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A conserved grain-associated immunosuppressive niche in Sudanese patients with mycetoma.

PLOS Neglected Tropical Diseases

Dear Dr. Kaye,

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We look forward to receiving your revised manuscript.

Kind regards,

Joseph M. Vinetz

Section Editor

PLOS Neglected Tropical Diseases

Joseph Vinetz

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Mohamed Osman, Helen Ashwin, Grant Calder, Peter O'Toole, Sahar M. Bakhiet, Ahmed M. Musa, Paul M. Kaye, and Ahmed Hassan Fahal. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Please avoid including the results or conclusions in the introduction section.

Please clearly state the objectives on the last paragraph of the introduction.

In the methods include country and city of the devices, software, and stains employed.

The design is appropriate and the population clearly stated. The sample size is small but that would be expected for such a rare disease. Ethical standards were followed. The statistical analysis is complex but adequate.

Reviewer #2: The study objectives are generally clear, aiming to characterize the spatial immune microenvironment in mycetoma; however, a clearly defined, testable primary hypothesis is lacking, and the work is better framed as exploratory. The study design is appropriate for spatial proteomic analysis, but the inclusion of only 11 out of 28 patients based on grain visibility and the use of manually selected ROIs introduce potential selection bias that should be more explicitly addressed. Ethical and regulatory requirements appear to be adequately met, with appropriate approvals and informed consent obtained.

Reviewer #3: Yes. See detailed comments.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis matches the methods section and are clearly presented. The figures are of great quality. Please include in this section what VISTA means instead of in the discussion section. Please include if any of the patients included had comorbidities (HIV, malnutrition, diabetes, etc.).

Reviewer #2: The analytical approach is appropriate and robust for the study design; however, the exploratory nature and limited sample size should be more explicitly acknowledged when interpreting the findings.

Reviewer #3: Could be improved. See detailed comments.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Please replace “chemotherapeutic agents” with antibiotics in line 314. Please replace “benign” with a more appropriate term.

Reviewer #2: The conclusions are generally supported by the data, particularly the identification of a peri-grain immune microenvironment enriched in CD66b⁺ARG1⁺VISTA⁺ cells. The limitations are acknowledged but could be more explicitly discussed, especially regarding sample size and potential bias.

Reviewer #3: Somehow. See detailed comments.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor revision

Reviewer #2: No further suggestions

Reviewer #3: As a neglected tropical disease, effective treatment of mycetoma requires a better understanding regarding the pathophysiology and virulence factors driving this disease. Therefore, the concept behind this manuscript and employing multiplex microscopy as well as spatial proteome analysis could address an interesting question on 1) the architecture and positioning of immune cells near the granuloma structure and 2) effector proteins shaping the immune cell response. Despite the importance and novelty, a few considerations and some experimental set-ups are required to tackle the speculations.

1. CD66b is a degranulation marker expressed by both neutrophils and eosinophils. Since eosinophils are long known to be involved and drive anti-inflammatory responses, it would be prudent that authors differentiate the two using microscopy. If neutrophils, therefore the near ROI should be CD15+/CD16+/CD14- and if eosinophils sigelc-8, CCR3, and IL-5Rα should be positive.

2. Authors are required to provide further proof that indeed the environment is immunosuppressive given that the major outcome of this manuscript, which is indeed interesting, is that the near ROI is an immunosuppressive niche. Therefore, I would suggest determining the expression of IL4, IL13, and IL14 either using ELISA or using microscopy. The latter might be more interesting since it could show if those cytokines are colocalized with CD66b.

3. Although it’s possible to infer that this immunosuppressive response is driven by the pathogen, authors should note that 1) the involvement of mycetoma in driving this response has yet to be fully confirmed and validated, and 2) this might be an appropriate immune response to the tissue destruction created by the grain structure and therefore maintaining homeostasis and healing. The latter is also possible since based on Figure 2, the expression of fibronectin and SMA are significantly lower in near compared to far ROI. Therefore, immunosuppressive immune response could be mounted to restore the shortage and restore homeostasis. But it also may imply that the proper immune cells restoring the homeostasis is not sufficient near ROI, which could question the very nature of the finding. That is why also inclusion of the alternatively activated cytokines- IL4/IL13/IL14- is important in this context.

4. Why authors did not include inflammatory proteins, including IL6, IL1β, IFNγ, and TNFα?

5. How the anti-inflammatory responses of actinomycetoma are reconciled with previous data suggesting that this complication is inflammatory as started by authors in the beginning of discussion?

Minor points

1. It would be prudent to provide microscopic images of all patients in supplementary files.

2. In the beginning of discussion section, since the data is premature, it is not recommended to mention that this CD66b hallmark of mycetoma is pathogenic, unless well-designed immunological studies are conducted to unravel this notion. The same is true for lines 364-366.

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Please avoid including the results or conclusions in the introduction section.

Please clearly state the objectives on the last paragraph of the introduction.

In the methods include country and city of the devices, software, and stains employed.

The design is appropriate and the population clearly stated. The sample size is small but that would be expected for such a rare disease. Ethical standards were followed. The statistical analysis is complex but adequate.

The analysis matches the methods section and are clearly presented. The figures are of great quality. Please include in this section what VISTA means instead of in the discussion section. Please include if any of the patients included had comorbidities (HIV, malnutrition, diabetes, etc.).

Please replace “chemotherapeutic agents” with antibiotics in line 314. Please replace “benign” with a more appropriate term.

Reviewer #2: The study objectives are generally clear, aiming to characterize the spatial immune microenvironment in mycetoma; however, a clearly defined, testable primary hypothesis is lacking, and the work is better framed as exploratory. The study design is appropriate for spatial proteomic analysis, but the inclusion of only 11 out of 28 patients based on grain visibility and the use of manually selected ROIs introduce potential selection bias that should be more explicitly addressed. Ethical and regulatory requirements appear to be adequately met, with appropriate approvals and informed consent obtained.

The analytical approach is appropriate and robust for the study design; however, the exploratory nature and limited sample size should be more explicitly acknowledged when interpreting the findings.

The conclusions are generally supported by the data, particularly the identification of a peri-grain immune microenvironment enriched in CD66b⁺ARG1⁺VISTA⁺ cells. The limitations are acknowledged but could be more explicitly discussed, especially regarding sample size and potential bias.

Reviewer #3: As a neglected tropical disease, effective treatment of mycetoma requires a better understanding regarding the pathophysiology and virulence factors driving this disease. Therefore, the concept behind this manuscript and employing multiplex microscopy as well as spatial proteome analysis could address an interesting question on 1) the architecture and positioning of immune cells near the granuloma structure and 2) effector proteins shaping the immune cell response. Despite the importance and novelty, a few considerations and some experimental set-ups are required to tackle the speculations.

1. CD66b is a degranulation marker expressed by both neutrophils and eosinophils. Since eosinophils are long known to be involved and drive anti-inflammatory responses, it would be prudent that authors differentiate the two using microscopy. If neutrophils, therefore the near ROI should be CD15+/CD16+/CD14- and if eosinophils sigelc-8, CCR3, and IL-5Rα should be positive.

2. Authors are required to provide further proof that indeed the environment is immunosuppressive given that the major outcome of this manuscript, which is indeed interesting, is that the near ROI is an immunosuppressive niche. Therefore, I would suggest determining the expression of IL4, IL13, and IL14 either using ELISA or using microscopy. The latter might be more interesting since it could show if those cytokines are colocalized with CD66b.

3. Although it’s possible to infer that this immunosuppressive response is driven by the pathogen, authors should note that 1) the involvement of mycetoma in driving this response has yet to be fully confirmed and validated, and 2) this might be an appropriate immune response to the tissue destruction created by the grain structure and therefore maintaining homeostasis and healing. The latter is also possible since based on Figure 2, the expression of fibronectin and SMA are significantly lower in near compared to far ROI. Therefore, immunosuppressive immune response could be mounted to restore the shortage and restore homeostasis. But it also may imply that the proper immune cells restoring the homeostasis is not sufficient near ROI, which could question the very nature of the finding. That is why also inclusion of the alternatively activated cytokines- IL4/IL13/IL14- is important in this context.

4. Why authors did not include inflammatory proteins, including IL6, IL1β, IFNγ, and TNFα?

5. How the anti-inflammatory responses of actinomycetoma are reconciled with previous data suggesting that this complication is inflammatory as started by authors in the beginning of discussion?

Minor points

1. It would be prudent to provide microscopic images of all patients in supplementary files.

2. In the beginning of discussion section, since the data is premature, it is not recommended to mention that this CD66b hallmark of mycetoma is pathogenic, unless well-designed immunological studies are conducted to unravel this notion. The same is true for lines 364-366.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Amir Arastehfar

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Dear Prof. Kaye,

We are pleased to inform you that your manuscript 'A conserved grain-associated immunosuppressive niche in Sudanese patients with mycetoma.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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.

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 Neglected Tropical Diseases.

Best regards,

Joseph M. Vinetz

Section Editor

PLOS Neglected Tropical Diseases

Joseph Vinetz

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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