PONE-D-20-35446

Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes

PLOS ONE

Dear Dr. Brox,

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.

Authors described a whole blood method to evaluate the impact of aspirin treatment on cytokine production by a a panel of TLR ligands. Although seemingly more physiological doses of asqírin were used the data confirms what has been reported before that prostanoids and NSAIDs are able to modulate cytokine production in leukocytes. The whole blood has its merits, but it is less amenable to investigate potential mechanisms of action for the reported effect.

There is no clear correlation between the impact of aspirin on cytokine production and PGE2 generation. Perhaps, the authors should include a correlation plot of cytokine x PGE2 with the data for each donor. The impact of Indomethacin and Celecoxib on PGE2 generation should also be included in the manuscript.

 Authors should reconcile the paradoxical effects of aspirin and PGE2 as both treatments raise the production of IL-10, induced by LPS, and IL-6, induced by ssRNA40 (although the effect of aspirin in SSRNA40-induced IL-6 shown in figure 3E, does seem to be reproduced in figure 6E).  Simply stating the different PGE2 receptors does not provide a compelling explanation for the observed effect. Authors should also include data on the effect of PGE2 on the production of IL-10 and IL-6 induced by other TLR ligands to demonstrated that this effect is specific to LPS and ssRNA40, respectively.  

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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: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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: This manuscript aimed to investigate the immunomodulatory role of physiological Acetylsalicylic acid concentrations in a human whole blood (WB) of infection-induced inflammation model.

The authors describe a WB assay using an array of toll-like receptor (TLR) ligands 1–9 in order to explore the immunomodulatory activity of Acetylsalicylic acid plasma concentrations in physiologically relevant conditions. Release of inflammatory cytokines and production of prostaglandin E2 (PGE2) were determined directly in plasma supernatant.

The study exploited the development and validation of an in vitro whole-blood model for the evaluation of immunomodulatory agents. The results show, accordingly the referenced WB-cytokine assay, that plasma concentrations of Acetylsalicylic acid significantly increased TLR ligand-triggered IL-1β, IL-10, and IL-6 production in a dose-dependent manner. In contrast, indomethacin did not exhibit this capacity, whereas cyclooxygenase (COX)-2 selective NSAID, celecoxib, induced a similar pattern like Acetylsalicylic acid, suggesting a possible relevance of COX-2. Accordingly, the authors found that exogenous addition of COX downstream product, PGE2, significantly antagonized most of the immunostimulatory activity of Acetylsalicylic acid.

In summary, the results indicate a potential role of PGE2 and COX-2 in mediating the immunostimulatory effects of ASA. The study opens an avenue to further investigation in order to unravel the complex mechanisms behind the immunostimulatory properties of physiologically relevant ASA concentrations.

Despite the results show support for the final conclusion, I recommend major and minor revisions.

Major issues:

1) Acetylsalicylic acid is a globally used non-steroidal anti-inflammatory drug (NSAID) in both men and women in the medical clinic. For this reason, I recommend the addition of healthy female donors in the study in order to compare effects in both sexes either a justification for not considering those samples.

2) One important set of data that should be showed is the cellular viability and the cellular composition in the WB-cytokine assay in all experimental groups. The cellular composition would give information of relative proportions of each type of leukocyte to ensure that WB-cultures correspond to the normal original range of leukocytes in male healthy donors. A flow cytometer analyses such as Annexin V and PI Apoptosis staining, could be done to evaluate viability. And a regular hematology analyzer would work to analyze the relative cell proportions. This set of data would better validate the results.

Minor issues:

1) As one of the stressline of the present study is the validation of an assay using WB samples for the study of immunomodulatory agents, which is new in this type of approach, I suggest the authors add a positive control group in each measurement of cytokines to know if the assay works properly. A simple model using an established cell line would show a worthy response. Besides, there are several references that the authors gave in the discussion that would work for this purpose. For example: Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdörfer B, Giese T, et al. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol. 2002;168(9):4531-7.

Barr TA, Brown S, Ryan G, Zhao J, Gray D. TLR-mediated stimulation of APC: Distinct cytokine responses of B cells and dendritic cells. European Journal of Immunology. 2007;37(11):3040-53.

2) Does the Tri-sodium citrate used to avoid coagulation in samples play an effect on Ca++ availability in the cultures? Is this anticoagulant the best choice for not interfering in the activity of AA-pathway enzymes?

3) Although is more practical to keep the graphs of Figure 1 as they are, I rather authors could group the cytokines into lower and higher levels, such as in Figure 1A put TNF-α and IFN-γ in the same graph and IL-6 and IL-1β in other graph, for example, to avoid scale issues. The values of cytokines as TNF-α and IFN-γ cannot be seen properly in several graphs due to high-range scale.

4) I suggest a better representation of the X axis in several graphs. For instance, in Figure 4 the treatments became very confused visually. This could be ameliorated with crosses (in case of treatment) and traces (in case of absence of treatment) in a lines and columns table-pattern bellow X axis.

5) On page 8 the sentence: "Significant higher cytokines concentration were only observed for TNF-α and IFN-γ upon stimulation by LPS and ssRNA40, respectively (Fig 4C, D)."

Should be replaced by: "Significant higher cytokines concentration were only observed for TNF-α and IFN-γ upon stimulation by LPS and ssRNA40, respectively (Fig 4C, E)."

6) On page 7 the sentence: "Similarly, a significant elevation of IL-1β was detected in the supernatant of WB cultures simulated with Flagellin and LPS (Fig 3C, D)."

Should be replaced by: "Similarly, a significant elevation of IL-1β was detected in the supernatant of WB cultures simulated with LPS and Flagellin (Fig 3C, D)."

7) In figure 5B the results display on X axis is from the highest to the lowest concentration of ASA (from left to right). It would be better if authors display this set of results as shown in the other graphs, that is from the lowest to the highest concentration of the agent (from left to right).

In an overall view, the manuscript is acceptable for publishing, presenting reasonable data support for the conclusions, appropriate statistical analysis and intelligible fashion and written in standard English. However, major and minor revisions are necessary.

Reviewer #2: The data presented by the article are relevant and demonstrate the possibility of developed a straightforward technique using human WB stimulated with different TLR ligands to investigate the immunomodulatory effects of in vivo relevant plasma concentrations of ASA.

Also, The authors could have numbered the lines to facilitate the identification of the questions for the review.

I believe that for acceptance of the manuscript the authors should consider correcting or clarifying the following points:

1 - In the introduction, the phrase “Two isoforms of COX identified: cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)” seems to be incomplete.

2 - In the results item, in “Development and validation of an in vitro whole-blood model for the evaluation of immunomodulatory agents”, in the results of figure 1, the authors used doses 1, 10, 100 and 1000 ng of Pam3CsK4. However, for the next experiment they used the dose of 500ng, and did not explain why he used the intermediate dose used in figure 1.

3- In the results of figure 1, the authors presented the results of 2 experiments, each performed in duplicate. For this experiment, n = 2? I believe that statistical analysis should occur with a n equal to or greater than 3. Could the authors clarify the n used?

4 - In the results of figure 2, the authors presented "At the same time, low concentration of Dexamethason (1 nM) upregulated IFN-γ release when stimulated with HKLM (mean = 158%), LPS (mean = 130%) or ssRNA40 (mean = 118%) followed by a significant decline after treatment with high concentration of 100 nM of Dexamethason (mean = 50% –2%)" . However, for LPS, there was no statistically significant difference for the authors to claim an increase of 130%. Was there no significant difference or lack of indication in the graph?

And, In the results of figure 2, the authors presented the results of 2 experiments, each performed in duplicate. For this experiment, n = 2? I believe that statistical analysis should occur with a n equal to or greater than 3. Could the authors clarify the n used?

5 - In the results of figure 5A, the authors should show statistical analysis regarding the production of PGE in the stimulated groups compared to the non-stimulated group.

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Reviewer #1: Yes: Luciana Boffoni Gentile

Reviewer #2: No

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Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes

PONE-D-20-35446R1

Dear Dr. Brox,

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,

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 #3: 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 #3: Yes

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

Reviewer #3: 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 #3: 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 #3: Yes

**********

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 #3: The manuscript reports valid and important questions about the physiological effects of aspirin on human blood cells that will allow a discussion after publication by the scientific community. In addition, the authors answered most of the questions raised by the reviewers. Based on that, I recommend accepting the manuscript for publication.

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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 #3: No