PONE-D-24-10014Prostaglandin D2 receptor 2 downstream signaling and modulation of type 2 innate lymphoid cells from patients with asthmaPLOS ONE

Dear Dr. Hohlfeld,

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 manuscript is focused on the "understanding of DP2 initiated ILC2 activity" (line 43). However, PGD2 and its metabolites were shown to activate other receptors, particularly PPARgamma and TP receptors, not to mention DP1. Authors dismiss the potential role of these other signaling pathways in the reported effects based on its "anti-inflammatory" (line 59) profile and the lower affinity of the metabolites (but not PGD2) compared with the affinity to DP2. Without selective DP1/TP/PPARgamma agonism/antagonism and/or expression data it is hard to dismiss offhand a potential role for this/these receptor/s in PGD2 and its metabolites on the activation of ILC2s. It is noteworthy that an essential cooperation between DP1 and DP2 in PGD2-induced LTC4 generation by eosinophils was shown before (https://doi.org/10.1111%2Fj.1476-5381.2010.01086.x). TP receptors have also been shown to activate in response to PGD2 (https://doi.org/10.1152/jappl.1989.66.4.1685, https://doi.org/10.1183/09031936.00061614) (although i don´t have any knowledge that PGD2 metabolites may have a similar effect). PPARgamma was also shown to be activated by PGD2 metabolites, although at a much higher concentration. I tend to agree that the reported effects are probably mediated by DP2, but the other possibilities should still be probably included in the introduction and most definitely in the discussion. Authors should also take into account the expression that seems to indicate that DP1 is not expressed by ILC2s while TP and PPARgamma are expressed. Authors may reconsider the assertion that differences in response to different agonists are attributable to how "ILC2 respond non-uniformly to PGD2 metabolites" and consider other receptors potentially involved and whether the impact of DP2 antagonist is due to inhibition of DP2 signaling, unopposed signaling by other receptor.

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Academic Editor

PLOS ONE

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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

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

Reviewer #1: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: 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: Thank you for the opportunity to review this manuscript. The work elegantly describes transcriptional differences in ILC2 from asthmatic patients treated with a DP2 antagonist and different PGD2 metabolites. This is relevant to the field as this lipid mediator plays an important role in the exacerbation of asthma, and has been thoroughly described as an important activator of ILC2. Overall, the paper is well written, the methodology is well executed, and the gates to identify ILC2 by FACS seem appropriate and well defined. However, some issues must be addressed by the authors.

- Major issue:

1. Delta12-PGJ2 and 15d-PGJ2 are both potent natural agonists of PPARgamma, and have been better described as such. Considering how the treatment with these specific metabolites ±fevipiprant showed strong effects that were rather different from the other treatments, it is not negligible that some of these observed effects are instead mediated by PPARgamma instead of DP2. I would strongly suggest the Authors perform one additional experiment using a PPARgamma antagonist, such as GW9662, to exclude this possibility. Given the known limitations of working with ILC2 and human samples, if this experiment is not feasible, I would suggest a directed analysis of RNAseq data, focusing on the PPARgamma signaling pathway, as a mean to assess whether this pathway is being in fact activated or not.

- Minor issues:

1. There have been some papers in the past decade describing the importance of some of these metabolites such as 15d-PGJ2 in murine models of asthma (doi: 10.3389/fimmu.2017.00740), and of PPARgamma and the activation of ILC2 (doi: 10.1038/s41385-020-00339-6; doi: 10.1038/s41467-021-22764-2). I would suggest the Authors consider mentioning these papers in the discussion, as I think it would benefit greatly from them.

2. In the discussion, lines 364 and 365, the Authors mention that “TCR expression on ILCs has not been described so far”. This is not entirely true, as there is at least one major report of such (doi: 10.1182/bloodadvances.2020002758); in fact, abortive gammadeltaTCR recombination has been taken into account as one likely theory for the origin of ILCs and their close relationship to T cells (doi: 10.3389/fimmu.2022.957711).

3. In the introduction, I would suggest the Authors to better clarify the findings of their previous publication. At times it felt like referring back to their previous work was fundamental to understanding the rationale of this paper. It is understandable that this work is a follow-up; however, minor reworks can help this body of work stand on its own, and less dependent on previous papers.

Overall, I think the paper is well written and offers interesting insight not only on the biology of ILC2 in asthma, but also on prostanoids and their importance on allergic inflammation. Other issues, such as the lack of data from healthy controls, the smaller sample size, and the fact that fevipiprant has been removed from clinical trials, have already been properly addressed by the Authors in the work itself.

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Reviewer #1: Yes: Lukas Bolini

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Prostaglandin D2 receptor 2 downstream signaling and modulation of type 2 innate lymphoid cells from patients with asthma

PONE-D-24-10014R1

Dear Dr. Hohlfeld,

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.

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Kind regards,

Bruno Lourenco Diaz, Ph.D.

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

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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

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

Reviewer #1: Yes

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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

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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

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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: The author addressed all the comments made in the previous round of review and I am satisfied with the implemented changes in the current version of the manuscript.

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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: Lukas Bolini

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