Estrogen exacerbates mammary involution through neutrophil-dependent and -independent mechanism
- NTU Institute for Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore
- Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore
- School of Basic and Applied Sciences, Dayananda Sagar University, India
- School of Computer Science and Engineering, Nanyang Technological University, Singapore
Figures
Figure 1 with 1 supplement
Estrogen accelerates mammary involution.
(A-C) Mice on the day of weaning (involution day 1-INV D1) were treated with vehicle control (Ctrl) or E2B for 48 hr before mammary tissues were collected for analysis; (A) (i) H and E stained …
Figure 1—figure supplement 1
Effect of neutrophil depletion on estrogen-induced cell death and adipocytes repopulation.
Mice at INV D1 were administered with isotype control (IgG) or anti-neutrophil antibody (Ly6G) and treated with Ctrl or E2B for 48h. (A) Neutrophil depletion attenuates E2B-induced adipocyte …
Figure 2 with 1 supplement
Estrogen regulates a multitude of neutrophil-dependent and –independent biological processes in involuting mammary gland.
Mice at INV D1 were treated with anti-Ly6G antibody (Ly6G) or isotype control (IgG). 24h later, they were treated with vehicle control (Ctrl) or E2B for 24h (Ctrl+IgG n=3, Ctrl+Ly6G n=3, E2B+IgG …
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Figure 2—source data 1
DESeq2 analysis result between Ctrl+IgG and Ctrl+Ly6G.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data1-v2.xlsx
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Figure 2—source data 2
DESeq2 analysis result between E2B+IgG and Ctrl+IgG.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data2-v2.xlsx
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Figure 2—source data 3
DESeq2 analysis result between E2B+Ly6G and Ctrl+Ly6G.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data3-v2.xlsx
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Figure 2—source data 4
DESeq2 analysis result between E2B+IgG and E2B+Ly6G.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data4-v2.xlsx
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Figure 2—source data 5
GO over-representation analysis result of DE genes between E2B+IgG and Ctrl+IgG.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data5-v2.xlsx
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Figure 2—source data 6
GO over-representation analysis result of DE genes between E2B+Ly6G and Ctrl+Ly6G.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data6-v2.xlsx
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Figure 2—source data 7
GO over-representation analysis result of DE genes found only in E2B+IgG.
- https://cdn.elifesciences.org/articles/57274/elife-57274-fig2-data7-v2.xlsx
Figure 2—figure supplement 1
Depletion efficiency of neutrophils with anti-neutrophil antibody Ly6G.
Mice at INV D1 was treated with anti-Ly6G antibody (Ly6G) or isotype control (IgG). 24 hr later, they were treated with vehicle control (Ctrl) or E2B for 24 hr. Flow cytometry analysis was performed …
Figure 3 with 2 supplements
Estrogen-induced Cxcr2 signalling in neutrophils plays a key role in neutrophil infiltration into the involuting mammary gland.
(A) Heatmap representation of estrogen-regulated genes associated to leukocyte migration and inflammation in neutrophils (≥3 and ≤ −3-fold); Experiment is conducted according to the description in Fi…
Figure 3—figure supplement 1
Putative S100A9 inhibitor Paquinimod promotes neutrophil infiltration.
Mice were treated at INV D1 with either E2B or E2B+PAQ for 48h. (A) Treatment with E2B and PAQ significantly induces neutrophil and monocyte infiltration into the involuting MG as compared to …
Figure 3—figure supplement 2
1H NMR Spectrum of the synthesized Paquinimod (PAQ): 1H NMR (CDCl3, 400 MHz) δ 7.43 (1H, dd, J = 8.6, 7.4 Hz), 7.29 – 7.10 (5H, m), 7.03 (2H, dd, J = 8.1, 5.5 Hz), 4.00 (2H, q, J = 7.1 Hz), 3.28 – 3.23 (3H+2H m), 1.30 (3H, t, J = 7.4 Hz), 1.23 (3H, t, J = 7.1 Hz).
1H NMR spectrum was recorded on a Bruker Avance 400 spectrometer.
Figure 4 with 3 supplements
Estrogen-induced adipocyte repopulation is associated with induction of adipogenic and tissue remodelling genes in neutrophils.
