PONE-D-22-30303Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cellsPLOS ONE

Dear Dr. Newton,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This article aims to study the regulation of the expression of BIRC2 and BIRC3 in bronchial epithelial cell lines cultured in the presence of pro-inflammatory cytokines (IL-1β or TNFα), in the presence or the absence of the anti-inflammatory compounds dexamethasone or budesonide.

The results shown are interesting but the article is very difficult to read and follow. This could be improved by an introduction in result paragraphs specifying the objectives of the experiments carried out. Moreover, concluding sentence at the end of the result paragraphs could help in the understanding of the results.

I did not understand why the authors analyzed the influence of INFγ (Fig S2D). Since INFγ is no longer used in later experiments and these results are not used for discussion, I believe they are not necessary (line 209-214).

Figure legends: could author indicate the number of experiments (instead of “N”)

Why the authors analyzed the regulation of BIRC2 and BIRC3 expression in pHBEC grown as both submersion culture or as highly differentiated cells? Did they expect different mechanisms of regulation in function of the differentiation state? It seems to me that the results are quite similar, whatever the cell line: A549, pHBEC-SC or pHBEC-ALI and also BEAS-2B and Calu-3. It should be possible to simplify the description of the results in order to avoid repetition and provide fluency (results paragraph 1 and 2).

Figure 3D showed that the IL-1β and TNFα-mediated overexpression of BIRC2 mRNA is prevented by IκBαΔN expression whereas it is not affected by PS-1145 (Figure 4A) that blocks NF-κB. Could authors explain these results. Moreover, PS-1145, as well as ML-120B reduced BIRC2 protein expression in TNF-α-treated cells while PS-1145 did not modify the level of expression of the mRNA. These results have to be explained.

A concluding sentence concerning the role of NF-κB in BIRC3 and BIRC2 expression at the end of the result paragraph 4 could greatly improve the understanding of the results.

Figure 5D. I think that there are errors in the indications of the presence or not of siGR and siCTL.

In the result paragraph 6, the authors analyzed the expression of BIRC3 and BIRC2 proteins in IL-1β or TNFα-treated cells in the presence of absence of cycloheximide. The author should introduce this paragraph and indicate the objectives of these experiments. What is the question addressed? Why did the authors add CHX 1 hours after the cytokine? CHX treatment prevented the IL-1β or TNFα-mediated increase of BIRC3 expression and in a lower extend BIRC2 expression and even decreased BIRC2 protein expression in TNFα-stimulated cells. Again, the authors should give the conclusion of the experiments to help in the understanding of the article. Then, the authors added CHX 6 hours after the cytokine. I did not understand why they did this experiment? What is the question addressed? These points could be precised in the manuscript.

In the last result paragraph, the author analyzed the mechanisms of BIRC2 and BIRC3 degradation? Since the degradation was only observed in the condition of TNFα + CHX treatment (Figure 6B), why did they analyze the condition of IL1β + CHX treatment and why did they analyze the role of proteasome inhibitor on IL1β- or TNFα-induced expression of BIRC3 and BIRC2 ? It is well known that NF-κB activation required proteasome-mediated degradation of IκBα. Since IL1β- or TNFα-induced expression of BIRC3 and BIRC2 is dependent on NF-κB, it seems logical that proteasome inhibitors block the induction of BIRC3 and BIRC2 expression.

The results show that BIRC2 and BIRC3 proteins is degraded 6-7 hours after stimulation of cells with TNFα but not after IL-1β stimulation. This could argue in a favor of a role of BIRC2 and BIRC3 in TNFR signaling pathway but not in IL-1βR signaling pathway. Since BIRC2 is an important cell signaling intermediate in TNFR signaling pathway, we may hypotheze that the degradation of BIRC2 is a feed-back regulatory mechanism in order to regulate TNFR signaling pathway in case of sustain stimulation of receptor. BIRC2 in an E3-ubiquitine ligase able to self-ubiquitinate and stimulate its own degradation and also able to promote the ubiquitinatation and degradation of BIRC3 (in case of Smac mimetic exposure). This point could be discussed.

Reviewer #2: In this excellent study, Thorne et al., from Newton’s group sought to dissociate the redundant roles of BIRC2 and BIRC3 in the regulation of inflammatory signaling in the presence or absence of glucocorticoids (GC) in physiologically relevant cells, airway epithelial cells. The authors used both pulmonary epithelial cell lines (A549, BEAS2B, Calu-3 cells) and primary human bronchial epithelial cells (pHBECs cultured either in submersion culture or ALI). To this end, responses to cytokines (TNF, IL1, IFNg) and GC (Dexamethasone, Budesonide) have been examined in detail. The role of NF-kB in BIRCs expression induced by cytokines has been demonstrated using various strategies such as siRNA, DN, and IKK inhibitors. The involvement of the glucocorticoid receptor in BRCs expression induced by GC has been also demonstrated using GR inhibitor or siRNA GR. The involvement of protein degradation in TNF effects on BRCs was explored and demonstrated using CHX and MG compound. The authors clearly demonstrated the differential regulation of BIRC3 and BIRC2 in epithelial cells by inflammatory cytokines and GC where the constitutive expression of BIRC2 is consistent with acute early events and the inducible expression of BIRC3 is more consistent with later onset roles. The authors also demonstrated that BIRC3 expression is induced by GC and suggested that BIRC3 plays key protective roles in inflammatory signaling. Overall, the study is well-designed, comprehensive and the Ms is well-written. Some minor issues are presented below.

