PONE-D-23-19772Mucin Producing In vitro Fish Mucosal Surfaces as a Model for Studying Host-Pathogen InteractionsPLOS ONE

Dear Dr. Lindén,

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 Mar 20 2024 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,

Maria del Mar Ortega-Villaizan

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. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript.

3. We note that your Data Availability Statement is currently as follows: "All relevant data are within the manuscript and its Supporting Information files. Raw MS data on O-glycan analyses are available on glycopost ( https://glycopost.glycosmos.org/) using the project ID: GPST000334 (CHSE-214) and GPST000335 (RTgillW1). MSMS data with tentative structures are available https://unicarb-dr.glycosmos.org/references/523."

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Additional Editor Comments:

The manuscript presented by Linden et al is of interest, however, major revisions should be performed before being aceptable for publication in PLOS One. Please follow the comments indicated by the reviewers.

[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: No

Reviewer #3: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: No

Reviewer #3: No

**********

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: No

Reviewer #3: 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

Reviewer #3: 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: This manuscript describes an interesting novel in vitro model based on modified fish cell lines for the study of mucin responses in mucosal epithelia. Its reading was inspiring and it will help to develop alternative methods to the in vivo ones.

There are some suggestions authors could follow in order to detail results and deepen discussion, which in my opinion could increase the study’s soundness.

-I wonder what the cell morphology in the in vitro membranes was, and if it changed after DAPT treatment. Could authors observe goblet cell morphology or cell polarization? Including a description on cell morphology, differentiation and polarization would be interesting. In fact, authors have included in the discussion section the issue of non-differentiation and non-polarization in unstimulated cultures on plastic surfaces, leaving this issue unanswered in the current model.

-Authors describe interindividual variation of GalNAz incorporation in terms of “relatively similar” or “larger variation” in each cell line. Statistical significance should be indicated in terms of an analysis of variance.

-L430 and following: Authors could include a comment on the need of semi-wet interfaces for fish in vitro membranes, since in vivo fish membranes remain immersed in the natural environment.

-L464-477: Have authors some hypothesis on the differential sialylation and fucosylation of the analyzed mucins compared to fish gill mucins? Do they have any suggestion in order to modulate the glycosylation pattern so that their mucin model may better replicate the in vivo pattern?

In the same line, PAS+ goblet cells with neutral mucins are usually present in fish mucosal epithelia. Authors could also discuss the absence of those in their in vitro model.

-Authors do not consider the differential involvement of secreted and membrane-bound mucins in their study. Especially when mucins from cell lysates are analyzed, such different mucin types in addition to intracellular immature mucins associated to the Golgi apparatus, should be considered.

Besides, some minor corrections should be implemented:

- L49: Please, change for “fish mucosal epithelia are”.

- L108: Correct “RTgutGC”.

- L166: Please, correct “The medium … was”.

- L169: Correct “mucosal surfaces” to lower case.

- L204-234: Use µL for microliters.

- L218: Describe what “conc HAc” is.

- L225: Shift to “control media”.

- L251: Correct “MS/MS”.

- L310: Please, place “…, in appearance, …” between comas.

- Table1: Empty cells should be filled with 0 or dash.

- L393: Please, rewrite. E.g. “…their relative abundance was estimated…”

- L411: Please, rewrite. E.g. “… is incorporated similarly in both cell lines and Alcian blue staining was also similar in both”

- L412: Please, change “which were all”.

- L419: Correct “models that mimic”.

- L420: Correct “often result in”

- L425: Please, delete “of mucus”.

- L500: Correct “models present”.

Reviewer #2: The manuscript title “Mucin Producing In vitro Fish Mucosal Surfaces as a Model for Studying Host” by Quintana-Hayashi and colleagues is about the possible use of fish cell lines as in vitro mucosal surface models. These models pretend to be an alternative to in vivo experiments and be a model to investigate the effect of pathogens and modulatory components on mucin production in fish.

I found this article really interesting and a good alternative to reduce the number of fish in in vivo experiments. However, I found the manuscript difficult to read because the material and methods are not well explained and therefore the workflow is confusing. In addition, I missed a proper control in many experiments and statistical analysis. For all of these reasons, I consider that is needed deep changes in order to be published.

