Tissue-resident macrophages have been ascribed various functions, including immune surveillance and phagocytosis of apoptotic debris (Davies et al., 2013). Depending upon the site of localization, these macrophages can exhibit additional tissue-specific behaviors, such as the regulation of surfactant by alveolar macrophages in the lung and of neuronal function by microglia in the brain (Haldar et al., 2014). These tissue-specific functions are thought to be driven in response to factors within the local tissue microenvironment. Tissue-resident macrophages are typically derived from embryonic precursors, and can maintain locally through self-renewal or be replaced by blood-derived monocytes, or a combination of both, depending on the tissue (Ginhoux and Guilliams, 2016; Hashimoto et al., 2013; Perdiguero et al., 2015; Sheng et al., 2015; Yona et al., 2013; Zhu et al., 2017). Recent studies of macrophage ontogeny and localization have highlighted the complex nature of macrophage heterogeneity and function at steady state (Chakarov et al., 2019). Further understanding of the diverse functions of tissue-resident macrophages will provide insights into the mechanisms through which these cells maintain tissue homeostasis and how they may contribute to tissue-specific disease.

Mammary gland development is regulated by numerous systemic and locally derived factors, including hormones, growth factors and cytokines (Hynes and Watson, 2010; Richert et al., 2000; Watson and Khaled, 2008; Watson et al., 2011). Tissue-resident macrophages are localized at distinct regions within the mammary gland and have been implicated in various stages of mammary gland development. Early studies demonstrated the presence of resident macrophages in close proximity to epithelial structures, along terminal end buds (TEBs) of developing ductal structures and in the adipose stroma (Gouon-Evans et al., 2000; Schwertfeger et al., 2006). More recent studies have demonstrated that macrophages are also located directly adjacent to the epithelial cells and can intercalate into the ductal epithelial layer (Jäppinen et al., 2019; Stewart et al., 2019; Dawson et al., 2020). Lineage tracing studies have demonstrated that macrophages in the mammary gland are initially derived from embryonic precursors (Jäppinen et al., 2019). Although embryonically derived cells can be found in mammary glands from adult mice, some turnover from blood-derived cells is also observed (Jäppinen et al., 2019). Resident macrophages have been functionally linked to mammary gland development. Analysis of mammary glands from mice that are deficient in Csf-1 demonstrated reduced ductal elongation and branching (Gouon-Evans et al., 2000). Furthermore, macrophages that are associated with epithelial ducts have been implicated in regulating stem cell activity (Gyorki et al., 2009) and collagen organization (O'Brien et al., 2010). In addition, macrophages contribute to mammary epithelial cell turnover during the estrous cycle (Chua et al., 2010) and can engulf apoptotic epithelial cells following damage (Dawson et al., 2020). Finally, macrophages contribute to epithelial cell death and participate in tissue remodeling during involution following lactation (Perdiguero et al., 2015; Dawson et al., 2020; Hughes et al., 2012).

Macrophages have been implicated in the repair and remodeling of the extracellular matrix (ECM). Macrophages can contribute directly to ECM remodeling through production of proteases and indirectly through modulating fibroblast function (Kim and Nair, 2019; Murray and Wynn, 2011). In the mammary gland, macrophages promote collagen fibrillogenesis around the developing terminal end bud (Ingman et al., 2006). Furthermore, collagen accumulation has been linked to macrophage recruitment during involution (O'Brien et al., 2010). Although these studies suggest a role for macrophages in the regulation of ECM within the mammary gland, the specific mechanisms through which macrophages might function in this context have yet to be elucidated. Recent studies have implicated a subpopulation of macrophages that can be identified by Lyve-1 as being involved in the regulation of ECM in the arterial wall and in the lung (Lim et al., 2018). Lyve-1 is a receptor for hyaluronan (HA), which is a negatively charged polysaccharide that consists of a repeating disaccharide structure and is an important component of the ECM. HA is important for maintaining tissue structure and hydration and is turned over at high levels under steady state (Gupta et al., 2019; McCourt, 1999). Hyaluronan synthase (Has2) is expressed by both mammary epithelial cells and stromal cells, and HA has been shown to contribute to epithelial branching in the mammary gland (Tolg et al., 2017). However, relatively little is known regarding the localization of HA throughout the adipose stroma and the mechanisms involved in modulating HA homeostasis in the mammary gland.

Here, we describe the localization patterns of a population of cells in the nulliparous mammary gland that is positive for both Lyve-1 and the macrophage marker F4/80. These cells are localized at distinct sites throughout the mammary gland, including in the adipose-associated stroma and in the mammary gland capsule region. Although these cells are also found in association with the epithelial-associated stroma, the majority of F4/80⁺ cells in this region are negative for the Lyve-1 marker. Further studies demonstrate that the F4/80⁺Lyve-1⁺ cells express high levels CD206, which is a marker that is typically associated with tissue reparative macrophages, and that these cells are associated with HA-enriched regions within the mammary gland. Using a shielded bone marrow chimera model, we also demonstrate that these cells are long-lived and capable of self-renewal, which are key traits of tissue-resident macrophages (Davies et al., 2013). Analysis of the adipose stroma following macrophage depletion reveals increased levels of HA and collagen, suggesting that macrophages are important for maintaining ECM homeostasis in the mammary stroma. These changes in the adipose stroma are not accompanied by significant alterations in ductal morphogenesis. These studies identify a distinct stromal macrophage subpopulation within mouse and human mammary glands. Furthermore, our findings suggest that macrophages are important for maintaining ECM homeostasis in mammary glands from virgin mice.

Here, we demonstrate the presence of a subpopulation of resident macrophages in the normal mammary gland that is specifically associated with ECM-rich connective tissue located within the stroma of both mouse and human mammary glands. Previous studies of resident macrophages in the mammary gland have focused primarily on macrophages that are located in close proximity to epithelial structures, and that demonstrated their presence adjacent to TEBs during ductal elongation and in close association with developing alveolar buds during pregnancy (Gouon-Evans et al., 2000; Schwertfeger et al., 2006). The use of mice that are deficient for Csf1, which lack mature macrophages, highlighted the importance of these cells in the context of mammary gland development (Gouon-Evans et al., 2000). More recent studies have also identified distinct myeloid cell populations, including CD11c⁺ antigen presenting cells, and Csf1r⁺ macrophages that are localized in close association with ductal epithelial cells (Stewart et al., 2019; Plaks et al., 2015). Macrophages that are localized within the mammary stroma have been reported, but recent studies have highlighted that these macrophages represent a subpopulation that is distinct from epithelial-associated macrophages (Gouon-Evans et al., 2000; Schwertfeger et al., 2006; Dawson et al., 2020). We demonstrate here that a distinct population of Lyve-1⁺ macrophages is associated with ECM-rich regions within the mammary stroma and the fibrous capsule of the mammary gland. Furthermore, we demonstrate that resident macrophages are important for maintaining homeostasis of HA, a key component of the ECM, within these regions. Further studies are warranted using models in which Lyve-1⁺ macrophages can be selectively depleted to define the importance of this subpopulation in ECM homeostasis within the adipose stroma.

