Dear Dr. Gadjeva,

Thank you very much for submitting your manuscript "Pseudomonas aeruginosa–induced nociceptor activation increases susceptibility to infection" 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.

This paper was sent to three reviewers, most of whom (due to scheduling conflicts) did not review the previous version but were aware of the reviewers comments and subsequent revisions made to this submission.

All three reviewers again noted the relevance of the work. All three felt the new controls provided were not adequately explained or presented. The associate editor in charge of this submission agrees with those comments and also thinks the first section of the results section needs more work to explain what experiment was done prior to explaining the results. Also the paradox that inducing more inflammation (in wild-type animals without to treatment to remove nerves in the eye) causes fewer neutrophils to be recruited is not particularly well explained. The extent of the work required for publication depends on whether the new controls were actually done in the manner requested by the three reviewers (and merely need to be explained better) or whether these controls need to be redone.

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.

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Sincerely,

William Navarre

Associate Editor

PLOS Pathogens

Alan Hauser

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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This paper was sent to three reviewers, most of whom (due to scheduling conflicts) did not review the previous version but were aware of the reviewers comments and subsequent revisions made to this submission.

All three reviewers again noted the relevance of the work. All three felt the new controls provided were not adequately explained or presented. The associate editor in charge of this submission agrees with those comments and also thinks the first section of the results section needs more work to explain what experiment was done prior to explaining the results. Also the paradox that inducing more inflammation (in wild-type animals without to treatment to remove nerves in the eye) causes fewer neutrophils to be recruited is not particularly well explained. The extent of the work required for publication depends on whether the new controls were actually done in the manner requested by the three reviewers (and merely need to be explained better) or whether these controls need to be redone.

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 overall premise of the study is novel. The authors have attempted to address one of the major issues raised by both reviewers regarding the absence of an appropriate control group for Figure 1. However, this was not executed well, and thus it is still not clear whether the data presented in Figure 1 represents nerve injury from PA exposure alone, or scratch + PA exposure combined.

Reviewer #2: In this manuscript, Lin et al. investigate the role of corneal nerves in modulating the innate immune response during Pseudomonas aeruginosa keratitis. The authors examine neuro-immune responses in the eye during bacterial infection. The studies are novel and interesting, but overall lack consistency in the data analysis and presentation, thus negatively impacting the quality of the manuscript. These weaknesses, among others, within the study create uncertainty over the findings. Below are comments that would strengthen the manuscript..

Reviewer #3: This is an interesting paper pointing to the interplay between corneal sensory nerves and the outcome of pseudomonas keratitis.

The novel aspect of the paper is the illumination of a new player-ie nerves- in the pathophysiology of infectious keratitis. It remains to be elucidated what is the clinical relevance of such findings, as patients with denervated corneas have generally poorer clinical outcomes after corneal infections

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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: Reviewer comment 1: One of the major concerns raised by both reviewers was the absence of a control (scratched) cornea group in the original submission. Given one of the major findings of the study is that exposure to PA is associated with lower sensory nerve density in the corneal epithelium, it is important that the appropriate controls were included. The authors have since conducted additional experiments that demonstrate no effect of the corneal scratch injury on nerve density, however, the description and design of this (important) control group experiment is inadequate.

Firstly: it is unclear what time point the images and graphical data presented in Supplementary Figure represent. Given the original Figure 1 contains a 0h, 24h and 48h time point, then at the very least all of these time points should be represented in the control experiment. It is surprising that the new control experiment did not contain groups exposed to PA. The ideal additional experiment to compare the nerve response would have included scratch + vehicle, vs scratch + PA at both 24h and 48h.

Secondly; in Supplementary Figure 1A, the Sham image appears to contain less nerves than the control image. The number of animals is described as N=4 in the legend, but there are approximately 5 datapoints for Control, and 10 datapoints for Sham in the graphs in Supp Figure 1b. Please clarify.

Lastly- a comment: three corneal scratches using a 25g needle would most certainly cause damage above and beyond only a minor disturbance of the lipid layer of the tear film (which ranges in thickness from 70-100nm).

Reviewer #2: I. A major concern is the lack of clarity to the methodology and proper controls for the experiments.

a. Sham scratch corneas have not been used as negative or sham controls for any experiments.

b. Cumulative values are used for some experiments, while others have only representative experimental data analyzed.

c. Although a significant decrease in corneal nerve density was seen at 24h and was most remarkable at 48h post-challenge, it is not clear why different time points after infection are used for analysis in different experiments instead. These inconsistencies in the time point of experiments make it difficult to assess the data.

II. The presentation of data points is not consistent throughout the figures in the manuscript.

a. Some of the graphs do not show individual data points and others have unequal numbers in different groups.

b. It is concerning that not all data points were not included in the analysis for some groups. Some of the experiments have unequal, small sample sizes and variable outcomes, which affects the assumption of equal variances in parametric tests like ANOVA and affects statistical power and error rates.

c. SD and SEM have been used in different figures without a clear rationale.

