PONE-D-20-23391

Prenylated quinolinecarboxylic acid compound-18 prevents sensory nerve fiber outgrowth through inhibition of the interleukin-31 pathway

PLOS ONE

Dear Dr. Ogura,

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 find enclosed the expert reviews on your manuscript. Although the results presented are considered important in the field, better validation of the inhibitory effect of PQA-18 on PAK2 is required. In particular some key results should be compared with other PAK-specific inhibitors, which are available. There are issues with regard to the presentation of results that need to be addressed. I hope you will be able to carry out the additional experiments in the time requested.

Please submit your revised manuscript by Nov 16 2020 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Ed Manser, PhD

Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: Yoshimi Homma’s lab had previously reported that PQA-18, a derivative of Polysphondylium pseudo-candidum isolated small molecule Ppc-1, acts as an immunosuppressant and a PAK2 kinase inhibitor, could alleviate atopic dermatitis in mouse model (ref. 21). In the current study, the authors are trying to understand the mechanism of action of PQA-18 in relieving the skin defects in atopic dermatitis.

The authors chose to examine the IL-31/IL-31R� pathway and provide data to show that PQA-18 decreased the cutaneous sensory nerve fibre density by inhibiting the expression of IL-31 receptor alpha in mouse skin. In cultured neuronal cells, PQA-18 inhibits IL-31 induced sensory nerve fibre outgrowth and neurite outgrowth by blocking IL-31R� activation of the PAK2/JAK2/STAT3 pathway. Lastly, PQA-18 selectively blocks PAK2 interaction with GIT2 and �-PIX, which are thought to activate PAK2.

That PQA-18 acts as a potent immunosuppressant and effectively improves the skin pruritus condition in mouse AD model are potentially important findings. Experimental data for that PQA-18 inhibits IL-31/IL-31R� pathway and prevents cutaneous nerve fibre growing is convincing. But the molecular mechanism described for PQA-18 inhibiting PAK2 kinase is less convincing. There are a several issues which the authors need to address.

Major points.

1. The authors believed that PAK2 is an important target of PQA-18 and based on MS data in Fig.7 they suggest that PQA-18 inhibited PAK2 activation by preventing its interaction with GIT2 / �-PIX. However more direct evidence is required to demonstrate PQA-18 blocks PAK2 interaction with �-PIX (GIT2 is likely indirect) using a direct binding assay.

2. Since both PAK1 and PAK2 are expressed in neuronal cells (PAK2 being ubiquitous) the authors should clarify if PQA-18 has similar effects on PAK1 kinase.

3. In Fig.2 (compare IL-31R� images of vehicle vs PQA-18) the data does not support the claim that PQA-18 inhibits IL-31Ra expression in cutaneous nerve fibres. Cf. as stated on page 2 “PQA-18 alleviates cutaneous nerve fibre density and the expression of IL-31 receptor α (IL-31Rα) in the skin of Nc/Nga mice.” and page 11 “These results suggest that PQA-18 ointment, but not FK506 ointment, alleviates excessive cutaneous nerve fibre density and expression of IL-31Rα in the skin of Nc/Nga mice”.

4. Do the authors think that PAK2 controls IL-31R� gene expression in mouse skin?

Minor points:

1) Images of “lesional-” panel in Fig.1A and B were adjusted brighter than other panels. All images should be taken under the same exposure condition. Figure 1 legend does not introduce what are blue channel and green channel representing.

2) Figure 1A and B, showed FK506 caused more than 40% reduction of PGP9.5+ and IL-31R� fibre density, why do the authors think this is not significant? (see page 11, lines 219-220 and 225-226)

3) Figure 1 legend: *p<0.01 should be *p<0.05

4) On page 18, this sentence is not clear. “PQA-18 significantly suppressed the development, while PQA-18 alone did not affect sensory nerve development.”

5) The authors need to clarify why DRG nerve fibre length in PQA-18 +rIL-31 treated DRG cells are shorter than PQA-18 alone and vehicle control in Fig.2 (see chart at bottom).

6) The authors need to explain why in Figure 4 PQA-18 inhibition of PAK2 and JAK2 phosphorylation is not dose dependent (over the conc used) however in Figure 5 PQA-18 inhibition of the neurite outgrowth is dose-dependent.

7) On page 9, line 169, addition of 40 pmol siRNA is in what volume of medium? It is maybe better to express as concentration (similar issue in Fig.6).

Reviewer #2: 

Ogura et al., (2016) showed that the prenylated quinolinecarboxylic acid derivative PQA-18  suppresses immune response, likely through inhibition of PAK2.  This was based on studies showing that there were changes in cofilin phosphorylation upon PQA-18  treatment (which could be via effects of PAK2 on LIMK1).  However the data presented then was not equivocal that  PQA-18  inhibits PAK2  directly, although other studies in KO mice to point to PAK1 and PAK2 regulating the immune system.