(A-C) Non-OVX mice were treated daily with either anti-Ly6G antibody (Ly6G) or isotype control (IgG) from 24h post-weaning (INV D1); (A) Flow cytometry analysis of mammary neutrophils (CD45+ CD11b+ …
Figure 4—figure supplement 1
Neutrophil depletion transiently reduces E2B-induced adipocyte repopulation during mammary involution.
Non-OVX mice were treated daily with either anti-Ly6G antibody (Ly6G) or isotype control (IgG) from 24 hr post-weaning (INV D1). Mammary tissue was collected for analysis at INV D3 and INV D4. (A) (i…
Figure 4—figure supplement 2
50% of estrogen-regulated ECM genes are abolished by neutrophil depletion.
Mice at INV D1 was treated with anti-Ly6G antibody (Ly6G) or isotype control (IgG). 24 hr later, they were treated with vehicle control (Ctrl) or E2B for 24 hr. Heatmap representation of genes …
Figure 4—figure supplement 3
Estrogen regulation of lipid metabolism during post-weaning mammary involution were attenuated with neutrophil depletion.
Mice at INV D1 was treated with anti-Ly6G antibody (Ly6G) or isotype control (IgG). 24 hr later, they were treated with vehicle control (Ctrl) or E2B for 24 hr. Heatmap representation of genes …
Figure 5 with 2 supplements
Estrogen stimulates the activity of lysosomal proteases that are critical for LM-PCD.
(A) qPCR validation of E2B-induced expression of Bid, Ctsb, and Tnf relative to Rplp0 identified from DESeq2 analysis (Figure 5—figure supplement 1) (Ctrl+IgG n = 3, Ctrl+Ly6G n = 3, E2B+IgG n = 3, …
Figure 5—figure supplement 1
Heatmap representation of estrogen-regulated genes associated with cell death from the GO over-representation analysis.
Mice at INV D1 was treated with anti-Ly6G antibody (Ly6G) or isotype control (IgG). 24h later, they were treated with vehicle control (Ctrl) or E2B for 24h. The genes plotted exhibit fold change of …
Figure 5—figure supplement 2
Estrogen-induced genes associated with cell proliferation are regulated in both mammary gland and neutrophil population.
(A-B) Mice at INV D1 was treated with anti-Ly6G antibody (Ly6G) or isotype control (IgG). 24 hr later, they were treated with vehicle control (Ctrl) or E2B for 24 hr; (A) Heatmap representation of …
Figure 6
Estrogen accelerates TNFα-induced cell death in vitro.
MCF7-caspase3(+) cells were treated with either vehicle control (1xPBS) or TNFα of varying concentrations. An hour later, cells were treated with either vehicle control (0.01% ethanol) or 10nM …
Figure 7
Estrogen regulation of inflammatory and apoptotic genes in nulliparous mammary tissue.
OVX nulliparous mice were treated with isotype control (IgG). 24 hr later, they were treated with either Ctrl or E2B for 24 hr. (A) Flow cytometry analysis of mammary neutrophils (CD45+ CD11b+ Gr1hi)…
Figure 8 with 1 supplement
Estrogenic stimulations of neutrophil gene expression and mammary cell death are mediated by ERα.
Mice at INV D1 was treated with vehicle control (Ctrl), ER-α agonist PPT, or ER-β agonist DPN for 48h before mammary gland were collected for analysis. (A) qPCR analysis of gene expression in …
Figure 8—figure supplement 1
Expression of Esr1 (ERα) is about 40 times higher than Esr2 (ERβ) in mammary neutrophils during mammary involution.
Mice at INV D2 was treated with either vehicle control (Ctrl) or E2B for 8h. Mammary neutrophils were isolated from tissue using Dynabeads bound anti-Ly6G antibody. Gene expression of Esr1 and Esr2 …
Figure 9
Estrogen exacerbates mammary cell death, inflammation and adipocytes repopulation through distinct mechanisms.
Estrogen induces Ctsb gene expression in the mammary cells leading to increased pro-CTSB which is cleaved and activated in lysosomes. The increased active sc-CTSB further activates CTSD and CTSL. …
Additional files
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Supplementary file 1
List of qPCR primers used.
- https://cdn.elifesciences.org/articles/57274/elife-57274-supp1-v2.xlsx
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Supplementary file 2
List of antibodies used.
- https://cdn.elifesciences.org/articles/57274/elife-57274-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57274/elife-57274-transrepform-v2.pdf
