PONE-D-21-23414

Promoting mechanism of serum amyloid A family expression in mouse intestinal epithelial cells

PLOS ONE

Dear Dr. Hayashi,

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Wendy Huang, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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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: The authors previously published their finding that SAA can be used better for biomarker as well as endoscopic mucosal activity in ulcerative colitis rather than well-used biomarkers, such as CRP. In this manuscript, they aimed to determine the major organs of SAA expression as well as the underlying mechanisms of SAA expression in the GI tract using the DSS mouse model. To improve it, there were several concerns in this manuscript.

Major

I would like to know that the experiment was repeated, especially DSS colitis. If it was repeated, please mention that in the text (Materials and Methods). The error bars of SD was relatively big. Showing each value as a dot must be the best approach for all the readers. Please show all the RT-PCR data in the graphs using dot plots.

Please explain how the authors determined the concentration of these cytokines for the detection of SAAs. Basically, the authors should have had several titrations (dilution) to optimize the experiment. The concentration which showed saturated expression of SAA must not be used.

In this study, the best option was to use human organoids to check the SAAs expression; even the authors used the DSS animal model in Figure 1. Please consider doing this experiment. So far, only using cell line and mouse organoid are very weak.

The biggest concern (issue) in this paper was the analyzing method for the RT-PCR data. What does “normalized SAA expression” mean? All the data looks like a relative expression somewhere. Each data sets have 1.0 in somewhere. Did the author set one data set 1.0, and then relative levels were presented? In the method section, it was mentioned that samples were normalized for (must be by) ACTB expression in each organ and organoid). This was the right way. However, the expression of SAAs was quite low. For example, if you have just 0.00001 for SAA1 in untreated, then they should not use the relative expression. If the quite low value was set as 1.0, then the obtaining relative expression value must be really high, and all the samples are messed up and cannot be compared. Based on this point, all the data did not completely make sense. The authors must learn this and re-analyze all the RT-PCR data correctly.

Minor:

It would be interesting if the author shows the expression levels of cytokine receptors using organoids (ideally both mouse and human organoids).

Please show fluorescent minus one for the immunohistochemistry data for Figures 3C and 4B)

Please provide the information about 5-ASA. Need to explain in more detail why the authors used this drug? What are the differences between NFkb inhibitors (BAY11-7082) and 5-ASA? The author mentioned that In previous reports, NF-κB was suppressed by 20–40 mM 5-ASA in HCT-116 and Caco-2 human colon cancer cell lines. Then what was the purpose of the use of 5-ASA? It sounds just like a very similar experiment.

Reviewer #2: In this manuscript, Wakai et al. present data which leads them to conclude that the expression of serum amyloid A (SAA), in particular SAA1, is regulated by the NF-kB signaling pathway through toll-like receptor (TLR) stimulation in ulcerative colitis (UC) mouse model. SAA is significantly expressed in the intestinal track of UC mouse model and a good inflammatory biomarker. However, the mechanism underlying SAA expression remains to be. Using a dextran sulfate sodium (DSS)-induced enterocolitis mouse model, the authors study SAA expression by various cytokines and TLR ligands. The pre-treatment with an NF-kB inhibitor, BAY11-7082, dampened the expression of SAA mRNA leads the authors to conclude that TLR stimulation induces SAA via NF-kB. This is an interesting topic; however, I found some of the data are relatively weak to support the conclusion with the following major concerns.

1) In Figure 3, the authors showed both LPS (through TLR4) and flagellin (through TLR 5) induced SAA1 and 3’s mRNA expression. However, the NF-kB inhibitor BAY11-7082 treatment was only done in flagellin (Fig 3B) but LPS (Fig 3A) stimulation. LPS is known to activate both canonical and non-canonical NF-kB signaling. Do the authors have any idea/data suggesting with pathways it is?

2) After pre-treatment with BAY11-7082, the NF-kB activities was not monitored. BAY11-7082 was known to inhibit IkBalpha phosphorylation, as well as directly inhibit functions on the NLRP3 inflammasome by blocking the sensor's ATPase activity. Therefore, showing the IkBa’s protein degradation and NF-kB nuclear translocation is necessary to support the inhibitory effects of SAA expression was through NF-kB signaling.

3) If the expression of SAA is regulated by NF-kB, is SAA a direct target gene of NF-kB or it is a secondary effect of NF-kB activation? If it is a direct target gene, what is the kB binding site(s)?

4) The authors only showed SAA mRNA expression level upon different cytokines and TLR ligands stimulation, how about the SAA proteins’ expression level? It will be interest to see the protein level as well.

5) In general, all the gene expression figures have huge error, especially Figure 1 and 2; some panels the error bar is larger than the bar graph itself. Figure 1A does not have p-value either. Box & Whisker plots with overlay of individual data points are needed to show the significance of the data.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-23414R1Promoting mechanism of serum amyloid A family expression in mouse intestinal epithelial cellsPLOS ONE

Dear Dr. Hayashi,

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 Feb 20 2022 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,

Wendy Huang, Ph.D.

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.

Additional Editor Comments:

In the revised manuscript, Wakai et al completed additional experiments and revisions to address many of the reviewers’ main concerns. There are three remaining minor issues that the authors should address:

In response to Reviewer #1, point #2, the authors stated that “We examined the expression of SAA with these cytokines in a concentration-dependent manner… and found no significant difference”. No data figure were shown. The authors should at least include in the revised Result text section the exact range of the concentrations tested that yield the negative result. For example, “IL-1β (1, 100, 1000 ng/mL) and IL-23 (5, 50, 500 ng/mL) were also tested and similarly returned no significant induction of SAA in the intestinal organoid culture.”

Related to Reviewer #2, point #1: On page 12 of the text, the text stated “Figure 3. Flagellin and lipopolysaccharide (LPS) promote SAA1-4 expression in small intestinal organoid via NF-kB”. However, Fig 3 only included NF-kB inhibition results in the Flagellin condition, but not the LPS treatment. Therefore, Reviewer #2 asked NF-kB inhibitor to be tested on LPS treated cells. As the authors noted on page 59 of the point-by-point, result of this additional experiment suggest LPS induced SAA expression is NF-kB independent (data not shown). Given this new information, the authors should consider moving the Figure 3A(LPS) panel to a separate figure and take “LPS” out of the Figure 3 title/legend etc – the current writing would give readers the wrong impression that LPS induced SAA is dependent on NFkB. Instead, LPS+NFkB inhibitor results should be shown under a separate figure - texts in the results section should include description of the negative result (e.g. In contrast to the flagellin pathway, LPS induced SAA expression is NF-kB independent.). Discussion should be revised to comment on potential non-NFkB dependent pathway that maybe involved in LPS induced SAA expression subject to future studies.

Related to the author’s response to Reviewer #2, point #2: For Figure 4B, western experiment missing loading control (e.g. beta-actin or GAPDH). Figure legend needs to state how many time this experiment had been performed independently showing similar results. Original un-cropped gel images/scans should be displayed.

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Reviewers' comments:

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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.

Promoting mechanism of serum amyloid A family expression in mouse intestinal epithelial cells

PONE-D-21-23414R2

Dear Dr. Hayashi,

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Kind regards,

Wendy Huang, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments: