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. 2013 Apr;9(4):459-75.
doi: 10.4161/auto.23164. Epub 2013 Feb 4.

ATG Proteins Mediate Efferocytosis and Suppress Inflammation in Mammary Involution

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Free PMC article

ATG Proteins Mediate Efferocytosis and Suppress Inflammation in Mammary Involution

Irina Teplova et al. Autophagy. .
Free PMC article

Abstract

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1(+/-) and Atg7-deficient mammary epithelial cells (MECs) produced 'competent' apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment. Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.

Keywords: ITGB5; MERTK; autophagy; dead cell clearance; ductal ectasia; efferocytosis; engulfment; inflammation; mammary involution.

Figures

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Figure 1. Autophagy is induced in the early phase of mammary involution. (A) GFP fluorescence on frozen sections of mammary glands from GFP-LC3 mice. (B) RT-PCR quantification of Becn1, Atg7 and Map1lc3α mRNA in mammary glands from wild-type mice. L (Lact), lactation; I (Inv), involution; d, days; h, hours.
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Figure 2. Monoallelic Becn1 loss or MEC-specific Atg7 deletion compromises autophagy induction in mammary involution. (A) EM micrographs of Becn1+/+ and Becn1+/− mammary glands on day 2 post-weaning. Red arrows point to autophagosomes. (B) Quantification of autophagosomes in Becn1+/+ and Becn1+/− mammary glands on day 2 post-weaning by EM. (C) RT-PCR quantification of Becn1 and Map1lc3α mRNA in mammary glands from Becn1+/− mice. (D–F) ATG7, LC3 and SQSTM1 IHC respectively on mammary glands from parous wild-type and Atg7F/F;WAP-Cre mice on day 2 post-weaning. (G) RT-PCR quantification of Becn1 and Map1lc3α mRNA in mammary glands from parous wild-type and Atg7F/F;WAP-Cre mice type. L, lactation; I, involution; d, days; h, hours. Parous mice: after second pregnancy and lactation
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Figure 3. Monoallelic Becn1 loss or MEC-specific Atg7 deletion delays mammary involution. H&E-stained sections of formalin-fixed and paraffin-embedded mammary tissues from parous wild-type, Becn1+/− and Atg7F/F;WAP-Cre mice on days 1–7 post-weaning. Parous mice: after second pregnancy and lactation.
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Figure 4. Monoallelic Becn1 loss or MEC-specific Atg7 deletion delays mammary involution. (A) Whole mammary gland mounts from parous wild-type, Becn1+/− and Atg7F/F;WAP-Cre mice on days 1–4 post-weaning. (B) Mammary gland repopulation by adipocytes (adipose tissue/total mammary tissue, %) during days 0.5–14 post-weaning.
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Figure 5. Involution delay in Atg-deficient mammary glands is associated with dead cell accumulation in alveolar lumens. (A) Cleaved CASP3 IHC on mammary glands from wild-type and Becn1+/− mice on involution days 1 and 4. (B) Quantification of post-lactational apoptotic bodies in mammary glands from wild-type and Becn1+/− mice during involution days 0.5–21. (C) Fraction of apoptotic bodies engulfed by living MECs in mammary glands from wild-type (blue line) and Becn1+/− (red line) mice during involution days 1–5. (D) Cleaved CASP3 IHC on mammary glands from wild-type and MEC-specific Atg7-deficient mice on involution days 1 and 4. (E) Quantification of post-lactational apoptotic bodies in mammary glands from wild-type and MEC-specific Atg7-deficient mice during involution days 0.5–21. (F) Fraction of apoptotic bodies engulfed by living MECs in mammary glands from wild-type (green line) and MEC-specific Atg7-deficient (yellow line) mice during involution days 1–5. (G) Apoptotic body distribution between non-engulfed (i.e., in alveolar lumens, L) and engulfed (i.e., inside MECs in alveolar wall, W) fractions in mammary glands from wild-type (blue and green bars), Becn1+/− (red bars) and MEC-specific Atg7-deficient (yellow bars) mice during days 2–4 post-weaning. **p 0.001, *p 0.01.
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Figure 6.Atg-deficiency impairs efferocytosis during mammary involution in association with decreased MERTK and ITGB5 levels, but without compromising PS exposure on apoptotic bodies or MFGE8 expression. (A) (Left panel) EM micrographs of Becn1+/+ and Becn1+/− mammary glands on day 2 post-weaning. Red arrows point to autophagosomes (engulfed by MECs in wild type, non-engulfed in Becn1+/− gland); (Right panel) Phagocytic index, i.e., fraction of engulfed over total number of autophagosomes in HPF. (B) Annexin V IHC on mammary glands from wild-type, Becn1+/− and MEC-specific Atg7-deficient mice on day 2 post-weaning. (C) MFGE8 IHC on mammary glands from wild-type, Becn1+/− and MEC-specific Atg7-deficient mice on day 3 post-weaning. (D) MERTK, ITGB5, CD14 and CD68 IHC on mammary glands from parous wild-type, Becn1+/− and MEC-specific Atg7-deficient mice at 48 h post-weaning.
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Figure 7. Involution delay due to Atg-deficiency is accompanied by persistence of inflammation, M2-macrophage infiltration, increased collagen deposition and ductal ectasia. (A) F4/80 IHC on mammary glands from wild-type, Becn1+/− and MEC-specific Atg7-deficient mice on days 5–21 post-weaning. (B) Macrophage quantification in mammary glands from wild-type, Becn1+/− and MEC-specific Atg7-deficient mice on days 5–21 post-weaning. (C) ARG1 IHC (left column); Mason trichrome staining (right column) on mammary glands from wild-type, Becn1+/− and MEC-specific Atg7-deficient mice on day 21 post-weaning. (D) Whole gland mount (left column) and H&E-stained sections (right column) of mammary glands from parous wild-type, Becn1+/− and MEC-specific Atg7-deficient mice.
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Figure 8. BECN1 or ATG7 knockdown in wild-type iMMECs compromises efferocytosis in vitro without affecting PS exposure on apoptotic bodies, but in association with decreased engulfment-related RAC1 activation. (A) Phagocytic index for stable Becn1+/− and Becn1+/− iMMEC lines in vitro. (B) Phagocytic index for wild-type iMMECs treated with scrambled or Atg (Becn1 or Atg7) siRNA. (C) Quantification of BECN1 and ATG7 knockdown in wild-type iMMECs. (D and E) FACS-based quantification of PS exposure on apoptotic bodies derived from wild-type iMMECs treated with scrambled or Atg (Becn1 or Atg7) siRNA. (F) Efferocytosis-induced RAC1 activation in wild-type iMMECs treated with scrambled or Atg (Becn1 or Atg7) siRNA.

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