Physiological TLR4 regulation in human fetal membranes as an explicative mechanism of a pathological preterm case
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Evaluation Summary:
This manuscript provides insight into the zone-specific regulation of inflammatory gene expression in the fetal membranes prior to labor at term. Specifically, the authors demonstrate distinct epigenetic and mi-RNA control of TLR4 signaling in the amnion and chorion, highlighting the role of this pattern recognition receptor in physiological labor. Overall, the experimental design and data analysis are suitable, though the study would benefit from the inclusion of the analysis of fetal membrane tissues from pregnancy complications and/or in vivo studies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The integrity of human fetal membranes is crucial for harmonious fetal development throughout pregnancy. Their premature rupture is often the consequence of a physiological phenomenon that has been exacerbated. Beyond all the implied biological processes, inflammation is of primary importance and is qualified as ‘sterile’ at the end of pregnancy. In this study, complementary methylomic and transcriptomic strategies on amnion and choriodecidua explants obtained from the altered (cervix zone) and intact fetal membranes at term and before labour were used. By cross-analysing genome-wide studies strengthened by in vitro experiments, we deciphered how the expression of toll-like receptor 4 (TLR4), an actor in pathological fetal membrane rupture, is controlled. Indeed, it is differentially regulated in the altered zone and between both layers by a dual mechanism: (1) the methylation of TLR4 and miRNA promoters and (2) targeting by miRNA (let-7a-2 and miR-125b-1) acting on the 3’-UTR of TLR4. Consequently, this study demonstrates that fine regulation of TLR4 is required for sterile inflammation establishment at the end of pregnancy and that it may be dysregulated in the pathological premature rupture of membranes.
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Author Response:
Reviewer #1 (Public Review):
[...]
- A notable shortcoming of the authors' interpretation is the generalization of their findings to preterm premature rupture of membranes (PPROM). As noted by the authors, term labor is considered a "sterile" process, which is particularly important in terms of the authors' findings since TLR4 in the fetal membranes may be responding to endogenous signals such as danger signals. However, a large proportion of PPROM cases are associated with microbial invasion of the amniotic cavity, and thus in this context TLR4 would be responding to bacterial products.
To bring in some new elements and address this reviewer’s concern, along with the potential extrapolation between physiological rupture and pathological rupture in the case of PPROM, we decided first to remove Figure 3C (expression …
Author Response:
Reviewer #1 (Public Review):
[...]
- A notable shortcoming of the authors' interpretation is the generalization of their findings to preterm premature rupture of membranes (PPROM). As noted by the authors, term labor is considered a "sterile" process, which is particularly important in terms of the authors' findings since TLR4 in the fetal membranes may be responding to endogenous signals such as danger signals. However, a large proportion of PPROM cases are associated with microbial invasion of the amniotic cavity, and thus in this context TLR4 would be responding to bacterial products.
To bring in some new elements and address this reviewer’s concern, along with the potential extrapolation between physiological rupture and pathological rupture in the case of PPROM, we decided first to remove Figure 3C (expression of TLR4 in the presence of LPS from bacterial origin) from the revised version of the manuscript. To address this comment, it is well known that the percentage of PPROM associated with microbial invasion are variable based on the weeks of gestation. In fact, early gestational ages are clearly linked to high-microbial-associated intra-amniotic inflammation prevalence (64.3% when <25 WGA) whereas this percentage subsequently decreases throughout gestation (Romero et al., 2015), reaching one-third at term, which better links with the gestational stage of the current study. Such observations support the fact that the TLR4 model in physiological rupture could be transposed—at least in part—to sterile PPROM and initiated by the presence of alarmins (i.e., HMGB1) and their binding to such type of receptors. Indeed, TLR4 is now well described as being stimulated by ligands other than LPS, such as HMGB1, a member of the DAMPs (Robertson et al., 2020). Furthermore, the quantification of TLR4 mRNA expression and protein in the case of PPROM without chorioamnionitis compared with term no labor without chorioamnionitis was already carried out (Kim et al., 2004), indicating an absence of clear link between the chorioamnionitis and TLR4 expression. Finally, in an animal model of PPROM, an article underlined the importance of TLR4 in preterm labor by using TLR4 mice mutants in a sterile context (Wahid et al., 2015).
