Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation
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Evaluation Summary:
This study examined the role of CEBP4 in resolution of immune inflammatory responses. The manuscript uses genetic and pharmacologic approaches to demonstrate requirement of CEBP4 for survival following LPS administration and outlines certain downstream details of the mechanism. However, certain conclusions pertaining to this mechanism are either weak or not fully clarified. Further, the study proposes that RNA-binding proteins CPEB4 and TTP play important roles in regulating inflammation-associated mRNA transcripts by binding to CPEs or AREs to promote RNA stability or degradation. There is general agreement that most of the claims in the paper appear reasonably well-supported by the experimental data. However, there are some concerns regarding the robustness and significance of the presented data and conclusions as indicated in the individual reviews that require revision.
(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
Chronic inflammation is a major cause of disease. Inflammation resolution is in part directed by the differential stability of mRNAs encoding pro-inflammatory and anti-inflammatory factors. In particular, tristetraprolin (TTP)-directed mRNA deadenylation destabilizes AU-rich element (ARE)-containing mRNAs. However, this mechanism alone cannot explain the variety of mRNA expression kinetics that are required to uncouple degradation of pro-inflammatory mRNAs from the sustained expression of anti-inflammatory mRNAs. Here, we show that the RNA-binding protein CPEB4 acts in an opposing manner to TTP in macrophages: it helps to stabilize anti-inflammatory transcripts harboring cytoplasmic polyadenylation elements (CPEs) and AREs in their 3′-UTRs, and it is required for the resolution of the lipopolysaccharide (LPS)-triggered inflammatory response. Coordination of CPEB4 and TTP activities is sequentially regulated through MAPK signaling. Accordingly, CPEB4 depletion in macrophages impairs inflammation resolution in an LPS-induced sepsis model. We propose that the counterbalancing actions of CPEB4 and TTP, as well as the distribution of CPEs and AREs in their target mRNAs, define transcript-specific decay patterns required for inflammation resolution. Thus, these two opposing mechanisms provide a fine-tuning control of inflammatory transcript destabilization while maintaining the expression of the negative feedback loops required for efficient inflammation resolution; disruption of this balance can lead to disease.
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Author Response:
Reviewer #1 (Public Review):
[...]
- It is mentioned that deconvolution was applied but it is unclear how and what the presented data actually corresponds to (Fig. S1A-D).
We have clarified the deconvolution methodology in the Materials and Methods.
- Several of the differences indicated as statistically significant in Fig. S2E (for example for INFg, inflammatory response), do not seem to indicate biologically meaningful differences.
We share the reviewer’s perspective and accordingly described these changes as “minor” in the text. We thought the information may have been interesting for some readers, even though it is not relevant for the main conclusions of this work, and added this descriptive result as supplementary information. We have now replaced these two panels by the GSEA descriptive information, showing …
Author Response:
Reviewer #1 (Public Review):
[...]
- It is mentioned that deconvolution was applied but it is unclear how and what the presented data actually corresponds to (Fig. S1A-D).
We have clarified the deconvolution methodology in the Materials and Methods.
- Several of the differences indicated as statistically significant in Fig. S2E (for example for INFg, inflammatory response), do not seem to indicate biologically meaningful differences.
We share the reviewer’s perspective and accordingly described these changes as “minor” in the text. We thought the information may have been interesting for some readers, even though it is not relevant for the main conclusions of this work, and added this descriptive result as supplementary information. We have now replaced these two panels by the GSEA descriptive information, showing more categories, including different degrees and directions of changes. New Fig. S2E.
- There is an inconsistency regarding how proposed targets are studied and the results seem inconsistent. For example, in figure 4E Dusp1 and Il1rn are validated as CPEB4 targets. In figure 4F-G the focus is then shifted to SOCS1. Then the results from SOCS1 are extrapolated to other targets in Fig. 4H but Fig 4H data do not seem to correspond to Fig S4C data (e.g. 4H indicates that Socs3 is less expressed in Cpeb4-/- after 6h while S4C indicates that these are essentially identical; for Tnfaip3, the opposite regulation is indicated in 4H as compared to S4C after 6h).
