PONE-D-19-28373

Interleukin-10 contributes to PGE2 signalling through upregulation of EP4 via SHIP1 and STAT3

PLOS ONE

Dear Dr. Mui,

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.

In particular, Rev 1 made several critical comments that have to be addressed before the manuscript can be reconsidered for publication including consistency of experimental systems (this requires at least additional controls and potential revision of conclusions), use of adequate controls for determining effects of Il10 on EP4 and role of LPS in that phenomenon, and, proper normalization of western blots as well as validation of nuclear localization-perturbing constructs. 

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Samiea et al describes a potential mechanism by which the anti-inflammatory activities of IL-10 are manifested in macrophages. The authors find that IL-10 induces upregulation of the prostaglandin E2 receptor EP4 and this requires both STAT3 and SHIP1. Using a variety of knockdown systems, the authors present data suggesting that IL-10-mediated upregulation of EP4 and subsequent EP4 signaling is required to elicit the anti-inflammatory activities of IL-10. While the manuscript is presented in a clear and logical fashion, the quality of the some of the figures makes the data difficult to interpret. Furthermore, there are important controls missing from some of the figures.

Specific comments.

1. The data presented in figure 1 suggests that IL-10 upregulates EP4 mRNA in peritoneal macrophages in the presence of LPS, and this is dependent on both SHIP1 and STAT3. The data in figure 2 suggests that IL-10 induces EP4 protein in a macrophage cell line and a bone marrow-derived macrophage line derived from SHIP1 knockout mice that has been reconstituted with SHIP1, and this is independent of LPS. Is the upregulation of EP4 mRNA in figure 1 also independent of LPS? The use of the different cell lines in the different figures makes the data difficult to interpret since IL-10 can have differential effects on different cell types. The authors should be consistent in the systems they use to test theit hypotheses.

2. Since it appears that LPS is not required for induction of IL-10-mediated EP4 protein induction, effects on (figure 2), the authors should consider including looking at the effects of IL-10 alone in their efforts to elucidate the IL-10/EP4 signaling axis.

3. How are the authors quantitating STAT3 activation in the STAT3 knockout cells in figure 4C? There is no STAT3 expressed in those cells.

4. The authors should show that the different point mutants of SHIP1 used for the data in figure 5 are indeed defective for nuclear translocation.

5. From figure 5B, it appears that LPS and LPS + IL-10 decrease the expression of the K331A mutant SHIP1. This needs to be clarified. Is it possible this decrease in the K331A SHIP1 level is sufficient to impair the induction of EP4?

6. The total levels of STAT3 are missing from figure 5 which should be used to normalize activation of STAT3, rather than Actin. Similarly, the levels of CREB, p85-PI3K and p70 S6K are required for normalization of the concomitant phosphor proteins in figure 6. The data is difficult to interpret without these important controls.

7. The data presented in figure 6 suggests that EP4 is required for IL-10 activity. The use of an alternate method to verify this would strengthen the manuscript. Macrophages from EP4 knockout mice or an EP4 antagonist could be used to confirm these data.

8. The western blots for EP4 in figures 3 and 4A are not clear, making it difficult to interpret the data.

9. There is no point to including an Actin loading control in Figure 4 if it is not used as a normalizer for quantitation.

Reviewer #2: Interesting study well supported by the experimental data, adding to the existing information on PGE2 and IL-10 Rs. The authors wrote aclear report on their data which add to the existing information on IL-10 signaling including now the upregulation of EP4 in macrophages

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Reviewer #2: No

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Interleukin-10 contributes to PGE2 signalling through upregulation of EP4 via SHIP1 and STAT3

PONE-D-19-28373R1

Dear Dr. Mui,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Michal Hetman

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

**********

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: (No Response)

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Reviewer #1: No