- It is a well-known concept that TLR4 is expressed by the fetal membranes and is responsive to LPS stimulation, and thus the confirmatory set of experiments performed by the authors do not seem to be as novel. Indeed, given that this study was focused on the "sterile" process of term labor, perhaps the utilization of danger signals that can interact with TLR4 would be more appropriate.
The choice to use LPS (Figure 3C) was only to confirm that TLR4 leads to a proinflammation activation in the amnion and choriodecidua, demonstrating the functional pathway after TLR4 activation in the fetal membranes environment. We completely agree these are not novel data; this is why we decided to remove this part of results in the revised version of the manuscript. Furthermore, we decided to not repeat the use of DAMPs (such as HMGB1) to stimulate the TLR4 pathway in this work because it was already published in the fetal membranes context (Bredeson et al., 2014). To be in accordance with your comments, we have modified the end of the results paragraph entitled ‘Combination of transcriptomic and methylomic results in the ZAM zone demonstrate that genes more expressed in the choriodecidua are linked to pregnancy pathologies’ to better justify the choice to focus on TLR4 global transcriptional regulation.
- The distinction between the ZAM and ZIM seems to have been lost among the TLR4-focused experiments, and thus it is unclear how these fetal membrane zones fit into the conceptual model proposed by the authors in the final figure.
The reviewer is correct here, so to avoid confusion between the ZIM and ZAM used, we decided to do the following:
- Read carefully all the successive paragraphs of the results to check for the presence of ‘ZAM specification’
- Add ‘ZAM’ in the legend of Figure 4. This information was present in the related text of the article.
- Update Figure 7 and its legend (model of regulation). We had ‘ZAM zone’ in the discussion part regarding Figure 7.
- The study is largely descriptive and would benefit from the addition of fetal membrane tissues from pregnancy complications such as PPROM and/or animal models in which premature rupture of the membranes has been induced.
We agree that animal models are available. Nevertheless, we considered that such models are far from the human reality. In fact, animal models are often used for fetal membrane studies, but they are different regarding pregnancy physiology, structure and uterine environment, which hamper their use. We used ‘term’ fetal membrane to decipher the physiological rupture of membrane and demonstrate the importance of the TLR4 actor. To bring some elements regarding this comment and the possible extrapolation between physiological rupture and pathological rupture in the case of PPROM, we decided to remove Figure 3C (expression of TLR4 in the presence of LPS from bacterial origin) to focus more on the physiological rupture of fetal membranes without the involvement of bacterial presence. Previous bibliographic data answer the reviewer’s question: Kim et al. (2004) well demonstrated that TLR4 mRNA levels are higher in PPROM (31.2 weeks of gestation) fetal membranes without chorioamnionitis than in term (39.1 week of gestation) ones without chorioamnionitis.
- The study focuses on the mechanisms of rupture of membranes, but does not provide an explanation as to how the regulation of TLR4 mediates the process of membrane rupture.
We agree with your comment; however, ‘how the regulation of TLR4 mediates the process of membrane rupture’ is not the topic of the manuscript. In addition, this has already been well established in previous publications. Nevertheless, we added a sentence in the introduction part between the lines 97-100 : ‘The mechanisms implying TLR4 in the physiological or pathological rupture of membrane in case of PPROM are well known. Triggering TLR4 will lead to NFκB activation, leading to an increase of the release of proinflammatory cytokine, concentration of matrix metalloprotease and prostaglandin, which are well established actors of fetal membrane rupture (Robertson et al., 2020).
Reviewer #2 (Public Review):
This is a well-conceived and executed paper that adds novel data to improve our understanding of rupture of the human fetal membranes. The new information presented not only addresses gaps in our understanding of normal parturition mechanisms but also the significant issue of preterm birth. The authors highlight the need to understand the understudied human fetal membranes to be able to understand its role in normal parturition but also to lower the rates of preterm birth. They not only establish the need to study this tissue but also to improve our appreciation for regional differences within it, using a comprehensive genetic approach. The authors provide data from a genome wide methylation study and cross reference this with transcriptome data. Using this new knowledge, they then zero in on a specific gene of interest TLR4. This receptor is already established as an extremely important receptor for preterm birth but little is known about its role in normal parturition. Strengths of this paper stem from the comprehensive data set provided, answering both the questions pertaining to the specific aims of this paper but also potentially future questions and providing potential focused targets of study. One example of this may be the common methylated genes that are found in both the ZIM and ZAM, illustrating not regional changes but gestational programming of this tissue.
