PONE-D-21-39358Granzyme K initiates IL-6 and IL-8 release from epithelial cells by activating protease-activated receptor 2PLOS 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

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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?

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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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: The manuscript by Kaiserman et al. “Granzyme K Activates PAR2” assesses a role for GzmK in PAR2 activation. Previous studies suggest a role for GzmK in PAR1 activation and proinflammatory cytokine release. The present study proposes a novel role for GzmK in PAR2 activation. Interestingly, although cleavage occurs at the canonical site similar to other trypsin, Ca2+ mobilisation is not initiated but rather induces ERK phosphorylation and b-

arrestin recruitment in epithelial cells. Historically Gzms have been thought to function exclusively as perforin-dependent, cytotoxic proteases.The present manuscript supports emerging evidence for a non-cytotoxic, extracellular roles for granzymes. A number of suggestions are listed below for improvement:

Major:

1. Introduction: Some key citations are missing eg. p4: “GzmK cleaves and activates PAR1 on endothelial cells and fibroblasts (Sharma et al., 2016; Turner et al., 2019)” should be amended to include Cooper et al., 2011. This should also be included in first line of Results “Previous studies have indicated that GzmA and GzmK can cleave and activate PARs (Suidan et al., 1994; Sower et al., 1996b; Sharma et al., 2016) given its relevance to the present article. Recent articles in Immunity, 2021 suggesting a role for GzmK in inflammaging as well as the recent paper citing a role for extracellular GzmB in maternally-transferred childhood asthma citing a mechanism involving PAR2 published in J Clin Invest (Qian et al, 2020) should also be mentioned possibly in the intro and/or discussion.

2. It is unclear as to why GzmB-mediated PAR2 activation was not explored given the high impact study published last year in the Journal of Clinical Investigation by Qian et al suggesting a key role for extracellular GzmB-mediated PAR2 activation of airway epithelial cells as the mechanism as to how maternal exposure to diesel exhaust particles predispose offspring to allergic airway disease.

3. Results are well-described although it is unclear as to why GzmB was dropped after PAR1. GzmB was also shown to induce IL-8 release via an unknown mechanism (Hiroyasu et al, Nature Comm, 2021). Given that GzmB was not assessed throughout the manuscript, it may be worth focussing exclusively on GzmA and K and dropping the GzmB data as it is incomplete. Alternatively, adding GzmB throughout for PAR2/IL6/8 studies could be added to give a more complete picture. The latter would require substantial revision.

4. Discussion Line 3 states “GzmA and GzmK cleave both PAR1 and PAR2, while GzmB cleaves only PAR1.” This contradicts the study by Qian et al (J Clin Invest, 2020) in which a substantial amount of evidence would suggest otherwise in a clinically-relevant model. This must be supported by data and a strong discussion if disputing this work given the impact. I would suggest removing the GzmB PAR2 work and any text dismissing GzmB-mediated PAR2 activation as this was not adequately assessed in the present manuscript.

Minor

1. Methods: Please include number of replicates where relevant.

2. Results: ‘Cleavage of full-length PAR1 and PAR2 by GzmA and GzmK’: Literature reports a sharp decrease in activity of the NanoLuc system below pH 8, whereas Gzms optimally function around pH 7. A brief mention of the potential limitations should be discussed.

3. Discussion: “In lung epithelial cells, GzmK treatment leads to the release of proinflammatory cytokines IL-6 and IL-8, but not TNFα or IL-1β, via a PAR2 and MEK-dependent mechanism.” Include reference.

4. Discussion: “GzmK treatment leads to the release of proinflammatory cytokines IL-6 and IL-8, but not TNFα or IL-1β, …” is inconsistent with the previous results section which mentioned GzmK treatment did not lead to IFN-y or IL-1β release (TNFα not mentioned). This sentence also mentioned that the mechanisms of IL-6 and IL-8 release were both PAR2 and MEK-dependent, however, the previous results section (and corresponding figure) only investigated this mechanism in the context of IL-8. It should be mentioned that GzmB can induce IL-8 release from keratinocytes (Hiroyasu et al, Nature Comm., 2021)

5. Figures, while one can interpret the results, are of low resolution/blurry in the PDF version.

6. Figures, 1a/b: Describe source of WT PARS. I may have missed where it was discussed in the text.

7. Figures, 2c/d: The experiment group trypsin + buffer is coloured grey instead of green as depicted in the legend.

8. Figures, 2-5: There are no statistics/p values included in the graphs despite significance values having been mentioned within the figure legends.

9. Figures 3: Since the same experimental groups are presented as in Figure 2, it would be ideal to keep colours/patterns consistent for treatments.

10. Figure 4: The figure legend is inconsistent with the presented images – there is no (c) or (d). As a result, the labels are also incorrect. There was no display of IL-8 release shown.

11. Figure 5: For clarity for the reader, include in brackets in the figure legend the identity of each PAR inhibitor, as done in text. Also, there was no mention in the figure legend of the MEKi (PD184352) or what the solid bars represent (treatment with GzmK vs activating peptide). Previous figures used the nomenclature GzmK-AI instead of AEBSF. For consistency, it should be the same.

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Reviewer #1: No

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Granzyme K initiates IL-6 and IL-8 release from epithelial cells by activating protease-activated receptor 2

PONE-D-21-39358R1

Dear Dr. Kaiserman,

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,

Karl X Chai, Ph.D.

Academic Editor

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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: 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

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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 authors have adequately addressed all of my concerns. The research offers further insights into the potential mechanism of action for granzyme K in PAR-2 activation.

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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

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