Investigations into the ontogeny, longevity and function of distinct tissue-resident macrophage populations have demonstrated that resident macrophages can be derived from embryonic populations and self-sustain within the tissue. These studies have also found that turnover of resident macrophages in adults can vary depending upon the tissue site (Ginhoux and Guilliams, 2016). Recent studies have demonstrated that macrophages in the mammary gland are initially seeded from embryonic precursors and that these macrophages are maintained into adulthood, with embryonically derived macrophages remaining detectable at 3 months of age (Jäppinen et al., 2019). Furthermore, the authors demonstrated that the embryonically derived population was associated with an F4/80^hi and CD206^hi profile. Interestingly, we found that the Lyve-1⁺ population also exhibits a F4/80^hi and CD206⁺ profile, although not all of the F4/80^hi and CD206⁺ populations were found to be Lyve-1⁺, providing further support for distinct resident macrophage populations in the mammary gland. Furthermore, analysis of BrdU incorporation revealed that the Lyve-1⁺ cells are capable of self-renewal. Studies of resident macrophages in the lung have demonstrated that peri-vascular Lyve-1⁺ macrophages are derived from Ly6C^hi monocytes (Chakarov et al., 2019). The majority of Lyve-1⁺ macrophages were maintained during the 6-week bone marrow chimera experiment, but a small population of donor-derived Lyve-1⁺ macrophages was also detected, suggesting a slow rate of turnover of this population from the blood. Therefore, it is possible that this population represents a combination of infiltrating and self-renewing macrophages.

Through co-localization studies, we found that the majority of Lyve-1⁺ cells were associated with HA-containing regions within the mammary stroma, including the fibrous capsule surrounding the mammary gland. The mammary gland capsule is comprised of a fascial plane that is likely to serve a structural function to encase and protect the fat pad. We do not yet know whether this structure also serves a barrier function that reduces pathogen infiltration, as has been shown to be the case for macrophage-enriched capsular structures surrounding intraperitoneal organs such as the liver (Sierro et al., 2017). Interestingly, while some F4/80⁺Lyve-1⁺ cells were found to be associated with the stroma in direct association with TEBs, the majority of F4/80⁺ cells in this region were negative for Lyve-1 staining. Results from the RNA-seq data suggest that the F4/80⁺Lyve-1^– population is enriched in pro-inflammatory factors when compared with the F4/80⁺Lyve-1⁺ population, suggesting a potential role in immunosurveillance. This finding is consistent with the localization of the F4/80⁺Lyve-1^– cells near the epithelial structures, which have the potential for pathogen exposure via the ductal lumen. It is important to note that the bulk RNA-seq analysis was performed on CD11b⁺ cells, thus this analysis probably does not include the recently described CD11c⁺ ductal macrophages (Dawson et al., 2020). It would be interesting to determine where the CD11b⁺F4/80⁺Lyve-1^– cells are localized in comparison with the CD11c⁺ ductal macrophages. In addition, although the majority of the F4/80⁺Lyve-1⁺ cells were positive for CD206, we also observed a separate population of CD206⁺Lyve-1^– cells, suggesting that CD206 expression is not limited to the Lyve-1⁺ macrophage population. Overall, these findings demonstrate the presence of distinct subpopulations of macrophages in the mammary gland that probably exhibit distinct functions that are based on their localization and exposure to tissue-specific signals.

Macrophages have been implicated in ECM repair and remodeling during the resolution phase of wound healing and in the tumor microenvironment (Condeelis and Pollard, 2006; Wynn and Barron, 2010). In the mammary gland, macrophages have been shown to modulate the organization of type I collagen around the TEB (Ingman et al., 2006). ECM regulation is complex and macrophages can impact levels of ECM within a tissue via a variety of mechanisms, including direct production of ECM molecules, expression of ECM-modulating proteases and regulation of ECM-producing fibroblasts. Gene-profiling studies have demonstrated that macrophages express genes that are associated with ECM remodeling, including ECM molecules and proteases (Etich et al., 2019). Macrophages have also been shown to produce proteases that can act directly on ECM factors, such as collagen, to maintain ECM homeostasis and suppress fibrosis (Lim et al., 2018). Finally, macrophages can impact fibroblast-mediated production of ECM, which contributes to the modulation of fibrosis (Wynn and Barron, 2010). Lyve-1⁺ macrophages have been suggested to repress fibrosis directly, in part through production of collagen cleaving proteases (Lim et al., 2018). Given that Lyve-1 is a well-documented receptor for HA (Banerji et al., 1999; Schwertfeger et al., 2015), we hypothesized that Lyve-1 expressing macrophages localize to HA-containing regions in the mammary gland. HA was found to be enriched around TEBs, in fibrous septae within the adipose stroma and in the capsule surrounding the fat pad. Despite the localization of HA within the TEB stroma, few Lyve-1⁺ cells were found within this region. However, Lyve-1⁺ cells were found to be associated with other HA-enriched regions, including the fibrous septae and the capsule. While the reasons for this are unknown, differences in the composition of the TEB-associated stroma and the adipose-associated stroma may drive expression of unique ECM receptors and/or recruit distinct macrophage subtypes that have different ECM binding affinities. Future studies are required to determine whether Lyve-1 expression is required for the localization of these macrophages to HA-enriched regions within the mammary stroma.

HA is a major component of connective tissue, and high molecular weight HA is important for maintaining tissue structure and hydration. HA turnover is estimated at approximately 15 g per day, suggesting high levels of turnover in tissues under steady state (Stern, 2004). The specific mechanisms involved in the regulation of HA turnover in normal tissues remain unknown, but macrophages have been implicated in HA catabolism on the basis of their ability to internalize HA via endocytosis (Underhill et al., 1993). We also found that macrophage depletion leads to increased collagen accumulation. Although macrophages have been linked to collagen degradation via production of MMPs, Mrc1-expressing macrophages have also been shown to internalize and degrade collagen in the dermis (Madsen et al., 2013). Further studies involving the quantification and analysis of different ECM components in the adipose stroma following macrophage depletion would provide additional insights into the mechanisms through which macrophages impact ECM homeostasis. It is important to note that pexidartinib, which was used to deplete macrophages in these studies through targeting CSF1R, inhibits additional kinases including c-Kit and, to a lesser extent, Flt3 (DeNardo et al., 2011). Thus, it is possible that in addition to macrophage depletion, inhibiting these pathways in other cell types may contribute to the observed stromal phenotypes. Our findings suggest that resident macrophages contribute to maintaining connective tissue homeostasis within the mammary gland, due in part to the regulation of ECM turnover. Nevertheless, further studies using additional methods of macrophage depletion, ideally using methods to delete Lyve-1⁺ macrophages selectively, are required to confirm these findings.

In the normal mammary gland, HA is primarily found in the high molecular weight form, and results from in vitro studies have suggested a potential role for HA in epithelial branching (Tolg et al., 2017). We demonstrate here that HA is also associated with fibrous regions within the adipose stroma and the mammary capsule. Given the role for HA in the structural support of tissues and hydration, it seems likely that HA represents a key structural component of the mammary gland. Surprisingly, although we found that depletion of macrophages by CSF1R inhibition during ductal development led to enhanced ECM accumulation in the mammary gland, we did not observe significant defects in ductal elongation or branching. Previous studies have demonstrated that macrophage deletion in a Csf1-deficient mouse model, in which macrophages are not present during the initial stages of ductal development, results in reduced ductal elongation and branching during puberty (Gouon-Evans et al., 2000). Interestingly, targeting of CD11c⁺ cells leads to enhanced ductal elongation in the mammary gland (Plaks et al., 2015). Furthermore, deletion of CD11b⁺ cells, which reduced the stromal macrophage population, also enhanced ductal elongation (Dawson et al., 2020). The differences between these findings and our results may be the result of many factors, including differences in methods of macrophage depletion, timing of depletion and efficacy. Notably, CSF1R treatment only achieved 60% depletion efficiency in our studies, which may not be sufficient to impact ductal development significantly. Furthermore, we assessed mammary glands following two weeks of depletion, rather than one week as performed in the CD11b depletion model (Dawson et al., 2020), which may impact our ability to detect differences in elongation. Together, these findings suggest that myeloid regulation of ductal development in the mammary gland is complex and that identifying and modifying specific macrophage subpopulations may be required to understand fully the mechanistic functions of macrophages in mammary gland development and homeostasis. Furthermore, studies of stromal macrophages during processes that typically involve alterations in ECM, such as involution and aging (Butcher et al., 2009; Li, 2005; Schedin and Keely, 2011), may highlight additional functional consequences of altered ECM on epithelial morphology in the mammary gland.

Alterations in ECM accumulation in the breast are associated with changes in breast density (Huo et al., 2015). Furthermore, increased density is an established risk factor for developing breast cancer (McCormack, 2006). Our studies suggest a potential correlation between the presence of ECM-modulating macrophages and ECM accumulation. Unlike the mouse mammary gland, the density of stroma in the human breast varies widely, with regions of adipocytes interspersed with areas of connective fibers of varying densities. Furthermore, stromal density varies widely between individuals. Interestingly, areas of high mammographic density in the stroma are associated with reduced numbers of CD206⁺ macrophages in human breast samples (Huo et al., 2015), consistent with the hypothesis that CD206⁺ macrophages impact ECM regulation in the normal mammary gland. Understanding what drives differences in macrophage localization in different regions of the mammary gland and in different individuals may provide insights into the mechanisms that contribute to breast density. Furthermore, understanding the mechanisms through which stromal composition is modulated in the normal mammary gland will ultimately lead to the development of strategies to modulate breast density and reduce breast cancer risk.

In addition to localization in the mammary gland, we also found that F4/80⁺Lyve-1⁺ cells accumulate in the peritumoral stroma surrounding mammary tumors. Given that the peritumoral regions of tumors are often associated with ECM modulation, this localization is consistent with the concept that these cells are involved in ECM regulation. The presence of Lyve-1⁺ macrophages has been documented in tumors, and these cells have been implicated in lymphatic vessel remodeling and tumor progression (Elder et al., 2018; Etzerodt et al., 2020). Interestingly, Lyve-1⁺ macrophages are lost during melanoma progression and melanoma tumor growth is enhanced in Lyve-1^–/– mice , suggesting that Lyve-1-expressing cells have a tumor restraining role during tumor growth (Dollt et al., 2017). Further studies are required to differentiate between the functional roles of Lyve-1⁺ macrophages and lymphatic endothelial cells in these models. By contrast, Lyve-1 expression is also associated with a specific macrophage subpopulation that was found to promote ovarian cancer progression and metastasis (Etzerodt et al., 2020). In addition, recent studies have also implicated macrophages expressing lymphatic-associated markers, including Lyve-1 and podoplanin, in mammary tumor metastasis through modulation of lymphatics (Elder et al., 2018; Bieniasz-Krzywiec et al., 2019). Although further studies are required to understand Lyve-1 macrophage function across different tumor types, these studies highlight the importance of identifying and understanding the functional contributions of distinct macrophage subpopulations in the context of both normal tissue homeostasis and tissue-specific disease.

Sequencing data have been deposited in GEO under accession codes GSE148207 and GSE148209.

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Author details

1. Ying Wang

   Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Thomas S Chaffee

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9052-0023

2. Thomas S Chaffee

   Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Ying Wang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5535-1535

3. Rebecca S LaRue

   University of Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, United States

   Contribution

   Formal analysis, Writing - review and editing

   Competing interests

   No competing interests declared

4. Danielle N Huggins

   Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, United States

   Contribution

   Supervision, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Patrice M Witschen

   Comparative and Molecular Biosciences Graduate Program, University of Minnesota, Minneapolis, United States

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

6. Ayman M Ibrahim

   1. Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane School of Medicine, New Orleans, United States
   2. Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

7. Andrew C Nelson

   1. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, United States
   2. Masonic Cancer Center, University of Minnesota, Minneapolis, United States

   Contribution

   Conceptualization, Supervision, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

8. Heather L Machado

   Department of Biochemistry and Molecular Biology, Tulane Cancer Center, Tulane School of Medicine, New Orleans, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

9. Kathryn L Schwertfeger

   1. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, United States
   2. Masonic Cancer Center, University of Minnesota, Minneapolis, United States
   3. Center for Immunology, University of Minnesota, Minneapolis, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft

   For correspondence

   schwe251@umn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9755-7774

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