1-I do not know the protocol that authors used to differentiate fish cell lines into mucus producing cells. In material and methods it seems that only with culturing the cells in snapwell cell culture for 2 weeks and after that with the inhibitor DAPT and IWP-2 is enough but in the results is suggested that authors have used other methods to differentiate cell for example: line 278-282 “We started by investigating morphology, adherence, and mucin production in the salmonid cell lines RTgill-W1, CHSE-214 and SSE-5 under standard conditions as well as under conditions that we have used in the successful development of mammalian in vitro mucin producing mucosal surfaces from epithelial cells, including treatment of the cells with goblet cell differentiating media and prolonged culture ”. Please clarify these issue.

2-The workflow of the experiments is confusing, for example in the results of the sections: The metabolic label GalNAz is incorporated into both RTgill-W1 and CHSE-214 based in vitro membranes, O-glycan analyses from CHSE-214 and RTgill-W1 and A. salmonicida binds to RTgill-W1 and CHSE-214 cells. I do not know the protocol that authors used to differentiate fish cell lines if it is the same that in the section RTgill-W1 and CHSE-214 cell lines form an adherent layer and produce mucins. Please clarify this issue. I suggest to draw a schema in order to clarify how the experiments are performed.

3-Material and methods are incomplete; I do not know how the experiments are made and I missed commercial references of many reactives in many sections. In addition, I did not find the microscope use for the images taken in figure 1 and Figure2. In figure 2 C and F, I do not know how authors obtain these data and there are not properly explain in the legend. Moreover, in many sections of material and method, authors said for example aliquots were dot plot, cell lysate aliquots or paraffin embedded sections… but they do not indicate which samples were analyzed and therefore makes more difficult to understand the experiments that they perform.

4- In addition, I missed a proper control of many experiments. For example, in figure 2 I do not understand why the not labeled membranes are stained. I must include a positive and negative control to compare the results obtained and include also in C and F graphs. In figure 3 andfFigure 4 I would include a control of CHSE and RTGill without differentiate as a proper control in addition to the media control.

5- In figure 1 and Figure2 authors should include in both legends the magnification of these pictures and the number in the scale. In figure 2 please include statistical analysis of the graphs.

6-The binding assay in my opinion is not enough to confirm this issue. authors should include more experiments to confirm the binding.

7-The results showed in table 1, in my opinion, the spectrum of the peaks of the chromatogram of O-glycans should be included in the manuscript. I do not understand why some samples are dot blot and others by gel band, please explain properly.

8- I found some confusing sentences that I detailed below:

-line 80-82” A decreased secretion could lead to an increased amount of mucus in the cells, conversely, an increased secretion could lead to a decreased amount of mucus in the cells”. please clarify.

-line 461-463” the lower inter-individual variation and more distinct GalNAz staining of

the RTgill-W1 in vitro membranes most likely makes 462 this a better model for studies comparing effects of different conditions/treatment on mucin production”. I do not understand the sentence “more distinct GalNAz staining”, in the graph seems that there are not much variation in the fluorescence intensity. Please clarify this sentence.

-line 474-476”. Although the majority of the rainbow trout mucins are sialylated in line with the results from the RTGill-W1 mucin glycans, approximately 30% of the glycans are sialylated (6), a feature we did not detect among the RTGill-W1 mucin glycans”. It is contradictory the information in the sentence, first authors observed sialylated mucins but at the end of the sentence said they did not detect. Please clarify. In addition, line 487-488 “The high abundance of sialylated glycans is likely the reason that Aeromonas salmonicida bound to both cell lines”. Please clarify.

Minnor things

Line 33, DAPT define acronym.

Line 111-112 cldn3 and il-1β, il-6, il-8 and tnfα define acronym

Line 154 WNT acronym

Line 218, conc HAc define acronym

Line 298 figure 1, I will specify to Fig 1A, F in order to reference the information which is included in the sentence.

Line 307 AB define acronym

line 367 the bottom, I believe that it is on the top. Please change it.

-Indicate at least for the first time define h as hours

-Line 426 HT29-MTX-E12 define acronym and the specie of the cell line

-Line 508 Neu Ac define acronym

Reviewer #3: *General comments: The current manuscript proposes the use of fish cell lines as a potential model platform for evaluating host/pathogen interactions on mucosal surfaces by mucin production.