In this new study it is noted that PQA-18 improves the skin pruritus condition in a mouse model.  In order to demonstrate that PQA-18 works by inhibiting PAK2 (and likely PAK1) some additional experiment is needed.  In the neuroblastoma cell line etc.. it would be important to validate their 'PAK2 inhibitor' a well characterized ATP mimetic of  which FRAX are the best characterized.  It should be noted that IPA-3 does not work in vivo and should not be used.

The effects of PQA-18 on the IL-31/ IL-31R� pathway are quite convincing, and maybe clinically important.  I highlight below some issues that need to be considered.

(1) The treatment with PAK2 SiRNA shown in Figure 6 leads to profound changes in neurite outgrowth while the PAK2 KD by western is not that convincing (A).  To resolve whether the modest changes in PAK2 levels the authors need to use Frax 597/ 1036, which will strongly suppress the p-PAK Ser141 signal (which can be compared to PQA-18).

(2)  In Fig 3C  the authors show Stat3 western blots.  However since with no IL31 (lane 1)there is no observed pStat3 signal (bands) they need to present a different panel (ie one of the other blots which has been used to obtain average but not shown). 

(3)  Table 1. Data on the PAK2 interacting proteins should be more complete, and the 'top' MS derived set should be listed according to either enrichment relative to control or intensity / sequence coverage.

(4)  The MS data which indicates that GIT2 and aPIX are present in the PAK complex is interesting (Fig 7). Based on current models, if PQA-18 inhibits PAK1/PAK2 directly one would expected that this would stabilize the PIX complex.  So this new data is interesting but should be supported with PAK2 IP & western data (for say aPIX).

(5)  In the raw data the p-JAK shows 2 strong bands which seem to be co-regulated.  What is the presumed identify of the top band?

(6)  In Figure 4 the anti-PAK2 data shows strong band at ~ 60 kDa with no other background bands.  By contrast the anti-PAK2 in Figure 6 shows several background bands - why is there such a large difference in the WB? Are different Abs used?

(7) The identity of the various antibodies used for analysis should be better defined in the figure legends (for example Fig 4) and ideally on the figures themselves.   cf.  The specifics of sites for phosphorylated sites in STAT3, JAK2, and PAK2.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-23391R1

Prenylated quinolinecarboxylic acid compound-18 prevents sensory nerve fiber outgrowth through inhibition of the interleukin-31 pathway

PLOS ONE

Dear Dr. Ogura,

Thank you for resubmitting your updated  manuscript to PLOS ONE.  Apologies for the delays over the holiday period. The reviewers have indicated that they consider their comments have been addressed through the addition of new data, new figures, updated legends, and substantial correction of text.

There is one key issue outstanding regarding the new Figure 6 that will require the authors expanding/rewriting that section, namely that they have used Frax597 at a sub-optimal concentration.  The reason for doing this might be to avoid 'off-target' effects of Frax597 which are documented (to avoid affecting other kinases).

In Figure 6 I note you observe ~ 50% inhibition of PAK2 pS141 in their cells using 1 uM Frax597 with more substantive effect in cell assay. Thus the author should have considered 2 and 5 uM Frax597 doses. Please revise the MS to reflect the reasons why the 1 uM maximum (50% inhibition) was chosen for the neurite outgrowth assay (and p-PAK2 tested I think after for 30 min, rather than longer times, cf. 24/48h).

Admittedly the literature is a mess with respect to the proper concentration and timing to block PAK activity in cells. While the in vitro Ki is ~ 10-50 nM the effective dosing of cells with Frax579 usually requires 1-5 uM (ie 100 times more).

Licciulli et al., (2013) which the authors quote indeed indicates that pS141/4 signal was suppressed the cellular inhibition in SC4 cells in the range 0.5 -1 uM after 2h treatment. However this may reflect sensitivity of SC4 or a purer source of Frax579. In a 2015 paper (Oncotarget. Jul 10;6(19):16981-97) the authors use a 2h treatment of 2 uM Frax597 to effectively block both pS144 /141 signals.

Please submit a revised version of the manuscript that addresses the this point.

Please submit your revised manuscript by Feb 25 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

We look forward to receiving your revised manuscript.

Kind regards,

Ed

Edward Manser, PhD

Academic Editor

PLOS ONE

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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: Authors have addressed most of the concerns I raised earlier.  In my opinion, provided the data on PAK inhibition in Figure 6 is better explained, the manuscript after minor revisions is therefore publishable.

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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

[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.

Prenylated quinolinecarboxylic acid compound-18 prevents sensory nerve fiber outgrowth through inhibition of the interleukin-31 pathway

PONE-D-20-23391R2

Dear Dr. Ogura,

Thank you for the changes presented in the new version of the MS.  I understand the reasoning for using the FRAX597 at lower conc. and the inhibition profile fits with published data in this cell line.   We’re pleased to inform you that your manuscript will be formally accepted for publication once it meets any outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing any technical 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,

Ed Manser, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: