Embryo-derive TNF promotes decidualization via fibroblast activation
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eLife assessment
The authors provide novel evidence for a connection between fibroblast activation and eutherian stromal decidualization. This important work substantially advances our understanding of decidua biology and its contribution to pregnancy. The authors are using solid evidence to support the findings. The methodology includes in vivo mouse and human stroma cells is broadly supports the claims with only minor weaknesses.
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Abstract
Decidualization is a process in which endometrial stromal fibroblasts differentiate into specialized secretory decidual cells and essential for the successful establishment of pregnancy. The underlying mechanism during decidualization still remains poorly defined. Because decidualization and fibroblast activation share similar characteristics, this study was to examine whether fibroblast activation is involved in decidualization. In our study, fibroblast activation-related markers are obviously detected in pregnant decidua and under in vitro decidualization. ACTIVIN A secreted under fibroblast activation promotes in vitro decidualization. We showed that arachidonic acid released from uterine luminal epithelium can induce fibroblast activation and decidualization through PGI 2 and its nuclear receptor PPARδ. Based on the significant difference of fibroblast activation-related markers between pregnant and pseudopregnant mice, we found that embryo-derived TNF promotes CPLA 2α phosphorylation and arachidonic acid release from luminal epithelium. Fibroblast activation is also detected under human in vitro decidualization. Similar arachidonic acid-PGI 2 -PPARδ-ACTIVIN A pathway is conserved in human endometrium. Collectively, our data indicate that embryo-derived TNF promotes CPLA 2α phosphorylation and arachidonic acid release from luminal epithelium to induce fibroblast activation and decidualization.
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Author Response
Reviewer #2 (Public Review):
The idea that decidualization is related to or evolved from wound healing, including fibroblast activation, is old, going back all the way to Creighton 1878 who pointed to the similarity between granulation tissue and decidual tissue, and is supported by the fact that embryo implantation is a compensated form of the endometrial lesion. Nevertheless, the mechanistic connection between FB activation and decidualization is an important fact necessary for understanding decidualization, a fact that is reflected in previous work, for instance, Kim et al., 1999 (Hum Reprod 14 Suppl 2), their reference 20, and Oliver et al., 1999 (Humn Reprod 14), their reference 56 a.o.m. More specifically, a recent single-cell study of in vitro decidualization has shown that a myofibroblast-like cell state is a …
Author Response
Reviewer #2 (Public Review):
The idea that decidualization is related to or evolved from wound healing, including fibroblast activation, is old, going back all the way to Creighton 1878 who pointed to the similarity between granulation tissue and decidual tissue, and is supported by the fact that embryo implantation is a compensated form of the endometrial lesion. Nevertheless, the mechanistic connection between FB activation and decidualization is an important fact necessary for understanding decidualization, a fact that is reflected in previous work, for instance, Kim et al., 1999 (Hum Reprod 14 Suppl 2), their reference 20, and Oliver et al., 1999 (Humn Reprod 14), their reference 56 a.o.m. More specifically, a recent single-cell study of in vitro decidualization has shown that a myofibroblast-like cell state is a transient state in the process of decidualization, i.e. decidual cells themselves are not so much activated fibroblasts, but rather decidual cells differentiate after endometrial stromal fibroblasts undergo a FB activation like process, and the decidual re-programming happens from these activated FB like states (Stadtmauer et al., 2021, Biol. of Reprod. 1-18).
Yes, the paper from Stadtmauer DJ and Wagner GP (2022) was cited in revised version.
The above assessment of how the current study fits into the conceptual landscape of mammalian reproductive biology does not diminish the importance of the paper under consideration. The study contributes a large amount of observational and experimental facts to the understanding of how FB activation and decidualization are related. The authors suggest, in particular, that blastocyst-derived TNF activates the cLPA- producing Arachidonic acid (AA), activating PGI2 and PPARd signaling pathway (more about this later).
Other major comments:
The authors suggest that luminal epithelial cells signal through the release of arachidonic acid (AA) in response to TNF. That is interesting and supported by in vitro experiments inducing decidualization and FB activation by AA. What makes this conclusion a little problematic is that it is known that luminal epithelial cells also express COX2/PTGS2 and thus the synthesis of prostaglandins is already starting in the LE and thus LE can also signal to the stoma via PGE2, PGI2 as well as PGL2 rather than AA directly. The in vitro experiments can not exclude the possibility that the ESF is producing some prostaglandin and then having an autocrine effect.
