may arise from rare Cx3cr1+ cells in the distal embryonic gland.

a, 3D image of a Cx3cr1GFP/+ mammary rudiment at E18.5 with enlarged optical sections of the distal (i) and nipple (ii) regions, immunostained for K5 (magenta), GFP (cyan) and MHCII (yellow). Image representative of 2 mice. Arrows indicate Cx3cr1+MHCII cells. b, 3D image of a Cx3cr1GFP/+ mammary gland at postnatal day (P) 4 with enlarged optical sections of the distal (i), mid (ii) and nipple (iii) regions, immunostained for K5 (magenta), GFP (cyan) and MHCII (yellow). Image representative of 2 mice. Arrows indicate Cx3cr1+MHCII cells. c, 3D image of an entire Cx3cr1GFP/+ mammary gland at P7 with enlarged optical sections of the distal (i), mid (ii) and nipple (iii) regions, immunostained for K5 (magenta), GFP (cyan) and MHCII (yellow). Image representative of 2 mice. Arrows indicate dendritic Cx3cr1+MHCIIlo/+ cells. All scale bars, 300 µm (overview) and 100 µm (enlargements).

Extended Data Fig. 6 Intravital microscopy of virgin and involuting mammary glands.

a, Photo of a mouse prepared for intravital imaging (left) and the exposed mammary gland after intraductal injection of fluorescent beads (right). b, Isolation of DM MHCII signal in 3D-IVM images from Elf5-GFP mice with labelling by anti-MHCII AF647 antibody (see Methods, 6 experiments). Left: raw GFP (magenta) and MHCII (cyan) signal. Middle: addition of GFP surface rendering (white). Right: GFP and masked duct-adjacent DM MHCII signal (yellow). Scale bar, 30 µm. c, Time-points from a 3D-IVM movie (Supplementary Video 3) in which precise laser damage was induced in the epithelium of a Cx3cr1GFP/+ mouse at 9 weeks. Images representative of 3 mice. Time hrs:mins. Scale bar, 20 µm. d, 3D-IVM of Elf5-GFP mammary tissue at 3 days involution (Supplementary Video 5) showing GFP (magenta) and anti-MHCII AF647 antibody (yellow). Left: overview. Right: enlarged 3D projections at time-points throughout phagocytosis and the outlined volume viewed from the side. Arrows indicate GFP+ cells within DMs. Images acquired every 5 mins. Images representative of 3 mice. Scale bars, 100 µm (left), 20 µm (enlargements).

Extended Data Fig. 7 DMs during mammary ontogeny (supporting information for Figs. 5 and 6).

a, MØ FACS profiles throughout postnatal development and DM/epithelial cell (CD45CD24+) ratio in virgin and pregnant glands. n=3, 5 weeks, preg d12.5 and preg d16.5 ; n=5, 9 weeks; n=6, lactation. Error bars, s.e.m. b, 3D images of 11 week-old adult and 16.5 d pregnant glands and enlarged optical sections, immunostained for K5 (magenta) and MHCII (yellow) and labelled for EdU (cyan). Mice were treated with EdU 2 hrs prior to collection. Images representative of 4 mice. Hollow arrow-head indicates an EdU DM. Arrow indicates an EdU+ DM. Scale bars, 100 µm (overviews) and 15 µm (enlargements). c, 3D image and enlarged optical section of Cx3cr1GFP/+ tissue at 4 days involution, immunostained for GFP and labelled for F-actin. Images representative of 2 mice. Dotted lines indicate the outer edge of the F-actinhi basal layer. Scale bar, 30 µm. d, Optical section from a 3D image of Elf5-GFP tissue at 3 days involution. Image representative of 3 mice. Arrows indicate large GFPlo alveolar cells surrounded by MHCII signal. Hollow arrow-heads indicate binucleated cells. Scale bar, 20 µm. W, weeks; d, days; lact, lactation. Source data

Extended Data Fig. 8 Depletion of DMs during involution.

a, FACS of CD11c-DTR mice at 1 day involution, treated with DT or PBS upon forced weaning at 14 d lactation (n=2 mice). b, MØ and DC frequency at 4 d involution by FACS after treatment as in a (n=4 mice, CD11c-DTR; n=10, control). Percent CD45+ cells normalized to controls. Control: CD11c-DTR mice treated with PBS or WT mice treated with DT. DC1, CD11bloCD24hi DCs; DC2, CD11b+CD24int DCs. Error bars, s.e.m. Two-way ANOVA with Sidak correction. ***P=0.0007, ****P<0.0001, ns P>0.9999 (Ly6C+CD11c MØs), P=0.0941 (DC2). c, Optical sections of CD11c-DTR tissue at 4 d involution after treatment as in (a). Images representative of 3 mice (PBS) and 4 mice (DT). Immunostaining for CC3 (cyan) and MHCII (yellow) and labelling for F-actin (magenta). Scale bars, 100 µm. d, FACS quantification at 4 d involution after treatment with AFS98 anti-Csf1r antibody or isotype control one day prior to weaning, at weaning and at 1 d involution (n=3 mice). Fold-change percent total cells relative to control mean. Monocytes CD64+F4/80+Ly6ChiMHCII. Error bars, s.e.m. Two-way ANOVA with Sidak correction. *P=0.0212, ***P=0.0008, ns: see Source data. e, H&E staining of tissue from d with alveolar lumen area outlined and quantified (n=3 mice). Error bars, s.e.m. Two-sided Student’s t-test *P=0.0261. Scale bars, 100 µm. f, 3D images of tissue from d immunostained for MHCII (yellow) and CC3 (cyan) and labelled for F-actin (magenta). Images representative of 3 mice. Scale bars, 20 µm. g, Optical sections from a 3D image of Cx3cr1GFP/+ tissue at 6 d involution, immunostained for GFP (yellow) and CC3 (cyan) and labelled for F-actin (magenta). Images representative of 2 mice. Arrows indicate CC3+ DMs. Scale bars, 15 µm. Source data

