-->PONE-D-26-02176-->-->The role and targeting potential analysis of angiogenesis-related target THY1 in DSS-induced acute colitis in mice-->-->PLOS One

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Additional Editor Comments:

Both the reviewers raised multiple issues that need to be addressed in the revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: Partly

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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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

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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 manuscript entitled “The role and targeting potential analysis of angiogenesis-related target THY1 in DSS-

induced acute colitis in mice” explores the role of THY1 in two aspects of DSS-induced colitis: alterations in colonocyte biology and changes in macrophage phenotypes. While the study apparently focuses on how THY1 regulates the phenotypic alterations of these cells that lead to colitis pathology, there are specific points which are not clear to me in terms of logic, as outlined below.

Major points

1- How well-established is the cell line-based model of IBD (lines 159-161)? Authors need to cite references.

2- How could the authors guarantee that the lentiviral vector-mediated delivery of the constructs could solely be targeted to colonocytes and not any stromal or immune cells around them? This is particularly important as THY1 expression on stromal cells has a crucial role in colitis pathogenesis (reference: https://doi.org/10.4049/jimmunol.212.supp.1326.5032).

3- The 2nd point of the results (lines 303-348) only shows that THY1 causes definite changes in macrophage phenotypes parallel to the aggravation of DSS-induced colitis in mice. How could the authors draw a causal inference in the statement “THY1 promotes the pathological process of DSS-induced colitis through inhibiting M2 macrophage polarization?” To establish such causal connection, authors need to experimentally deplete macrophages or alter macrophage phenotypic states under THY1-OE or THY1-silenced conditions, and repeat the same experiments in vivo.

4- In the same point, the authors seemingly check several different aspects (cytokines, ROS, angiogenesis markers) related to macrophages. However, there is no definitive proof that the changes in these markers due to THY1 manipulations are solely, or at least majorly, related to their altered output from macrophages in vivo. In addition, why they check so many aspects together without any apparently clear hypothesis is not understandable.

5- Again, in the coming sections, authors assess colonocyte apoptosis and angiogenesis marker levels without any clear connection to the previous sections. The entire story looks like an ensemble of experiments without any proper logical link among them.

6- Although the authors check multiple phenotypic effects of THY1 manipulations on colonocytes, how each of them affects colitis has not been checked. So the entire set of findings by large stays correlative.

7- Why do the authors check ROS-related phenotypes (ROS, MDA, GSH etc) in cell supernatants? It would have been more physiologically relevant if they had done the experiment with cell homogenates/lysates.

8- None of the experiments involve DSS-conditioning of the THP-1 cells, and only involves co-culturing them with colonocytes of different phenotypes. This violates the physiology of colitis, where macrophages also encounter DSS, and are likely affected by it to some extent as well (reference: https://doi.org/10.1691/ph.2016.6688).

Minor points

1- It would have been preferable if the authors would have shown the time kinetics of THY1 expression in colon along the course of DSS treatment in mice and afterwards.

2- For apoptosis assay, how could the authors resuspend the cells in 1 microliter binding buffer? I suspect it is a typing error, and needs to be taken care of. Furthermore, didn’t they use any strategy for gating live/dead cells differentially in their flow cytometry data for macrophages?

3- If colonocytes were the target, why did the authors opt for lentivirus injection intravenously?

4- Why didn't the authors opt for showing the intestines from mice groups photographically? It might have been more convincing than putting up a graph.

5- A much better way to represent the data in fig. 3 would have been to gate on the CD11b+ cells, followed by the assessment of CD86 and CD206 simultaneously in them.

6- For cleaved caspase-3 immunoblots, total caspase-3 level needs to be demonstrated as well.

7- Was VEGF levels quantified with or without Golgi-stop/Golgi-plug treatment?

8- For mice experiments, plotting individual data points is always preferable. In addition, it is better to plot SEM rather than SD for showing data corresponding to biological replicates.

9- Did the authors perform normality tests before doing parametric statistical tests?

10- In the cases where authors have claimed “THY1 does so..” and on, should be reframed as “colonocyte-expressed THY1 does so…” or “THY1 on colonocytes does so….”. This will specify the findings.

Reviewer #2: The study by Pengliang Zhang et al claim to identify a novel role of THY 1 in DSS induced colitis. They found out that THY1 promotes inflammatory angiogenesis that exacerbate the disease. The methods and models employed enrich the study and the data gathered collectively reinforces the key hypothesis of the study. The hard work is commendable. However some clarifications are needed.

1.) The study doesn’t appear to be unique and the first of it’s kind as evident from the sentence “In this study, we first revealed the crucial role of the angiogenesis-related target THY1 in the development of acute colitis”. One similar study “The Journal of Immunology, Volume 212, Issue 1,1326_5032, https://doi.org/10.4049/jimmunol.212.supp.1326.5032” published in 2024 had a similar objective and conclusion. Many more can be cited. Hence, it appears that the authors reinforces and substantiate the earlier studies.

2.) Use of a variety of techniques and models often overwhelm the readers. The design could have been much simplified and succinctly presented to make reading and assimilation easier.

3.) Although the authors state the use of AI for improving English, the English still need ample revision.

4.) In the co-culture experiment, choice of human cell lines NCM460 and THP-1 is not accurate. A co-culture of Mice primary intentional epithelial cells and peritoneal macrophages would have been more appropriate to establish and validate the in vitro model

5.) What is the natural ligand for THY1 and are there any inhibitors of THY1 that may have been used alongside or instead of sh-THY1?

The study presents a detailed evaluation of the effect of THY1 in colitis, which needs substantial refinement for enhanced clarity in light of the points raised.

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Reviewer #1: Yes: Soumyadeep Mukherjee

Reviewer #2: No

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The role and targeting potential analysis of angiogenesis-related target THY1 in DSS-induced acute colitis in mice

PONE-D-26-02176R1

Dear Dr. Zhang,

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.

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Kind regards,

Subhasis Barik

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

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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: I Don't Know

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-->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

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-->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: All comments from the previous round have been addressed. I recommend the manuscript for acceptance in its current form.

Reviewer #2: The revised manuscript is fair enough for acceptance. The authors have responded to the reviewer comments with clarity.

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Reviewer #1: No

Reviewer #2: No

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