PONE-D-21-13350

Upregulation of a nuclear factor-kappa B-interacting immune gene network in mice cochleae with age-related hearing loss

PLOS ONE

Dear Dr. Maeda,

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.

Please submit your revised manuscript by Aug 28 2021 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: http://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,

Vasu Punj

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that Figure 5 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 5 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. 

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: Yes

**********

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: In this manuscript, the authors have compared the expression of immune-related genes between young and old mice with manifested hearing loss and identified differentially expressed genes (DEGs), which expression could be associated with age-related hearing loss (ARHL). Among the significantly upregulated DEGs, eight genes (Ccl5, Cxcl10, Cxcl9, Il1a, Lta, Ltb, Ptgs2, and Tnf) were proposed transcriptionally regulated by NF-kB, which Nfkb1 subunit is also upregulated in aged cochleae. In addition, seven other immune-related genes (Casp1, IL18r1, IL1B, Card9, Clec4e, Ifit1, and Tlr9) were significantly upregulated at the advanced stage ARHL.

Although there is another recently published similar study (Su Z, et al. 2020, PeerJ. 2020;8:e9737. PubMed PMID: 32879802) analyzing the expression of a much higher number of the genes (731 genes) and showing the association of cochlear inflammation with ARHL, this study also represents a valuable contribution in clarification the role of inflammaging in development of ARHL.

Although the results are compelling and very relevant, there is still room for improvement.

Major issues

1. Keeping that authors showed significant upregulation of NF-kB in the aged cochlear tissue and predicted its involvement in transcriptional regulation of eight significantly upregulated DEGs (Ccl5, Cxcl10, Cxcl9, Il1a, Lta, Ltb, Ptgs2, and Tnf), it is not clear why they did not show the expression of these genes on different ages (3-month-old, 6-month-old, 9-month-old and 12-month-old mice) as it was shown for nine genes selected based on the preliminary data from next-generation sequencing (RNAseq).

2. Although the authors referred in the discussion part that the hearing status is getting to worsen as animals are older, they should assess the hearing status of animals at all analyzed ages (at least at 7-weak, 6- month and 12- month age) in parallel with the gene expression analysis. That will help understand whether the up-regulation of NF-kB-regulated genes is associated with the development of AHR or is only manifested in the oldest animals (12-month age).

3. Immunohistochemical analysis of cochlear sections from mice at different ages should be also extended for some of NF-kB -regulated significantly upregulated DEGs, as it was done for IL-18r1 and IL-1B.

4. Considering the role of macrophages in the cochlear inflammatory response and the immune capacity of cochlear supporting cells, co-staining inflammatory mediators and cell-specific markers will contribute to identifying the primary cellular source of up-regulated cytokines and immune-related factors associated with ARHL.

Minor issues

1. Although authors called on their preliminary data, they should clarify how many genes were analyzed by next-generation sequencing (RNAseq). This statement will also put weight on the paper, considering that a similar study analyzing more than 700 genes has already been published.

2. There is a terminology issue that should be solved.

a) Interleukins and chemokines are cytokines as well, and they should not be listed separately from cytokines. In that sense, the sentence:

“The targeted genes were those encoding cytokines, chemokines, and interleukins, their receptors and signaling molecules, and genes involved in acute, chronic, and intracellular inflammatory responses (lines: 118-120)”,

should be modified like this:

“The targeted genes were those encoding cytokines (including chemokines and interleukins), their receptors and signaling molecules, and genes involved in acute, chronic, and intracellular inflammatory responses.

b) IL-18 receptor accessory protein (IL18RAP, or IL-18Rβ) is the other subunit or IL-18R, which means it is not a cytokine. In this context, it is correct to name together IL1B and IL18rap as pro-inflammatory molecules or factors than cytokines.

3. IL-18 is mistakenly written instead of IL-1B (line 199).

4. “Kyoto Encyclopedia of Genes and Genomics” (line 256) should be replaced by an abbreviation that has already been introduced.

5. Table 1. The prefix “up” is wrongly positioned, indicating Myd88 instead of Nfkb1.

Reviewer #2: In this manuscript by Uraguchi K et al titled “Upregulation of a nuclear factor-kappa B-interacting immune gene network mice cochleae with age-related hearing loss” , the authors have used gene expression analysis and bioinformatics analyses to demonstrate upregulation of inflammatory pathways in a C57BL/6 mouse model for ARHL. Further, they have used immunohistochemistry to show expression of IL-1B and IL18r1 in aged mouse cochlear tissue which is consistent with their gene expression analysis.

The experiments in this manuscript are technically sound and their conclusions are reasonable and these studies may be of interest for researchers trying to elucidate molecular basis for ARHL. A few minor comments:

1. It will be useful to have a list of up regulated and down regulated genes together with the volcano plot, which by itself is not very useful. The entire list of 84 genes on the array could be included in supplementary table.

2. Table 1 may need to be formatted- in the reviewer’s copy the “Regulation” column was not properly aligned with the gene name. For example for gene FASL, the “up” in the regulation column was not in the same row, this has happened for several genes.

3. Fig 4: the Y axis is not labeled

4. They need to carefully proofread the manuscript. For example:

Line 170: “The KEGG pathway is the annotation….” This sentence could be rephrased as “ The KEGG pathway is the annotation of functional gene pathways involving a group of genes”

Line 351: “The DEGs of these immune-related genes…” could be rephrased as “The differential expression of these immune-related genes…”

**********

6. 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: Yes: Sasa Vasilijic

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Upregulation of a nuclear factor-kappa B-interacting immune gene network in mice cochleae with age-related hearing loss

PONE-D-21-13350R1

Dear Dr. Maeda,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Vasu Punj

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

**********

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)

**********

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: Yes: Sasa Vasilijic