Minor comments:

- While the authors suggested a protective role of BIRC3, some studies showed a correlation between BRC3 and asthma severity (PMID: 27304223). Further discussion is needed.

- While most of the supra-additive effects of IL1b and GC were seen at 24 hr (Fig. 2A), most of the western blot studies (Figs 3C, 5B and FC) were conducted at 6 hr where not clear supra-additive effects were observed. Some justification is needed.

- Figures: Fig 5D, western blot: Experimental conditions under bands, 3, 6, and 9 are not clear. Are those triplicates? Conditions with IL1B only were not presented while stated in the Fig 5D legend.

- Introduction section: Second paragraph, the opposite/contradicting role of BIRCs on the regulation of NF-kB in canonical (line 54-64) versus non-canonical pathways (line 64-67 where the term BRC2/3 inhibition is not clear) is a little bit confusing.

- Due the high number of data and variables in this study, the addition of a summary table highlighting all the findings in one place will enhance the clarity of this study.

- Similarly, a graph depicting the conclusion of the authors should help reinforcing this critical concept and findings generated by this study.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-30303R1Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cellsPLOS ONE

Dear Dr. Newton,

Thank you for submitting your manuscript to PLOS ONE. One of our reviewers still has raised concerns regarding the article, specifically citing difficulties in following the manuscript and issues with readability.

The reviewer has pointed out that the current version of the manuscript is difficult to follow, making it challenging to read. Additionally, the reviewer has noted that some results are repeated in different figures, and has suggested that the article could be further simplified.

In general, the reviewer has recommended that several figures could be deleted to improve clarity and conciseness, and has raised concerns about contrasting results between Figs. 2A, B and Fig. 6.

Please review the comments from the reviewer and make the necessary changes as requested.

Please submit your revised manuscript by May 27 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Aristóbolo M Silva

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors answered to my concerns. However, the article is still difficult to follow. I agree that replication of the results adds robustness but it also brings heaviness and makes the article difficult to read.

1. Figure S1 and Figure 3B both demonstrated that IL-1β and TNFα both induced the activation of NF-κB in the concentrations used in the study. I think that the supplementary Figure 1 can be removed. Then, the authors analyzed the contribution of NF-κB in IL-1β and IL-1β + DEX-induced BIR2 and BIRC3 mRNA expression by using RELA-directed siRNA or IκBα super-repressor. They showed that IL-1β and IL-1β + DEX-induced increased of BIRC2 and BIRC3 mRNA are, at least partly, NF-κB dependent. Then, they confirmed that the increase in BIRC3 protein expression also depended on NF-κB (Figure 3 and S6). The same results were observed in Figure 3C and S6. Could authors simplify the text by describing the two Figure in the same sentence. Since IL1B and IL1B + DEX did not enhanced BIRC2 protein expression (as demonstrated in Figure 2), I think that the authors could simplify the Figure 3 and remove the analysis of BIRC2 protein expression.

2. In Figure 4A, the authors confirmed that NF-κB is involved in the induction of BIRC3 mRNA and protein expression in IL1B, TNFα, IL1B + DEX and TNF + DEX-treated cells by using the IKK2 inhibitor PS-1145. By contrast, PS-1145 did not decrease the induction of BIRC2 mRNA in response to the stimuli. The analysis of BIRC3 protein expression confirmed the involvement of NF-κB. Again, since IL1B and IL1B + DEX did not enhanced BIRC2 protein expression (as demonstrated in Figure 2), I think that the authors could simplify the figure and remove the analysis of BIRC2 protein expression. Since the conclusions of the result paragraph 3 and 4 (Figure 3 and 4) are the same, e.i. the induction of BIRC3 mRNA and protein expression in IL1B, TNF, IL1B + DEX and TNF + DEX treated cells depended on NF-kB, the two paragraphs could be combined.

Line 342: “Dexamethasone-induced BIRC3 mRNA expression was not changed by PS-1145”. I do not agree with this sentence. Although statistically non-significant, the modest induction of BIRC3 in response to dexamethasone (in the left and right panels) in decreased by PS-1145 (Figure 4).

3. The authors analyzed the stability of BIRC2 and BIRC3 proteins in TNFα and IL-1β treated cells (Figure 6). Cells were first treated with the agents for 1 hours, then incubated in the presence or absence of CHX. As expected, IL1b or TNF both enhanced BIRC3 protein expression. While 6 hrs of treatment with IL1B or TNFα did not modify the BIRC2 protein expression in Figure 2A and B, it seems increased BIRC2 protein expression in Figure 6B (quantitative analysis, lower panel)? Is that significant? Could authors explain these results?

4. Figure 8C, left panel reproduced the results of Figure 6B. The authors could simplify and remove this panel. Since the authors demonstrated that IL-1β did not destabilize BIRC2 and BIRC3 protein in Figure 6B, the analysis the importance of proteasome inhibitor on destabilization of BIRC proteins in IL-1β treated cells is not relevant and this can be removed.

Reviewer #2: The authors have adequately addressed all the comments. No further comments.

The authors have adequately addressed all the comments. No further comments.

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Reviewer #1: No

Reviewer #2: No

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Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells

PONE-D-22-30303R2

Dear Dr. Newton,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Aristóbolo M Silva

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

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