PONE-D-21-14708

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

PLOS ONE

Dear Dr. Elzinga,

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.

The major issues relate to the presentation of protein expression data, quality of figure(s) or including the rationale and discussion for studies.  Rest of the issues are relatively minor and can be addressed to further strengthen the manuscript.

Please submit your revised manuscript by Jul 31 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,

Pradeep Dudeja

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. 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.

[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: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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

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 is an interesting study looking at the differential gene expressions in intestinal epithelial cells in static and semi-wet interface with mechanical stimulation (SWMS) culture conditions, particularly in terms of mucus production. The manuscript is written very well and the results are presented appropriately. I have only minor comments.

* Please include the relevance and purpose of mechanical stimulation in the introduction.

* It is not clear if the results represent more than one experiment.

* The quality of Fig.4 is not helpful for the readers. Please replace them. The authors may consider adding a simple cellular stain such as crystal violet or a nuclear dye for better visualization.

* Please include SGLT1 (SLC5A1) in the discussion on glucose metabolism.

* TEER studies can be supplemented with flux of paracellular probes such as FITC-dextran.

* There is scope for shortening the discussion; it is lengthy in the present form.

* Considering the manuscript’s focus on characterizing the mechanisms of mucin production, it is worth demonstrating the mucus layer morphologically/ e.g. by immunohistochemistry in static versus SWMS cultures.

* I missed cytoskeleton-associated changes which might be important in view of mechano-stimulation.

Reviewer #2: The manuscript by Elzinga et al. adds on to previous knowledge regarding the mucus production of HT29-MTX-E12 cells grown under semi-wet interface with mechanical stimulation. Although the work provides insights into this model for in vitro culture to study mucus, the following needs to be addressed to improve the overall impact of the work.

Major concerns

1. The authors bring up a point that this model capture human intestinal (colonic) mucus in vitro, however, the levels of other MUCs were significantly low in this model. Does this occur in vivo? Then why do the authors think this will be physiologically relevant? Discuss and explain the drawbacks etc.

2. Figure 1 A, how about MUC1 and MUC4? Please include other MUC proteins as well. Why were dot blots conducted instead of standard western blot? There should be enough protein for this assay please explain why. Also include western blot results which is normalized to a loading control such as GAPDH and Actin HSP etc. with a representative blot and densitometric analysis with statistics conducted of at least n>4. Please include at least n=3 when plotting data (Figure 1B) in addition.

3. It would be nice if the authors could supplement Figure 3 with FITC dextran data in addition to the TEER data.

4. Figure 4, what do the authors need the reader to observe? The photograph of the trans-well membrane provides no information pertaining to the amount of mucus etc. Please use a staining to show the amount of MUC2 or goblet cell mucins by using PAS staining immunofluorescence etc. Also, always add a scalebar to all microscopy images.

5. It wasn’t clear, which was the mechanism behind increased MUC2 production in HT29-MTX-E12 cells grown under SWMS conditions? The authors have not speculated this aspect. For example. The transcription factors were unchanged however, there was an increase in IL-33 production. Could this be one of the mechanisms? Please add all this and improve the discussion section

6. Please summarize key findings of the paper in the final paragraph of the introduction section.

7. Please remove MUC2/MUC2 etc. from the graphical abstract.

Reviewer #3: In this article, Elzinga et al. examined the Semi-Wet interface with Mechanical Stimulation (SWMS) culture conditions with the commonly used HT29-MTX cells. This culture model was selected as it represents a simple, low cost method to examine intestinal mucus production. HT29-MTX cells grown in SWMS conditions exhibited elevated levels of MUC2, decreased levels of MUC5AC and MUC5B and alterations in several transmembrane mucins compared to cells cultured conventionally. These cells were enriched for cell cycle and DNA replication related pathways. Interestingly, NOTCH1 and ATOH1 were both significantly upregulated and TFFs (1,2, 3) were significantly downregulated by SWMS conditions. The authors also noted decreased pH and increased glucose consumption with SWMS conditions. With this work, the authors have provided mechanistic insights into mucus production in HT29-MTX cells using the SWMS culture system. Overall, the manuscript is well written and follows a logical flow. The conclusions are well supported by the findings and the discussion is appropriate. As a result, I have only minor comments.

1. Line 43: This is a very broad statement and adding more references in support of this claim would strengthen the introduction.

2. Line 49: Paneth cells which produce lysozyme are found in the small intestine, but not normally in the colon (with the exception of IBD (PMID: 11851832). As result, the statement that colonic mucus contains lysozyme is misleading. Please remove lysozyme from the sentence.

3. In Line 65, the authors state that organoids “are supposed to resemble the in vivo colonic mucosal layer more closely in terms of mucus composition”. The term “supposed to” seems vague; please remove “supposed to” since the MUC2 in organoids does resemble colonic mucus in terms of protein and glycosylation pattern.

4. Supplemental Figure 1 and 3 depict important findings and the manuscript would be improved if they were incorporated as main figures. Additionally Figure 4 seems more appropriate as a supplemental figure.

5. The changes in ion transporters is intriguing. NHE3 (SLC9A3) has been shown in mice to be the major regulator of intestine pH- was NHE3 altered in the HT29-MTX AI cells?

6. Were there any changes in other major goblet cell markers (AGR, CDX2, MEP1β, FCGBP, etc.)?

7. Although the main focus of this paper is on a simple model of the intestinal epithelium (HT29-MTX cells), the discussion would benefit from inclusion of how SWMS conditions could be employed in organoid studies to create more robust models in this system as well.

**********

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: 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.

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

PONE-D-21-14708R1

Dear Dr. Elzinga,

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,

Pradeep Dudeja

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: (No Response)

Reviewer #2: Yes

**********

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

Reviewer #1: (No Response)

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: (No Response)

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: (No Response)

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: (No Response)

Reviewer #2: All concerns have been addressed and the authors have modified the revised version accordingly.

The revised manuscript is 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