III. Another concern are the immunofluorescent corneal whole-mount images used for quantification and representation. The corneal nerves seem to have different caliber/thickness in different images, giving an impression that the images have been taken at different depths in the cornea – the thinner nerves are from sub-basal plexus and the thicker nerves are deeper stromal nerves. The authors mention that projections of z-stacks were taken but the thicker nerves are not visible in all the images (Figures 1A, 2D, 4B and 6A) with scale bar 20um, and thinner nerves from the subbasal plexus are not visible in others.

IV. The Nav1.8 Cre+ DTA are mice are important, but certain studies need to be conducted in these mice. Levels of CGRP, CA-flux, and CGRP-induced upregulation of ICAM-1+ neutrophils needs to be tested in these mice.

V. There are several issues with the RTX treated mice that need to be addressed. It is not realistic that the corneal nerves in these mice decrease at the levels show, while corneal sensation remains intact. Looking at the corneal micrographs, only stromal nerves are being shown. These studies need to be repeated. This also coincides with the fact that RTX treatment had no effect on inflammatory responses. Given that all data in these mice, except corneal nerve staining show no effect, the most likely explanation could be that the corneal staining for nerves was not assessed appropriately. To verify this, 2 additional markers for neurons should be used to confirm presence/absence. In addition, the level of TRPV1+ nerves should be assessed with and without RTX. For Fig 6A, overlay of b3-tubulin and CGRP should be shown and not CGRP alone.

VI. In general, the conclusion that loss of sensory nerves results in improved innate immune responses to Pseudomonas aeruginosa are not supported by the data provided.

VII. Another main issue is that while the effect of TRPV1 and Nav 1.8 that does not directly imply that the effect is pain related as TRPV1 can result in other symptoms in patients as well.

Reviewer #3: Fig. 1. Please clarify if the no infection group has been scraped or not. If not, than this control should be added. Similarly, corneal sensitivity should be measured also in the scraped not infected mice. This is because scraping will inevitably damage the subbasal nerve plexus.

Fig. 4 B does not look convincing. Three whole mounts per group are not enough to draw conclusions and they should be at least 5 per group. In addition pictures do not seem to capture the same anatomical area: vehicle looks like sub-basal plexus, while vehicle infected and RTX controlateral look like stromal nerves.

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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: Reviewer comment 2: I request that the authors respond to the point (13) in their Response to Reviewers, as it was not addressed. This point is particularly important, given the variable density of corneal nerves across the cornea. They state in Figure 4B that “Three whole mounts per group per treatment” were analyzed, but no details about the number of images per cornea is included.

“13. Methods: how many images per cornea were used for nerve analysis and Ly6G+ cells? Were images collected from the central or peripheral cornea?”

Reviewer comment 3: Below is my original comment (point 14, including the author’s response in italics).

“14. If mice were anaesthetised for the Capsaicin eye wipe test, how did they respond by wiping their eyes? What type of anaesthesia was used?

Authors response: Mice were not anaesthetized for this assay. There is no statement to this end in the methods section.”

Reviewer follow-up response: The response by the authors is not adequate. In the Methods section (page 22, Capsaicin Eye Wipe Test”; the authors state clearly that the mice were anaesthetised ( see quoted paragraph below). Please clarify (again) how an eye wipe test was performed in anaesthetised mice.

“Capsaicin eye wipe test. The capsaicin eye wipe test was performed three weeks after RTX treatment. Animals were acclimated to the behavioral testing environment three days before commencing the behavioral test that were performed within the same room as their cage to reduce stress. While anaesthetized, mice were gently restrained, 3 mM of a capsaicin solution (Sigma Aldrich, St. Louis, MO, USA) was applied to the left cornea of the mouse. Mice were video recorded during each session immediately after capsaicin application [50]. Masked observers counted the number of wipes (via the ipsilateral forepaw) within a minute after application. Normal facial grooming behavior was not included.

Reviewer- Minor issues:

1. Capsaicin is spelled incorrectly as “Capsaisin” on several occasions, particularly in Figures and legends.

2. Figure legend 4B “couterlateral” is mis-spelled

3. Again, inconsistencies in presentation of data for the same parameters (i.e. nerve density in Fig 1 presented as individual data points and column graph, nerve density in Fig 4B presented as means +/- SEM (?) with no individual data points visible.

4. Images in Supp Figure 3 are poor quality: the sections are provided to demonstrate differences in Ly6G+ cells in the limbus however there appears to be similar cell density for both RTX and Baseline Control (which presumably corresponds to “Vehicle” in the graph?). Datapoints are not visible in the graph when columns are colored black, so it is not easy to interpret the data.

Reviewer #2: I. Introduction: The rationale that Pseudomonas aeruginosa results is a painful disease is supported by a single case report. Generally, this condition is not thought to be painful, precisely due to the loss of corneal nerves. More robust supportive data should be cited in the introduction.

II. Introduction: Last line in paragraph 1; it is not clear how this relates to the current study in pain.

III. Introduction: Many citations, while appropriate are incorrectly cited in terms of content. (e.g., ref 10 discusses polymodal receptors, ref 2 discussions nerve density and not sensitivity, refs 3-6, some also include herpes zoster and not only herpes simplex, ref 10 not all superficial nerves derive from the stroma, a separate superficial source supplies toe epithelium directly). In addition, not original articles have always been cited. Rather more recent review articles were cited.

IV. Introduction, page 5, first sentence: This is inflammatory pain and not neuropathic.

V. Introduction, page 5, first paragraph, last sentence: There are many additional published articles on this topic, and the statement that only a few articles on neuro-immune responses in viral and bacterial infections have been published is not correct. It would apply to bacterial infections though.

VI. Introduction: No clear hypothesis has been provided.

VII. Methods: The company, city, state have only been provided for some reagents and are missing for many others. There is in general a high level of inconsistency in the methods section.

VIII. Methods: The markers being used to assess neutrophils are not specific to neutrophils (e.g. CD11b and LYGG can be expressed by a number of other cells). Additional markers are required to conclusively and robustly identify neutrophils.

IX. Methods: For flow cytometry, live/dead marker, isotype controls, and tissue only controls are required in methods section.

X. Methods: The strain background and source for the Nav1.8Cre+ DTA mice are missing as are of all mice used.

XI. Photographs of corneas at different time points after infection have not been included in the manuscript, which would have provided a better clinical evaluation of the cornea.

XII. For image quantification, the authors mention two methods – Neuron J and custom-made-algorithm using Fiji software. It is not clear why different methods were used for the same purpose and what exactly is being calculated - length or area with µm/pixel. The same software and the same parameters should be used for all images.

XIII. Methods: why are two different methods for sorting neutrophils used?

XIV. Methods: Immunohistochemistry requires isotype controls

XV. In the methods section, “While anaesthetized, mice were gently restrained, 3 mM of a capsaicin solution (Sigma Aldrich, St. Louis, MO, USA) was applied to the left cornea of mouse. Masked observers counted the number of wipes (via the ipsilateral forepaw) within a minute after application.” Please explain the effect of anesthesia in this experiment. The application of anesthesia will affect the response as previously highlighted by the reviewers. It will result in inaccurate measurements.

XVI. The quality of images in Figure 2A need to be improved and the black arrows in 6294 need to be explained in the figure legends.

XVII. “Pseudomonas aeruginosa keratitis is known to be an intensely painful disease” and “Pseudomonas aeruginosa infection reduces neuronal network and corneal pain sensation” seem to be contradicting each other. Please elaborate.

XVIII. Fig 1A+B, please add scratch alone controls for the same time points.

XIX. It is not clear whether the bacterial burdens determined in different experiments were due to changes in adherence or clearance of the bacteria.

XX. The time points for quantification of nerve density in sham scratch corneas in supplementary figure 1 is not mentioned. Why was it not done at both 24h and 48h after injury? I assume that epithelial scratches, however superficial, will affect the nerve density at these earlier time points and thus this needs to be considered in the results.

XXI. Fig 2D, corneal nerves are present throughout the cornea. As such, panel D is not sufficient to conclude that cells are in close proximity of nerves as they would be regardless of location. This would require in vitro co-culture of neurons and neutrophils.

XXII. In supplementary figure 3, the staining does not seem specific. How were the sections selected for quantification?

XXIII. In supplementary figure 4 and 7, it seems odd that there are different number of data points in different groups as low as 2.

XXIV. Several previous comments by reviewer 1 have not been addressed and need to be addressed. I agree with those comments (e.g. #12, #13).

XXV. There were numerous errors/typos in the manuscript, please correct.

Reviewer #3: Capsaisin should be changed to capsaicin throughout the ms.

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Reviewer #2: No

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Dear Dr. Gadjeva,

We apologize for the delay in processing your manuscript.  We encountered difficulties in the review process at multiple levels, which prevented us from coming to a decision in a timely manner.  At PLOS Pathogens, we strive to review all manuscripts as rapidly as possible, and we obviously failed at this with your manuscript.  We thank you for your patience. 

We are pleased to inform you that your manuscript 'Pseudomonas aeruginosa–induced nociceptor activation increases susceptibility to infection' has been provisionally accepted for publication in PLOS Pathogens.

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Best regards,

Alan Hauser

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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Reviewer Comments (if any, and for reference):