Yes, we agree with you. It is possible that PGI2 and PGE2 from luminal epithelial cells may also induce fibroblast activation. Based on the data from in situ hybridization, COX-2, mPGES, PGIS and PPARδ are mainly expressed in subluminal stromal cells at mouse implantation site on day 5 of pregnancy (Lim et al, 2000; Ni et al, 2002; Wang et al, 2004). Therefore, PGI2 from stromal cells should be the dominant one compared to that from luminal epithelial cells. In the future, we will examine the effects of AA on COX-2, mPGES and PGIS in luminal epoithelial cells.
Lim H, Dey SK. PPAR delta functions as a prostacyclin receptor in blastocyst implantation. Trends Endocrinol Metab. 2000 May-Jun;11(4):137-42.
Ni H, Sun T, Ding NZ, Ma XH, Yang ZM. Differential expression of microsomal prostaglandin e synthase at implantation sites and in decidual cells of mouse uterus. Biol Reprod. 2002 Jul;67(1):351-8.
Wang H, Ma WG, Tejada L, Zhang H, Morrow JD, Das SK, Dey SK. Rescue of female infertility from the loss of cyclooxygenase-2 by compensatory up-regulation of cyclooxygenase-1 is a function of genetic makeup. J Biol Chem. 2004 Mar 12;279(11):10649-58.
344: here the authors report that PGE2 has no effect on FB activation marker expression, but the problem with that is, that (at least in human ESF), progesterone is causing a change in the expression of the PGE2 receptors from EP4 to EP2, and it is only the EP2 receptor that activates cAMP/PKA pathway.
Yes, we agree with you. PGES is highly expressed in stromal cells at implantation site. Previous studies also show that PGE2 is important during decidualization. In our study, PGES showed no significant changes after stromal cells were treated with AA. PGE2 also had no significant effects on fibroblast activation. Therefore, we focused on PGI2-PPAR pathway. It is possible that PGE2 may regulate decidualization through an alternative way rather than fibroblast activation.
The fact that the authors show an effect of PGI2 is interesting because PGI2 receptors are among the strongest expressed PTG receptors in mammalian ESF. Prostacyclin receptor is a GPCR rather than a nuclear receptor. So the question is really why the authors have not pursued the role of prostacyclin receptor and instead have focused on PPARd?
Yes, we agree with you. When mouse stromal cells were treated with AA, there was no significant change for the protein level of prostacyclin receptor (Figures 4E, 4F). When mouse stromal cells were treated with the agonist SELEXIPAG of prostacyclin receptor, the markers of fibroblast activation showed lower changes compared with treatments with PPARδ (Figure 3D). Therefore, we focused on PPARδ. Yes, we agree with you. Although prostacyclin receptor is less responsive than PPARδ in activating fibroblast activation, it should contribute to fibroblast activation. In the future, we will pursue the effect of prostacyclin receptor on fibroblast activation. Thank you very much for your suggestion.
Reviewer #3 (Public Review):
This manuscript postulates that uterine stroma cells undergo a stage of activation between the resting state and the differentiated decidual state in order to support embryo implantation. Using in vivo mouse and in vitro mouse and human stroma cells they demonstrate that during decidualization the stroma cells express the marker genes for activated stroma. They then trace an axis from the embryo-producing TNF to prostaglandin production and activin A that is required for this process. They propose data to show that activation of the stroma is altered in infertility due to fetal trisomy 16.
The strengths of this manuscript are:
- This is a comprehensive study using both in vivo and in vitro studies and in both mouse and human stroma cells.
- The experiments use a combination of ligands, agonists, and inhibitors to map the signaling axis regulating stroma activation.
- The data shown support the conclusions in this manuscript.
The weaknesses of this manuscript are:
- The conclusion that Acitvin A is the regulator of stroma activation as mentioned by this manuscript is correlative. What is needed is a knockdown of Activin A and then assess stroma activation to prove Activin A is the major regulator and not one of many TGFb family members.
Yes, the data from Activin A knockdown were provided.
- The use of uterine epithelial cells is problematic. The in vitro co-culture approach is not a state-of-the-art co-culture. Removal of epithelial cells from the uterus results in loss of the epithelial phenotype. If the manuscript used an epithelial organoid stroma cell coculture approach it may better reflect the role of the epithelial cells in this process. Otherwise, it is not clear that the epithelial cells are actual participants in the signaling axis. The treatments could be directly on the stroma cells.
Yes, we agree with you. According to your suggestions, we established a culture system for epithelial organoid. When the epithelial organoids were treated with TNF, a similar response was obtained compared with in vitro cultured mouse epithelial cells.
- Ishikawa cells are endometrial cancer cells. They do not really reflect uterine epithelium and it is not clear that any epithelial cell could be substituted for these cells.