We thank the reviewer for the positive and constructive comments regarding the article. Following all the reviewers’ comments, we now have an improved version.
Reviewer #3 (Public Review):
Manuscript by Belville et al describes the significance of epigenetic and transcription associated changes to TLR4 as a mechanistic event for sterile inflammation associated with fetal membrane weakening, specifically in the zone of altered morphology. This manuscript is timely in an understudied area of research.
The authors have taken an extensive set of experiments to derive their conclusions.
However, it is unclear why the focus is on TLR4. Although LPS is a ligand for TLR4, gram negative infections are rare in PPROM but mostly genital Mycoplasmas. The methylome and transcriptome analysis does not necessarily warrant examination of a single marker. A clear rationale would need to be included.
We would like to thank the reviewer for their comments regarding the article. For the last part of the public review, we would like to underline the following:
-The choice of focusing on TLR4 is explained in the article text between lines 161 and 165 by the following sentences: ‘Of all the genes classified in these processes, TLR4 was the only one represented in all these biological processes and, therefore, seems to play a central role in parturition at term. To validate this in-silico observation and pave the way for describing TLR4’s importance, immunofluorescence experiments were first conducted to confirm the protein’s presence in the amnion and choriodecidua of the ZAM (Figure 3B)’. Furthermore, this choice arises from analysis described in Figure 3A, which underlines that the four GO terms most represented have only one common gene: ‘TLR4’. The combination of two high-scale studies does not permit us to individually characterize how each gene is regulated. Nevertheless, the focus on TLR4 provides an original and interesting hypothesis on how a specific layer regulation between the amnion and choriodecidua could be cellular realised in the ZAM’s weaker zone. Finally, because the high-scale study results are public, this type of analysis could be conducted on other candidate genes.
-Throughout the text, we changed all the ‘E. Coli’ to ‘Gram-negative bacteria’. Furthermore, as found in the literature, genital mycoplasma are considered ‘Gram-negative bacteria’. We focused on the ‘sterile inflammation phenomenon’, and to support the hypothesis concerning the importance of TLR4, we realised a supplementary transcriptome ‘ZAM heatmap’, which confirmed a sur-expression of DAMP in choriodecidua, S100A7, A8 and A9, for example, which are well-known ligands of TLR4 (given below as an image).
Heatmap of genes differentially expressed in the ZAM zone in relation to the sterile inflammation phenomenon.
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Evaluation Summary:
This manuscript provides insight into the zone-specific regulation of inflammatory gene expression in the fetal membranes prior to labor at term. Specifically, the authors demonstrate distinct epigenetic and mi-RNA control of TLR4 signaling in the amnion and chorion, highlighting the role of this pattern recognition receptor in physiological labor. Overall, the experimental design and data analysis are suitable, though the study would benefit from the inclusion of the analysis of fetal membrane tissues from pregnancy complications and/or in vivo studies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Belville et al. investigated the epigenetic regulation of inflammation in the zone of altered morphology (ZAM) and zone of intact morphology (ZIM) of the fetal membranes before labor at term. The authors first utilized human fetal membrane samples to undertake methylomic and transcriptomic surveys and identify the biological processes enriched in hyper- and hypo-methylated genes in the ZAM/ZIM of the chorion and amnion. The combined analysis of these surveys identified several key processes, all of which involved TLR4. The presence of TLR4 in human fetal membranes was confirmed using immunofluorescence and in vitro LPS stimulation. The authors then demonstrated hypermethylation of TLR4 in the amnion, but not the chorion, together with increased expression of the inhibitory mi-RNAs miR-125b-1 and let-7a-2. …
Reviewer #1 (Public Review):
Belville et al. investigated the epigenetic regulation of inflammation in the zone of altered morphology (ZAM) and zone of intact morphology (ZIM) of the fetal membranes before labor at term. The authors first utilized human fetal membrane samples to undertake methylomic and transcriptomic surveys and identify the biological processes enriched in hyper- and hypo-methylated genes in the ZAM/ZIM of the chorion and amnion. The combined analysis of these surveys identified several key processes, all of which involved TLR4. The presence of TLR4 in human fetal membranes was confirmed using immunofluorescence and in vitro LPS stimulation. The authors then demonstrated hypermethylation of TLR4 in the amnion, but not the chorion, together with increased expression of the inhibitory mi-RNAs miR-125b-1 and let-7a-2. Based on their findings, the authors propose a mechanism whereby TLR4 expression is epigenetically regulated in the amnion and chorion, providing evidence of tissue-specific control of this receptor as part of the inflammatory process required for labor.