I think that the strengths of the study are the methods to differentiate cells and induce mucin production, which are quite clear and described in detail. Furthermore, considering the historical lack of tools and platforms at the phenotypic level in fish, this study also contributes to the in vitro characterization of the immune response in epithelial barriers. However, regarding its weaknesses, I think that a more quantitative or semi-quantitative evaluation of the different parameters evaluated was lacking, especially related to the production of mucins (Figure 1) and the capacity of the cell lysates to bind A. salmonicida (Figure 4). Finally, before proposing final acceptance of the manuscript there are aspects to improve, which I detail in the specific comments:

*Specific comments:

Title: The first part of the title could change from "Mucin Producing In vitro Fish Mucosal Surfaces" to "In vitro Fish Mucosal Surfaces Producing Mucins". This would improve the understanding of the title.

Keywords: "cell line" should be added

Abstract:

-Line 23: After "prevent disease and reduce antibiotic use." There is a sentence missing that connects this idea with the "Fish epithelia secrete mucus". For instance "To deal with this problem, fish epithelia that secrete mucus, which is mainly composed of highly glycosylated mucins, are a potential target for evaluation."

-Line 25: After "pathogens encounter." add "Therefore," the aim.. .

-Line 41: Change "These models thus" to "Thus, these models"

Materials and Methods:

-Line 138: Add "," after "flask"

-Line 175: In "H2O", the 2 must be in subscript.

-SSE-5 should be eliminated from the manuscript since due to their problems in adhering, the authors did not carry out the tests with these cells nor did they obtain data.

-Line 259: Performing blotting to determine the binding capacity of cell lysates to A. salmonicida does not seem to be the most sensitive test or the one that allows the best analysis. Considering the capacities that the authors have described, the test should have been carried out in 96-well plates with adherent capacity to determine OD, this would have allowed a quantitative experiment with better comparisons between the samples.

Results

-Line 277: RTgill-W1 and CHSE-214 cell lines form an adherent layer and produce mucins. The authors, in addition to showing the images (and describing them), should perform a semi-quantitative analysis (in software such as ImageJ) of the intensity and coverage of the labeling between the different samples. This will allow comparisons between cell lines, inducers and mucin production to be better established.

-From 278 to 286: This paragraph is just methodology again, it should be moved to that section (modifying it).

-From 327 to 331: Just methodology again, it should be moved to that section (modifying it)

-Seccion "The metabolic label GalNAz is incorporated into both RTgill-W1 and CHSE-214 based in vitro membranes". In figure 2, C and F should be merged and thus statistically evaluate the fluorescence intensity between RT-gill and CHSE214.

-From 348 to 350: Methodology again, it should be moved to that section (modifying it).

-Line 396: "A. salmonicida binds to RTgill-W1 and CHSE-214 cells" should be modified to the ability of the cell lysates to bind to the bacteria. For example: RTgill-W1 and CHSE-214 cell lysates binds A. salmonicida".

Discusssion:

Lines 485 and 487: Change "Aeromonas salmonicida" to "A. salmonicida"

Figures

In general, the resolution of the manuscript images that contain cells is low (even in the downloaded versions). This must be improved to achieve publication of the study.

References: Authors should consider reducing the percentage of self-citations (currently 60%). Of the 45 citations in the text, 27 are from one of the authors involved in this study. Multiple references in the same sentence should be eliminated and others from different groups incorporated, for example in the introduction.

**********

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: No

Reviewer #2: No

Reviewer #3: Yes: Byron Morales-Lange

**********

[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.

Attachments

Attachment

Submitted filename: Reviewers comment_PlosOne.docx

In vitro Fish Mucosal Surfaces  Producing  Mucin as a Model for Studying Host-Pathogen Interactions

PONE-D-23-19772R1

Dear Dr. Lindén,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Maria del Mar Ortega-Villaizan

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

Reviewer #2: 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

Reviewer #2: Yes

**********

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

**********

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: Regarding the cell polarization matter, authors should feel free to include it in the discussion. I was curious about cell morphology and polarization observed during the experiment but did not pretend that it should be deleted. In fact, I encourage authors to deepen this amazing issue in future works.

In regard to the modulation of mucin glycosylation patterns of the in vitro mucosal models, authors address this topic reasonably by ssuggesting gene editing. In fact, since I do not work with salmonid species, my initial question on PAS+ neutral goblet cells in fish gills might have been out of context. I apologize.

In my opinion the M&M section and the provided figures include all necessary details and their quality and contrast are adequate.

Reviewer #2: The authors have incorporated the corrections I made to the manuscript and therefore I have been able to properly understand their work and consider it acceptable for publication.

**********

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

Reviewer #2: No

**********