Thank you very much for your comments. It is true that Ishikawa cells should be different from in vivo endometrial epithelial cells. However, several studies showed that Ishikawa cell line possess apical adhesiveness to JAR trophoblast cells and expresses many of the same enzymes and structural proteins found in normal human endometrium (Castelbaum AJ et al, 1997).. Because both estrogen and progesterone receptors are expressed in Ishikawa cells, Ishikawa cells show a good response to both estrogen and progesterone (Castelbaum AJ et al, 1997). Therefore, Ishikawa cells are used as a model for receptive endometrial epithelial cells (Hannan NJ et al, 2010).
Castelbaum AJ, Ying L, Somkuti SG, Sun J, Ilesanmi AO, Lessey BA. Characterization of integrin expression in a well differentiated endometrial adenocarcinoma cell line (Ishikawa). J Clin Endocrinol Metab 1997; 82:136-142.
Hannan NJ, Paiva P, Dimitriadis E, Salamonsen LA. Models for study of human embryo implantation: choice of cell lines? Biol Reprod. 2010; 82:235-245.
Lessey BA, Ilesanmi AO, Castelbaum AJ, Yuan L, Somkuti SG, Chwalisz K, Satyaswaroop PG. Characterization of the functional progesterone receptor in an endometrial adenocarcinoma cell line (Ishikawa): progesterone-induced expression of the alpha1 integrin. J Steroid Biochem Mol Biol. 1996; 59:31-39.
- The activation of stroma cells in the fetal trisomy 16 experiments at the end is very superficial. Data should show that these cells decidualize with decidual markers. This appears to be an experiment to show the translational value of the signaling axis. This experiment, again, is not well developed, does not add much to the manuscript, and should be omitted.
Yes, we agree with you. The description on human trisomy 16 was deleted.
In summary, the concept of stroma cell activation as part of decidualization is nicely developed and will add to the field. Normally investigators consider decidualization a mesenchymal to epithelial transition while some consider it stromal activation. This manuscript demonstrates that stroma cell activation is a critical part of the process of decidualization.
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eLife assessment
The authors provide novel evidence for a connection between fibroblast activation and eutherian stromal decidualization. This important work substantially advances our understanding of decidua biology and its contribution to pregnancy. The authors are using solid evidence to support the findings. The methodology includes in vivo mouse and human stroma cells is broadly supports the claims with only minor weaknesses.
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Reviewer #1 (Public Review):
This manuscript provides evidence of a new pathway of endometrial decidualization through COX2-PGI2-PPARδ through fibroblast activation in response to embryo- derive TNFα, conserved in both mice and humans. This is an interesting finding that sheds light on the understanding of the decidua contribution to pregnancy.
The major inconvenience is how the authors perform the experiments and how they expose their findings. The work needs to be completely rewritten, starting with a clear abstract, with an interconnected introduction, using a defined objective, explaining which organisms/tissues/cells are used in each experiment, the sample size and replicates should be specified in each experiment, and ending with a strong conclusion.
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Reviewer #2 (Public Review):
The idea that decidualization is related to or evolved from wound healing, including fibroblast activation, is old, going back all the way to Creighton 1878 who pointed to the similarity between granulation tissue and decidual tissue, and is supported by the fact that embryo implantation is a compensated form of the endometrial lesion. Nevertheless, the mechanistic connection between FB activation and decidualization is an important fact necessary for understanding decidualization, a fact that is reflected in previous work, for instance, Kim et al., 1999 (Hum Reprod 14 Suppl 2), their reference 20, and Oliver et al., 1999 (Humn Reprod 14), their reference 56 a.o.m. More specifically, a recent single-cell study of in vitro decidualization has shown that a myofibroblast-like cell state is a transient state in …
Reviewer #2 (Public Review):
The idea that decidualization is related to or evolved from wound healing, including fibroblast activation, is old, going back all the way to Creighton 1878 who pointed to the similarity between granulation tissue and decidual tissue, and is supported by the fact that embryo implantation is a compensated form of the endometrial lesion. Nevertheless, the mechanistic connection between FB activation and decidualization is an important fact necessary for understanding decidualization, a fact that is reflected in previous work, for instance, Kim et al., 1999 (Hum Reprod 14 Suppl 2), their reference 20, and Oliver et al., 1999 (Humn Reprod 14), their reference 56 a.o.m. More specifically, a recent single-cell study of in vitro decidualization has shown that a myofibroblast-like cell state is a transient state in the process of decidualization, i.e. decidual cells themselves are not so much activated fibroblasts, but rather decidual cells differentiate after endometrial stromal fibroblasts undergo a FB activation like process, and the decidual re-programming happens from these activated FB like states (Stadtmauer et al., 2021, Biol. of Reprod. 1-18).