Overall, the authors' findings provide novel insight into the mechanisms controlling the expression of TLR4 in the fetal membranes prior to term labor. The confirmation that TLR4 plays an important role in the inflammatory processes associated with term labor further highlights that TLR4 may contribute to "sterile" inflammatory processes rather than only those involving microbes and their products. The combined use of methylomic and transcriptomic surveys to cross-validate the authors' findings is a notable strength of the study. However, there are some aspects of the study that require further consideration.
1. A notable shortcoming of the authors' interpretation is the generalization of their findings to preterm premature rupture of membranes (PPROM). As noted by the authors, term labor is considered a "sterile" process, which is particularly important in terms of the authors' findings since TLR4 in the fetal membranes may be responding to endogenous signals such as danger signals. However, a large proportion of PPROM cases are associated with microbial invasion of the amniotic cavity, and thus in this context TLR4 would be responding to bacterial products.
2. It is a well-known concept that TLR4 is expressed by the fetal membranes and is responsive to LPS stimulation, and thus the confirmatory set of experiments performed by the authors do not seem to be as novel. Indeed, given that this study was focused on the "sterile" process of term labor, perhaps the utilization of danger signals that can interact with TLR4 would be more appropriate.
3. The distinction between the ZAM and ZIM seems to have been lost among the TLR4-focused experiments, and thus it is unclear how these fetal membrane zones fit into the conceptual model proposed by the authors in the final figure.
4. The study is largely descriptive and would benefit from the addition of fetal membrane tissues from pregnancy complications such as PPROM and/or animal models in which premature rupture of the membranes has been induced.
5. The study focuses on the mechanisms of rupture of membranes, but does not provide an explanation as to how the regulation of TLR4 mediates the process of membrane rupture.
-
Reviewer #2 (Public Review):
This is a well-conceived and executed paper that adds novel data to improve our understanding of rupture of the human fetal membranes. The new information presented not only addresses gaps in our understanding of normal parturition mechanisms but also the significant issue of preterm birth.
The authors highlight the need to understand the understudied human fetal membranes to be able to understand its role in normal parturition but also to lower the rates of preterm birth. They not only establish the need to study this tissue but also to improve our appreciation for regional differences within it, using a comprehensive genetic approach. The authors provide data from a genome wide methylation study and cross reference this with transcriptome data. Using this new knowledge, they then zero in on a specific gene …Reviewer #2 (Public Review):
This is a well-conceived and executed paper that adds novel data to improve our understanding of rupture of the human fetal membranes. The new information presented not only addresses gaps in our understanding of normal parturition mechanisms but also the significant issue of preterm birth.
The authors highlight the need to understand the understudied human fetal membranes to be able to understand its role in normal parturition but also to lower the rates of preterm birth. They not only establish the need to study this tissue but also to improve our appreciation for regional differences within it, using a comprehensive genetic approach. The authors provide data from a genome wide methylation study and cross reference this with transcriptome data. Using this new knowledge, they then zero in on a specific gene of interest TLR4. This receptor is already established as an extremely important receptor for preterm birth but little is known about its role in normal parturition. Strengths of this paper stem from the comprehensive data set provided, answering both the questions pertaining to the specific aims of this paper but also potentially future questions and providing potential focused targets of study. One example of this may be the common methylated genes that are found in both the ZIM and ZAM, illustrating not regional changes but gestational programming of this tissue. -
Reviewer #3 (Public Review):
Manuscript by Belville et al describes the significance of epigenetic and transcription associated changes to TLR4 as a mechanistic event for sterile inflammation associated with fetal membrane weakening, specifically in the zone of altered morphology. This manuscript is timely in an understudied area of research.
The authors have taken an extensive set of experiments to derive their conclusions.
However, it is unclear why the focus is on TLR4. Although LPS is a ligand for TLR4, gram negative infections are rare in PPROM but mostly genital Mycoplasmas. The methylome and transcriptome analysis does not necessarily warrant examination of a single marker. A clear rationale would need to be included.
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