Estrogen exacerbates mammary involution through neutrophil dependent and independent mechanism

  1. Chew Leng Lim
  2. Yu Zuan Or
  3. Zoe Ong
  4. Hwa Hwa Chung
  5. Hirohito Hayashi
  6. Smeeta Shrestha
  7. Shunsuke Chiba
  8. Lin Feng
  9. Valerie CL Lin

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Abstract

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumour growth through the activity of neutrophils. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in Balb/c mice with and without neutrophil depletion showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation through neutrophil-dependent and neutrophil-independent mechanisms. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCL2-CXCR2 signalling. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was mediated by neutrophils-independent upsurges of cathepsins and their lysosomal leakages that are critical for lysosome-mediated cell death. Notably, these multifaceted effects of estrogen are unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.

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  1. eLife

    ###Reviewer #3

    This paper reported that estrogen can accelerate mammary involution by exacerbating mammary inflammation, inducing programmed cell death, and promoting adipocytes repopulation, that the effects of estrogen on the expressions of genes during mammary involution are majorly mediated by neutrophils, and that estrogen promotes mammary LM-PCD independent of neutrophils by inducing the expression and activity of lysosomal cathepsins and other pro-apoptotic markers such as Bid and Tnf. These findings are potentially interesting, and could expand the functions of estrogen. However, there is a lack in mechanistic insight into these observations.

    I. The mechanism underlying estrogen-induced cell death needs to be further explored. For example, what kind of player(s) connects estrogen with cell death? Whether TNF-alpha plays a role in linking estrogen to cell death? Is there any enrichment of cell death genes associated with the estrogen treatment in RNA-Seq data? Why the artificial MCF-7/Caspase-3 cells were used? The results about MCF-7/Caspase-3 cells showed that estrogen promoted TNF-alpha-induced apoptosis, rather than lysosomes-associated cell death. Maybe the authors should try MCF-10A cells as the model.

    II. Based on the data on Figure 4, it is not so convincible to conclude that neutrophils are involved in adipocytes repopulation during mammary involution normally, please see also Issues#3. The authors need to re-consider the relationship between these data and the conclusion. Maybe they should re-describe these results or modify the conclusion.

    III. The most interesting finding is that estrogen does not trigger the similar biological actions in age-matched nulliparous mammary tissue. However, this study does not figure out the molecular mechanism underneath the difference between the functions of estrogen in involutional and nulliparous mammary tissues. At least, the author should discuss about the potential possibilities.

    Other issues:

    1. Quantification in Figure 1B should indicate the fractions, for example, No. cells of total or area. The data in Figure 1C, except Csn2, were not described in the content, and these data should be associated with adipogenesis. As for Figure 1D, no any description was presented about Ly6G, and in fact, it was described in the second part of Results section. Supplemental Figure 2 was mentioned in the content before Supplemental Figure 1. The first part of results was very important for readers to understand the paper, but these problems confuse the readers.

    2. In Page 13, Line 219, "E2B treatment alone without the antagonist (E2B+DMSO) lead to an expected 1.57-fold increase (p=0.0082) in mammary neutrophils as compared to the Ctrl+DMSO". Should "1.57-fold" be "2.57-fold" or something other? It is not the case based on the data in Figure 3Ci.

    3. In Figure 4B, upon neutrophil depletion, Cebpb and Cebpd were already increased, which could limit their further enhancement when treated with E2B. As for Adig and Egr2, it seemed that they also apparently increased. In Figure 4D, the data had the similar problem to those in Figure 4B. No description about Figure 4E and 4F was found in the content. Overall, these data put it in question that estrogen-induced adipocyte repopulation is associated with the induction of adipogenic and tissue remodeling genes through neutrophils.

    4. In Page 19, Line 305, "This suggests that the up-regulation of Ctsb expression by E2B is a direct event independent of STAT3 activation". These data in Figure 5B could not demonstrate that Ctsb expression is the direct event of E2B. In Figure 5D, why the lysosomal pellet fractions showed no lysosomal proteins, such as catheptins. In Figure 5A, at least the protein level of TNF-alpha should be measured, because it was very important for the functions of E2B, based on the data in Figure 6.

    5. In Figure 6, TNF induces p-STAT3 while Fig 5A shows E2 induces TNF expression (mRNA), but no p-STAT3 was increased in Fig 5B. The increased mRNA does not mean the increased protein. Please measure the TNFa proteins in Fig 5A (See Issue#4). The MCF-7/Casp3 model seems not to well support the conclusion. The data in Figure 6 are about typical apoptosis not the lysosomes-associated cell death involved in the functions of estrogen as revealed in this study.

  2. eLife

    ###Reviewer #2

    In this study, Chew Leng Lim et al determined the diverse effects of Estrogen exposure on neutrophil infiltration, inflammation responses, cell death and adipocytes repopulation in mice models. While the authors revealed some new findings, this study suffers from obvious defects, including overdependence on the use of chemical inhibitors, lack of in-depth mechanistic investigation as well as unfocused research topics.

    Major concerns:

    1. In addition to neutrophil, estrogen exposure also induced macrophage infiltration, while "neutrophil" deletion by using anti-Ly6G antibody obviously reduced the infiltration of macrophage (Fig S1). Therefore, the role of macrophage in Estrogen exposure-induced biological responses should be deeply determined.

    2. Since anti-Ly6G antibody also reduced macrophage infiltration significantly, it is very likely macrophage play a pivotal role in Estrogen exposure-regulated gene expressions and cellular phenotypes. Therefore, the conclusion that 88% of estrogen-regulated genes are mediated through neutrophil is not solid. This point should be addressed by specific deletion of macrophage and neutrophil, individually.

    3. The source of CXCL1/CXCL2 upon estrogen exposure should be further investigated. In Fig 8, the authors indicated that CXCL1 and CXCL2 are produced by existing neutrophil. Further evidence to support this should be provided.

    4. Many biological and small molecule inhibitors, including anti-Ly6G antibody, PAQ (S100a9 inhibitor), CXCR2 antagonist SB225002, etc, have been frequently used in this study. However, the effects and specificity of some of these agents have not been well validated during the study. The use of genetic mice models for critical signaling pathways is highly suggested.

    5. Figure 5, The critical role of CTSB in the activation of CTSD/CTSL and induction of LM-PCD upon E2B treatment should be validated by downregulation of CTSB expression pharmacologically or genetically.

    6. The data presented in Figure 7 based on the analysis in a single cell line is not reliable.

  3. eLife

    ###Reviewer #1

    Dr. Valerie Lin discovered some interesting links between estrogen signals and the differentiation and programmed death of mammary cells, as well as the formation of pro-inflammatory microenvironment, which facilitate post-partum mammary involution and presumably parity-associated breast cancer. They also demonstrated that mammary gland-infiltrating neutrophils emerge as a major immune cell participant during this process.

    1. Dr Shengtao Zhou reported that ERβ has potent antitumor effects, which suppress lung metastasis by recruiting antitumor neutrophils to the metastatic niche. It is recommended to carefully address whether estrogen specifically target ERα or ERβ on post-weaning mammary cells and infiltrating neutrophils in this setting.

    2. Perhaps a missing point is whether estrogen-induced mammary cell death subsequently cause inflammation (presumably augmenting neutrophil accumulation), since several cell death modalities has been associated to inflammation via the release of danger molecules.

    3. Besides lysosome-mediated PCD, can estrogen induce other cell death modalities, such as pyroptosis, necroptosis, ferroptosis, which are all caspase 3/7/8-independent? It is important to make a clear conclusion.

    4. Since the synthetic estrogen regulate the differentiation associated genes of fat cells and accelerated LM-PCD. Does estrogen affect lipid metabolic pathways? How does this metabolic remodeling affect cell death and differentiation of adipocytes and the function of neutrophils? By carefully going over the Seq data, the authors may add more important discussions.

  4. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on April 30, 2020, follows.

    Summary

    Lin and colleagues aim to explore how estrogen promotes post-partum mammary involution and increases the risk of parity-associated breast cancer. Previous studies have unraveled different molecular mechanisms of mammary gland involution (see a previous summary PMID: 30448440). The authors highlighted that estrogen causes mammary involution by stimulating the accumulation of neutrophils, sculpturing the pro-inflammatory microenvironment, facilitating adipocytes differentiation, and causing lysosome-related programmed death of mammary cells. These findings are interesting, but further efforts are needed to nail down some of the major conclusions and to clarify the underlying mechanisms.

    Essential Revisions

    1. A major concern is about several discoveries on neutrophils. The contribution of neutrophils during estrogen-induced mammary involution should be cautiously defined with solid experimental evidence. Do other immune cell populations, such as macrophages, actively participate in this process? Does Ly6G antibody efficiently deplete neutrophils, rather than masking the labeling of Gr1 antibody (for validation)? As neutrophils have short half-life, secret lots of inflammatory mediators, and quickly replenish from BM progenitors, this point is important. The possible coordinations between neutrophils and other immune cells (e.g. macrophages, monocytes), and their relative importance at different stages of mammary involution can be examined and discussed (see a previous summary PMID: 24952477). In addition, more evidences are needed to prove whether the recruitment of neutrophils depends on a positive-feedback loop of CXCL1 and CXCL2. Quality controls are needed for the application of inhibitors.

    2. Another major concern is about estrogen-triggered mammary cell death. How do ERα, ERβ or death receptor-mediated signals contribute to this process? Does estrogen-induced programmed cell death exclusively rely on lysosome leakage and related effector molecules? Have the authors tested the existence of other cell death modalities? Does estrogen-induced cell death augment local inflammation and perhaps the accumulation of immune cell populations?

    3. The authors may consider to rephrase/weaken some of their claims and reorder the display of some of their results.

  5. Version published to 10.1101/2020.04.03.023341 on bioRxiv