We apologize for not explaining with enough detail the choice of targets shown in the different experiments. We did not want to focus on a particular target but rather on the global perspective, and in showing these data we try to be representative. For example, in Fig. 4D the primary data for Dusp1, Socs1 and Zfp36 are visually very clear whereas Il1rn, Socs3 and Tnfaip3, although statistically highly significant, they are less evident. Therefore, we plotted examples of both extremes of the identified genes to give the reader an accurate perspective of the primary data. Then in Fig. 4E, we included the quantitative validation of examples with both behaviors, plus Txnip as a positive control. The choice of Socs1 in Figs. 4F,G was more technical. For the western blot, we needed a protein that was expressed at reasonable levels and for which we had a suitable Ab available. SOCS1 fulfilled both criteria. For Tnfaip3, the 6 h and 3 h time points were swapped in the KO in S4C, we have corrected the error. Please note that, while Figs. 4H and S4C are based on the same data, one represents RPKM and the other the differential expression between WT and Cpeb4 KO macrophages. We have included a zoom-in of S4C to show the differences (new Fig. S4D).
In figure 4J cyclin B1 3'UTR with or without CPEs is evaluated while it would have been logical to focus on endogenous targets studied in other panels in figure 4.
We apologize if we failed to fully explain the rationale behind this experiment. This experiment was designed to test whether we could recapitulate the behaviors of the endogenous transcripts with only CPEs and AREs. Thus, we generated “synthetic” 3’UTRs containing only the desired cis-acting elements (CPEs and/or AREs). To this end, we used small 3’ UTRs that have been extensively characterized previously (Piqué et al 2008) and that we were sure did not contain additional Cis-acting elements. Thus, for the CPEs, we used the 3’ UTR of Cyc B1, which is only 21 nt long and basically consists of only 3 CPEs. The fact that it is derived from Cyc B1 is anecdotal. Endogenous genes with long 3’ UTRs would have potentially included a multitude of other Cis-acting elements.
In aggregate, the link between CPEB4 and targets which resolve the immune response can be better substantiated. The hypothesis from the authors is that these proposed downstream targets of CPEB4 underlie the resolution of the LPS-response. Although this is a plausible hypothesis, it should be noted that there are no experiments showing this.
This hypothesis is based on the identified targets, the changes in their decay rates in KO macrophages, and the phenotype of KO animals and cells. We agree with the reviewer that these do not provide direct evidence. However, given the number of CPEB4 targets and the need to reproduce temporal expression patterns, some sort of rescue would not be technically feasible. We have acknowledged the correlative nature of the model in the discussion.
Yet, although this may be technically very difficult, the conclusions could be strengthened by e.g. studying the role of CPEs for the endogenous genes of interest studied in Fig. 5D-E. This could be important as there is ample co-variance between not only AREs and CPEs (as indicated in Fig 5C) but also a range of other RNA elements which may also affect the stability of mRNA.
Please note that naïve-motif-discovery did not identify any other significantly enriched motif. In addition, we can reproduce the differential stability with a CPE/ARE containing “synthetic” 3’ UTR. The 3’UTR length is, however, another factor that further modulates mRNA stability - in general, not necessarily in response to LPS. This is why we perform point mutations in Fig. 5H,J. Identifying additional motifs that functionally interact with CPEs in the endogenous mRNAs (average 500 nt in length) would be almost impossible unless we had a specific motif to be mutated.
Finally, the authors present a model for their findings (Fig. 6A). The model well illustrates the findings of the paper although the data supporting activation of anti-inflammatory factors depending on CPE appears to be a weak link.
This model is based on the integration of the results of this work with existing literature. The key point that we wanted to raise is that previous studies have considered AREmediated deadenylation and mRNA destabilization as a binary “end-point” event. In the light of our results, we postulate that the extent of deadenylation and subsequent mRNA destabilization can be modulated, both in extent and time, or even reverted, by the balance between CPEs and AREs. We have clarified this concept in the Discussion.
Reviewer #2 (Public Review):
Cellular mRNA stability is regulated by a complex set of features including transcript polyA tail length, codon optimality, and internal elements that recruit RNA-binding proteins to promote RNA degradation or stabilization. In this manuscript by Suñer et al, the authors show that two RNA-binding proteins, CPEB4 and TTP, act in an opposing manner to regulate the stability of mRNA transcripts in macrophages and help regulate the inflammatory response. Specifically, CPEB4, a cytoplasmic polyadenylation element (CPE) binding protein, binds to CPEs present in the mRNAs of anti-inflammatory genes in macrophages to help stabilize these transcripts and promote inflammation resolution. The authors show that CPEB4 is upregulated in the blood of sepsis patients and in lipopolysaccharide (LPS)-challenged macrophages, and that CPEB4 KO mice have increased cytokine levels and an exacerbated inflammatory response that impairs their survival of sepsis. These results link inflammation response and resolution to CPEB4 levels in macrophages.
Next, the authors show that Cpeb4 mRNA levels are regulated by the LPS-activated p38a MAPK, where KO or inhibition of p38a in macrophages resulted in stabilization of the Cpeb4 mRNA. RNA-immunoprecipitation and RNA half-life measurements suggest this p38a-dependent stability results from the differential binding of AU-rich element (ARE)-binding proteins HuR and TTP to the Cpeb4 mRNA (which contains AREs), either stabilizing (HuR binding) or destabilizing (TTP binding) the transcript.
Finally, the authors use co-immunoprecipitation, RT-qPCR, and RNA half-life measurements to show that CPEB4 and TTP regulate the stability of mRNA transcripts that play key roles in LPS response and inflammation. These transcripts contain CPEs and AREs, which can be differentially regulated by the binding of CPEB4 and TTP, to promote stability or decay, respectively. Although the effects of CPE:ARE ratio on endogenous mRNA stability in cells appears somewhat complex, luciferase reporters bearing different combinations of CPEs and AREs suggest these elements help to directly determine the stability of mRNA transcripts during inflammation response.
Overall, this thorough work proposes that RNA-binding proteins CPEB4 and TTP play important roles in regulating inflammation-associated mRNA transcripts by binding to CPEs or AREs to promote RNA stability or degradation. While most of the claims in the paper appear reasonably well-supported by the experimental data, I do have some concerns on the robustness and significance of the presented data in some cases. The authors succeed in making a generally compelling case that CPEB4 plays a key role in regulating mRNA stability to impact inflammation resolution, but some of the individual claims identified, that appear to be more weakly supported by the presented data, should be addressed and/or clarified.
We thank the reviewer for his/her comments and specific points. We have included the requested clarifications as specified in the point-by-point responses.
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Evaluation Summary:
This study examined the role of CEBP4 in resolution of immune inflammatory responses. The manuscript uses genetic and pharmacologic approaches to demonstrate requirement of CEBP4 for survival following LPS administration and outlines certain downstream details of the mechanism. However, certain conclusions pertaining to this mechanism are either weak or not fully clarified. Further, the study proposes that RNA-binding proteins CPEB4 and TTP play important roles in regulating inflammation-associated mRNA transcripts by binding to CPEs or AREs to promote RNA stability or degradation. There is general agreement that most of the claims in the paper appear reasonably well-supported by the experimental data. However, there are some concerns regarding the robustness and significance of the presented data and conclusions as …
Evaluation Summary:
This study examined the role of CEBP4 in resolution of immune inflammatory responses. The manuscript uses genetic and pharmacologic approaches to demonstrate requirement of CEBP4 for survival following LPS administration and outlines certain downstream details of the mechanism. However, certain conclusions pertaining to this mechanism are either weak or not fully clarified. Further, the study proposes that RNA-binding proteins CPEB4 and TTP play important roles in regulating inflammation-associated mRNA transcripts by binding to CPEs or AREs to promote RNA stability or degradation. There is general agreement that most of the claims in the paper appear reasonably well-supported by the experimental data. However, there are some concerns regarding the robustness and significance of the presented data and conclusions as indicated in the individual reviews that require revision.
(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):
Resolution of immune responses are essential for organism health. In this manuscript, Suner et al. studies the role of CPEB4 during the response to LPS, which underlies sepsis. In the initial part of the manuscript the authors clearly demonstrate, using an animal model, that CPEB4 is necessary for survival following LPS stimulation. The authors then, using a combination of genetic and pharmacological approaches show that CPEB4 is induced in monocytes/macrophages during the LPS response via the previously described mechanism involving p38alpha, HuR and TTP. Consequently, it is argued that inability to induce CPEB4 hampers resolution of the LPS-response. This part of the study is in general well supported although some of the statements relative to the informatics analyses could be clarified.
1. It is …Reviewer #1 (Public Review):
Resolution of immune responses are essential for organism health. In this manuscript, Suner et al. studies the role of CPEB4 during the response to LPS, which underlies sepsis. In the initial part of the manuscript the authors clearly demonstrate, using an animal model, that CPEB4 is necessary for survival following LPS stimulation. The authors then, using a combination of genetic and pharmacological approaches show that CPEB4 is induced in monocytes/macrophages during the LPS response via the previously described mechanism involving p38alpha, HuR and TTP. Consequently, it is argued that inability to induce CPEB4 hampers resolution of the LPS-response. This part of the study is in general well supported although some of the statements relative to the informatics analyses could be clarified.
1. It is mentioned that deconvolution was applied but it is unclear how and what the presented data actually corresponds to (Fig. S1A-D).
2. Several of the differences indicated as statistically significant in Fig. S2E (for example for INFg, inflammatory response), do not seem to indicate biologically meaningful differences.
As CPEB4 affects mRNA-stability the authors next strive to identify mRNAs which are targets for CPEB4-dependent post-transcriptional regulation. Following an unbiased approach, the authors propose that CPEB4 is critical for regulation of anti-inflammatory factors that in turn would underlie resolution of the immune response. While being a logical continuation of the studies, this part of the manuscript has several weaknesses:
1. There is an inconsistency regarding how proposed targets are studied and the results seem inconsistent. For example, in figure 4E Dusp1 and Il1rn are validated as CPEB4 targets. In figure 4F-G the focus is then shifted to SOCS1. Then the results from SOCS1 are extrapolated to other targets in Fig. 4H but Fig 4H data do not seem to correspond to Fig S4C data (e.g. 4H indicates that Socs3 is less expressed in Cpeb4-/- after 6h while S4C indicates that these are essentially identical; for Tnfaip3, the opposite regulation is indicated in 4H as compared to S4C after 6h). As it seems that 4H and S4C are based on the same data it is hard to understand. In figure 4J cyclin B1 3'UTR with or without CPEs is evaluated while it would have been logical to focus on endogenous targets studied in other panels in figure 4. In aggregate, the link between CPEB4 and targets which resolve the immune response can be better substantiated.
The hypothesis from the authors is that these proposed downstream targets of CPEB4 underlie the resolution of the LPS-response. Although this is a plausible hypothesis, it should be noted that there are no experiments showing this.In the final part of the manuscript the authors examine how the interplay between RNA elements for HuR/TTP (ARE) and CPEB4 (CPE) interact to modulate mRNA levels during the LPS-response. It is suggested that it is the relative number of AREs vs CPEs that determine the pattern of regulation following LPS-stimulation. Although this part of the studies left some questions untouched (e.g. why are AREs not active when there is no CPE; or do these patterns of regulation correspond to modulation of poly-A tails) the conclusions are intriguing and supported by the data. Yet, although this may be technically very difficult, the conclusions could be strengthened by e.g. studying the role of CPEs for the endogenous genes of interest studied in Fig. 5D-E. This could be important as there is ample co-variance between not only AREs and CPEs (as indicated in Fig 5C) but also a range of other RNA elements which may also affect the stability of mRNA.