The above assessment of how the current study fits into the conceptual landscape of mammalian reproductive biology does not diminish the importance of the paper under consideration. The study contributes a large amount of observational and experimental facts to the understanding of how FB activation and decidualization are related. The authors suggest, in particular, that blastocyst-derived TNF activates the cLPA-producing Arachidonic acid (AA), activating PGI2 and PPARd signaling pathway (more about this later).
Other major comments:
The authors suggest that luminal epithelial cells signal through the release of arachidonic acid (AA) in response to TNF. That is interesting and supported by in vitro experiments inducing decidualization and FB activation by AA. What makes this conclusion a little problematic is that it is known that luminal epithelial cells also express COX2/PTGS2 and thus the synthesis of prostaglandins is already starting in the LE and thus LE can also signal to the stoma via PGE2, PGI2 as well as PGL2 rather than AA directly. The in vitro experiments can not exclude the possibility that the ESF is producing some prostaglandin and then having an autocrine effect.
344: here the authors report that PGE2 has no effect on FB activation marker expression, but the problem with that is, that (at least in human ESF), progesterone is causing a change in the expression of the PGE2 receptors from EP4 to EP2, and it is only the EP2 receptor that activates cAMP/PKA pathway.
The fact that the authors show an effect of PGI2 is interesting because PGI2 receptors are among the strongest expressed PTG receptors in mammalian ESF. Prostacyclin receptor is a GPCR rather than a nuclear receptor. So the question is really why the authors have not pursued the role of prostacyclin receptor and instead have focused on PPARd?
-
Reviewer #3 (Public Review):
This manuscript postulates that uterine stroma cells undergo a stage of activation between the resting state and the differentiated decidual state in order to support embryo implantation. Using in vivo mouse and in vitro mouse and human stroma cells they demonstrate that during decidualization the stroma cells express the marker genes for activated stroma. They then trace an axis from the embryo-producing TNF to prostaglandin production and activin A that is required for this process. They propose data to show that activation of the stroma is altered in infertility due to fetal trisomy 16.
The strengths of this manuscript are:
1. This is a comprehensive study using both in vivo and in vitro studies and in both mouse and human stroma cells.
2. The experiments use a combination of ligands, agonists, and …Reviewer #3 (Public Review):
This manuscript postulates that uterine stroma cells undergo a stage of activation between the resting state and the differentiated decidual state in order to support embryo implantation. Using in vivo mouse and in vitro mouse and human stroma cells they demonstrate that during decidualization the stroma cells express the marker genes for activated stroma. They then trace an axis from the embryo-producing TNF to prostaglandin production and activin A that is required for this process. They propose data to show that activation of the stroma is altered in infertility due to fetal trisomy 16.
The strengths of this manuscript are:
1. This is a comprehensive study using both in vivo and in vitro studies and in both mouse and human stroma cells.
2. The experiments use a combination of ligands, agonists, and inhibitors to map the signaling axis regulating stroma activation.
3. The data shown support the conclusions in this manuscript.The weaknesses of this manuscript are:
1. The conclusion that Acitvin A is the regulator of stroma activation as mentioned by this manuscript is correlative. What is needed is a knockdown of Activin A and then assess stroma activation to prove Activin A is the major regulator and not one of many TGFb family members.
2. The use of uterine epithelial cells is problematic. The in vitro co-culture approach is not a state-of-the-art co-culture. Removal of epithelial cells from the uterus results in loss of the epithelial phenotype. If the manuscript used an epithelial organoid stroma cell coculture approach it may better reflect the role of the epithelial cells in this process. Otherwise, it is not clear that the epithelial cells are actual participants in the signaling axis. The treatments could be directly on the stroma cells.
3. Ishikawa cells are endometrial cancer cells. They do not really reflect uterine epithelium and it is not clear that any epithelial cell could be substituted for these cells.
4. The activation of stroma cells in the fetal trisomy 16 experiments at the end is very superficial. Data should show that these cells decidualize with decidual markers. This appears to be an experiment to show the translational value of the signaling axis. This experiment, again, is not well developed, does not add much to the manuscript, and should be omitted.
In summary, the concept of stroma cell activation as part of decidualization is nicely developed and will add to the field. Normally investigators consider decidualization a mesenchymal to epithelial transition while some consider it stromal activation. This manuscript demonstrates that stroma cell activation is a critical part of the process of decidualization. -