Extended Data Fig. 9 MØ population dynamics throughout tumorigenesis.

a, FACS plots from an MMTV-PyMT tumour showing gating strategy (n=4 mice) b, FACS plots of MØs (CD45+Ly6GCD64+CD24lo) in age-matched WT FVB/N mammary glands, tumours and tumour-adjacent hyperplastic tissue from MMTV-PyMT, MMTV-Wnt1 and MMTV-Neu mice (n=4 mice per model). c, Quantification of myeloid cell frequencies by FACS in pooled WT controls, tumours and hyperplastic tissue. Eos: eosinophil, Neut: neutrophil, Mono: monocyte. Values are averages (n=6 mice, WT; n=4 mice, others). d, Quantification of MØ subsets throughout tumorigenesis in MMTV-PyMT/Cx3cr1GFP/+ mice, corresponding to Fig. 7b (n=2 mice). e, Immunostaining of MMTV-PyMT tumour tissue for TMEM119. Images representative of 2 mice. Control panel shown below. Scale bar, 10 µm. Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Whole-mount 3D confocal imaging of DMs in lactation. An animation of a whole-mount 3D confocal image of mammary tissue at 14 d lactation (Fig. 3d), immunostained for K5 (Magenta) and MHCII (yellow), and labelled for F-actin (pink).

Supplementary Video 2

Intravital imaging of steady-state DM behaviour. An animation of 3D-IVM of mammary ducts in an Elf5–GFP mouse with immunolabelling by fluorescently conjugated anti-MHCII antibody (Fig. 4a–c). The movie cycles over a 6-h time span with images acquired every 10 min. GFP, magenta; masked DM MHCII, yellow (see Extended Data Fig. 6b and Methods); and stromal MHCII, cyan. Time in h:min (n = 6 mice).

Supplementary Video 3

Intravital imaging of DM response to epithelial damage. An animation of 3D-IVM of a mammary duct in a Cx3cr1GFP/+ mouse (Extended Data Fig. 6c). The movie cycles through time points prior to damage showing the arrangement of GFPhi DMs (yellow) around a duct, then views an optical section through DMs before and after precise multiphoton laser damage at 4 h (bolt symbol). Images were acquired every 3 min (n = 3 mice).

Supplementary Video 4

Intravital imaging of DM response to apoptosis. 3D-IVM of a mammary duct in a nine-week-old Elf5–GFP mouse with immunolabelling by fluorescently conjugated anti-MHCII antibody (Fig. 4e). GFP, magenta; Masked DM MHCII, yellow. Apoptotic cells are labelled with PI (white). Images were acquired every 5 min (n =3 mice).

Supplementary Video 5

Intravital imaging of DM behaviour during involution. 3D-IVM of alveoli at 3 d involution in an Elf5–GFP mouse with immunolabelling by fluorescently conjugated anti-MHCII antibody (Extended Data Fig. 6d). GFP, magenta; MHCII, yellow. Images were acquired every 5 min (n = 3 mice).

Supplementary Video 6

DM-like TAMs form a dendritic network within mammary tumours. An animation of a whole-mount 3D confocal image of MMTV–PyMT/Cx3cr1GFP/+ tumour tissue immunostained for GFP (yellow) and labelled with DAPI (white; Fig. 7c; n = 4 mice).

Source data

Source Data Fig. 1

Statistical source data

Source Data Fig. 2

Statistical source data

Source Data Fig. 3

Statistical source data

Source Data Fig. 4

Statistical source data

Source Data Fig. 5

Statistical source data

Source Data Fig. 6

Statistical source data

Source Data Extended Data Fig. 1

Statistical source data

Source Data Extended Data Fig. 2

Statistical source data

Source Data Extended Data Fig. 3

Statistical source data

Source Data Extended Data Fig. 7

Statistical source data

Source Data Extended Data Fig. 8

Statistical source data

Source Data Extended Data Fig. 9

Statistical source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dawson, C.A., Pal, B., Vaillant, F. et al. Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling. Nat Cell Biol 22, 546–558 (2020). https://doi.org/10.1038/s41556-020-0505-0

Download citation

Further reading