Finally, the authors present a model for their findings (Fig. 6A). The model well illustrates the findings of the paper although the data supporting activation of anti-inflammatory factors depending on CPE appears to be a weak link. -
Reviewer #2 (Public Review):
Cellular mRNA stability is regulated by a complex set of features including transcript polyA tail length, codon optimality, and internal elements that recruit RNA-binding proteins to promote RNA degradation or stabilization. In this manuscript by Suñer et al, the authors show that two RNA-binding proteins, CPEB4 and TTP, act in an opposing manner to regulate the stability of mRNA transcripts in macrophages and help regulate the inflammatory response. Specifically, CPEB4, a cytoplasmic polyadenylation element (CPE) binding protein, binds to CPEs present in the mRNAs of anti-inflammatory genes in macrophages to help stabilize these transcripts and promote inflammation resolution. The authors show that CPEB4 is upregulated in the blood of sepsis patients and in lipopolysaccharide (LPS)-challenged macrophages, …
Reviewer #2 (Public Review):
Cellular mRNA stability is regulated by a complex set of features including transcript polyA tail length, codon optimality, and internal elements that recruit RNA-binding proteins to promote RNA degradation or stabilization. In this manuscript by Suñer et al, the authors show that two RNA-binding proteins, CPEB4 and TTP, act in an opposing manner to regulate the stability of mRNA transcripts in macrophages and help regulate the inflammatory response. Specifically, CPEB4, a cytoplasmic polyadenylation element (CPE) binding protein, binds to CPEs present in the mRNAs of anti-inflammatory genes in macrophages to help stabilize these transcripts and promote inflammation resolution. The authors show that CPEB4 is upregulated in the blood of sepsis patients and in lipopolysaccharide (LPS)-challenged macrophages, and that CPEB4 KO mice have increased cytokine levels and an exacerbated inflammatory response that impairs their survival of sepsis. These results link inflammation response and resolution to CPEB4 levels in macrophages.
Next, the authors show that Cpeb4 mRNA levels are regulated by the LPS-activated p38a MAPK, where KO or inhibition of p38a in macrophages resulted in stabilization of the Cpeb4 mRNA. RNA-immunoprecipitation and RNA half-life measurements suggest this p38a-dependent stability results from the differential binding of AU-rich element (ARE)-binding proteins HuR and TTP to the Cpeb4 mRNA (which contains AREs), either stabilizing (HuR binding) or destabilizing (TTP binding) the transcript.
Finally, the authors use co-immunoprecipitation, RT-qPCR, and RNA half-life measurements to show that CPEB4 and TTP regulate the stability of mRNA transcripts that play key roles in LPS response and inflammation. These transcripts contain CPEs and AREs, which can be differentially regulated by the binding of CPEB4 and TTP, to promote stability or decay, respectively. Although the effects of CPE:ARE ratio on endogenous mRNA stability in cells appears somewhat complex, luciferase reporters bearing different combinations of CPEs and AREs suggest these elements help to directly determine the stability of mRNA transcripts during inflammation response.
Overall, this thorough work proposes that RNA-binding proteins CPEB4 and TTP play important roles in regulating inflammation-associated mRNA transcripts by binding to CPEs or AREs to promote RNA stability or degradation. While most of the claims in the paper appear reasonably well-supported by the experimental data, I do have some concerns on the robustness and significance of the presented data in some cases. The authors succeed in making a generally compelling case that CPEB4 plays a key role in regulating mRNA stability to impact inflammation resolution, but some of the individual claims identified, that appear to be more weakly supported by the presented data, should be addressed and/or clarified.
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