Dear Prof. Lee,

Thank you very much for submitting your manuscript "Periodontitis promotes bacterial extracellular vesicle-induced neuroinflammation in the brain and trigeminal ganglion" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Thank you for submitting your work to PLOS Pathogens and I am sorry for the delay getting the reviews back to you. The Reviewers found the work of interest but there are some major issues that need to be addressed with additional experimental evidence. In particular, I agree with Reviewer 1 that specificity of the response needs to be addressed. Do other EVs have the same response as Aa EVs and are they trafficked similarly? These controls need to be demonstrated. Also, is the "dose" of EVs that are applied in this model in any way physiological? For example, how does the dose of Aa EVs compare to the concentration of bacteria (CFUs/ml) used to prepare the EVs?

These issues must addressed before further consideration of the manuscript.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Dana J. Philpott

Academic Editor

PLOS Pathogens

David Skurnik

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Thank you for submitting your work to PLOS Pathogens and I am sorry for the delay getting the reviews back to you. The Reviewers found the work of interest but there are some major issues that need to be addressed with additional experimental evidence. In particular, I agree with Reviewer 1 that specificity of the response needs to be addressed. Do other EVs have the same response as Aa EVs and are they trafficked similarly? These controls need to be demonstrated. Also, is the "dose" of EVs that are applied in this model in any way physiological? For example, how does the dose of Aa EVs compare to the concentration of bacteria (CFUs/ml) used to prepare the EVs?

These issues must addressed before further consideration of the manuscript.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This research article regarding periodontal pathogen EV-induced neruoinflammation by Ha et al is an interesting and in-depth study of the ability of extracellular vesicles (EVs) from the pathogen Aggregatibacter actinomycetemcomitans (Aa) introduced via the oral route to induce inflammation in the brain in a mouse model. The authors utilize two administrative routes via the oral cavity (gingival soaking and injection) and investigate neuroinflammatory responses using signaling pathway-deficient (MyD88 KO) and immune-competent (WT) mice. They followed the trafficking of the EVs using fluorescently labeled EVs and in situ imaging. This work follows on two very closely related studies from this group that already demonstrated bEV trafficking and exRNA delivery to the brain (2020, 2019), which reduces the novel aspects of this report. The major critical areas concern dosage and specificity as detailed below. At this point in these studies, it is expected that such questions need to be addressed in order to move the field forward in a significant way.

Reviewer #2: The study suggested that EVs derived from periodontopathogens such as Aa might be involved in pathogenic pathways for neuroinflammatory diseases, neuropathic pain, and other systemic inflammatory symptoms as a comorbidity of periodontitis. It was interesting and novelty.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Major comments:

1. A serious question is the matter of physiological dosing. The authors do not address experimentally whether the EV dose they are administering via the ginigival route is at all physiologically reasonable. How does the amount of EVs entering the gingiva compare to the amount produced by Aa? Is it an amount that is absurdly high such that it would never be attained by typical Aa colonization in the gingival tissue? This study claims to test the hypothesis whether periodontitis facilitates the migration of oral bEVs to brain to induce neuroinflammation (lines 252-3), yet the experimental model is not periodontitis, it is application of purified EVs by injection or oral gel. Notably, the authors claim (lines 87-92): "Previously we showed that EVs of Aa and their RNA cargo could cross the blood-brain barrier (BBB) and increase proinflammatory cytokine expression in the mouse brain [20,21]. This was the first direct evidence that blood-borne bEVs can cross the mouse BBB and cause neuroinflammation. However, it remained unclear whether EVs originated from periodontopathogens could enter the brain through leaky gum during PD development." However, the study here does not actually address whether the EVs that are made 'during PD development' traffic in the same manner or with the same immunoinflammatory results as those that have been isolated and introduced in the quantity that they were during these experiments.

2. Another critical question that is not addressed by these experiments is specificity. Obviously, there are numerous commensal species and strains of Gram-negative oral bacteria other than Aa which are also making EVs. Why would those EVs not traffic similarly through the BBB and generate neuroinflammatory disease? Such qualifications should also be considered to modify statements such as (Lines 349-351) "These findings suggest certain inflammatory disorders may be associated with the translocation of bEVs to systemic organs. Therefore, the ability of bEVs to circulate throughout the body may help in understanding autoimmune disorders, up to 50% of which lack a definite etiological cause [73]." There must be much more to generate specificity of transport and overcoming barriers in tissues, otherwise all tissues and sites would be flooded with bEVs that were produced by the human microbiome and all would cause pathologies due to PAMPs carried by the bEVs.

Reviewer #2: 1. Why choose 24h for osteoclastic activity?

2. Line 949-950, After 24 h, gingival tissues were stained for TRAP activity, the representative images of TRAP-positive cells 951 being shown (scale bar: 200 μm). It was unreasonable to analyse TRAP staining in gingival tissues.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: 1.Lack of animal ethics approval number.

2.How to exclude the influence of unilateral molding on the opposite side?

3.The image resolution was low.

4.Quantitative statistics are required for positive TRAP staining cells.

5.p value should be in italics.

6.*p≤0.05 was wrong.

7.Scale bar was missing in Fig 6. D.

8.The volume of injected Aa EVs and Aa EV-soaked gel should be demonstrated.

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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: No

Reviewer #2: No

Figure 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. 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 us at figures@plos.org.

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Dear Prof. Lee,

We are pleased to inform you that your manuscript 'Periodontitis promotes bacterial extracellular vesicle-induced neuroinflammation in the brain and trigeminal ganglion' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Moreover, please add the suggested addition to the transmission electron microscopy methods (more details on staining procedure).

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Dana J. Philpott

Academic Editor

PLOS Pathogens

David Skurnik

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have addressed the major concerns. A reasonable vesicle dose has been established. Content has been slightly edited to reflect key deficits regarding specificity. No further modifications are needed except the minor modification to the methods-- see below.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: There is insufficient detail regarding the Transmission Electron Microscopy methodology. Staining methods are missing.

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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