Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution

Abstract

SUMMARYMammary gland development is dependent on macrophages, as demonstrated by their requirement during the expansion phases of puberty and pregnancy. Equally dramatic tissue restructuring occurs following lactation, when the gland regresses to a state that histologically resembles pre-pregnancy through massive programmed epithelial cell death and stromal repopulation. Postpartum involution is characterized by wound healing-like events, including an influx of macrophages with M2 characteristics. Macrophage levels peak after the initial wave of epithelial cell death, suggesting that initiation and execution of cell death are macrophage independent. To address the role of macrophages during weaning-induced mammary gland involution, conditional systemic deletion of macrophages expressing colony stimulating factor 1 receptor (CSF1R) was initiated just prior to weaning in the Mafia mouse model. Depletion of CSF1R + macrophages resulted in delayed mammary involution as evidenced by loss of lysosomal-mediated and apoptotic epithelial cell death, lack of alveolar regression and absence of adipocyte repopulation 7 days post-weaning. Failure to execute involution occurred in the presence of milk stasis and STAT3 activation, indicating that neither is sufficient to initiate involution in the absence of CSF1R + macrophages. Injection of wild-type bone marrow-derived macrophages (BMDMs) or M2-differentiated macrophages into macrophage-depleted mammary glands was sufficient to rescue involution, including apoptosis, alveolar regression and adipocyte repopulation. BMDMs exposed to the postpartum mammary involution environment upregulated the M2 markers arginase 1 and mannose receptor. These data demonstrate the necessity of macrophages, and implicate M2-polarized macrophages, for epithelial cell death during normal postpartum mammary gland involution. KEY WORDS: TGF, Delayed involution, STAT3, Macrophage programming, Perilipin, MouseMacrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution (Clarkson et al., 2004;Lund et al., 1996;Schedin et al., 2004;Stein et al., 2007;Strange et al., 1992). Anticipated roles for involution-associated macrophages have focused on later stage activities, including phagocytosis of cell debris, milk clearance and protease production related to ECM restructuring, events that occur 3-4 days postweaning, after the peak in apoptosis (Atabai et al., 2007;O'Brien and Schedin, 2009;Schwertfeger et al., 2006). To address potential roles for macrophages in postpartum mammary gland involution, macrophages expressing CSF1R were systemically depleted prior to weaning. Unexpectedly, in the absence of macrophages, postpartum involution processes, including MEC death and adipocyte repopulation, failed to occur. Injection of bone marrow-derived macrophages (BMDMs) directly into mammary glands of macrophage-depleted mice at the time of weaning restored involution, confirming a requirement for macrophages in postpartum involution. MATERIALS AND METHODS Mouse br...

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

“…Therefore, the loss of ZnT2 in adipocytes may mirror insulinlike effects on lipogenesis that stunt ductal growth (60) or may affect the synthesis or release of paracrine factors, such as adiponectin and collagen VI (61) from adipocytes that initiate epithelial/mesenchymal interaction and facilitate ductal expansion (55). Finally, macrophages are also important for mammary gland development, as they are recruited to the epithelial cells to support proliferation for ductal outgrowth (30,62) by supplying growth factors, cytokines, and chemokines (63) or by engulfing apoptotic cells that interfere with epithelial proliferation (64). The lack of cell proliferation in ZnT2-null mice suggests that loss of ZnT2 in macrophages may not be able to support normal epithelial and fibroblast proliferation.…”

Apoptosis and Clearance of the Secretory Mammary Epithelium

“…Macrophages from MAFIA mice express a modified Fas receptor that, in response to a dimerization-inducing small molecule (AP20187), triggers Fas-mediated apoptosis. Depletion of macrophages immediately prior to weaning impaired apoptosis within the secretory mammary epithelium, despite milk stasis and STAT3 activation [105]. These results demonstrate that macrophages are necessary to initiate apoptosis in the mammary epithelium.…”

Nucling, a Novel Apoptosis-associated Protein, Controls Mammary Gland Involution by Regulating NF-κB and STAT3

“…Reduction in M2 Macrophage Recruitment in Mammary Gland Involution in Nucling-KO Mice-Macrophages with an alternatively activated M2 phenotype have been shown to play a role in involution (14). We also previously reported that Nucling affected the number of Kupffer cells in the liver (22).…”

“…Therefore, we attempted to elucidate the contribution of Nucling to M2 macrophage recruitment during mammary gland involution. To achieve this, we examined the expression of Arginase-1 and Ym1, markers of M2 macrophages (14,27). At 36 h of involution, protein levels of Arginase-1 were similar in WT and Nucling-KO mice.…”

“…Furthermore, a recent study demonstrated that mammary gland involution was characterized by the recruitment of macrophages with M2 characteristics, which is crucial for epithelial cell death and adipocyte repopulation (14).…”