-->PONE-D-26-04172-->-->Fasciola hepatica Excretory-Secretory Products Enhance Remyelination and Reduce Neuroinflammation in the Cuprizone –Induced Multiple Sclerosis Model-->-->PLOS One

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PLOS One

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Additional Editor Comments:

Based on the reviewers' suggestions, the paper needs major revision. The reviewers' comments can be found below.

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: Manuscript No. PONE-D-26-04172

Fasciola hepatica Excretory-Secretory Products Enhance Remyelination and Reduce Neuroinflammation in the Cuprizone-Induced Multiple Sclerosis Model.

General opinion.

This is an interesting work that demonstrate the therapeutic potential of F. hepatica ESP in ameliorating the MS-pathology in a mouse model. The manuscript has certain merit; however, it requires major modifications to be suitable for publication in PLOS One.

Major Comments

1. The number of animals per group was 7. Please provide evidence of power analysis.

2. Why two different concentrations of Cuprizone (0.7% w/w first and then 0.2% w/w) for inducing demyelination? The more frequent cuprizone concentration in standard rodent chow is 0.2%, which is associated with extensive and reproducible demyelination. Higher concentrations (0.4%-0.6%) are only necessary in aged mice, which is not the case because the mice were 6-week-old. Please provide a rationale for this.

3. Despite the excretory-secretory products are prepared in sterile conditions this does not guarantee that the antigen is endotoxin-free. How did you assured that antigens were endotoxin-free?

4. Please, provide a proper rationale for F. hepatica ESP amount used in injecting animals (10ug). In the reference cited (Lume ME et al 2014), the F. hepatica ESP were produced by culturing parasites for only 8 hours vs. 24 hours used for you, which definitively can lead to a change in the antigen mixture composition and thereby, the dose used by Lume in a diabetes model with NOD mice could not necessarily works in your cuprizone MS model. Why didn’t you assay a dose-response experiment? Why don’t you administer the antigen based on body weight instead a single a unique amount?

5. It would be recommendable to perform additional experiments aimed to determine how T-cells (Th1, Th17 and Treg) were affected by the treatment with F. hepatica ESP as well as also investigate the effect of antigen on cytokines associated to MBP. This is an autoantigen closely related to the activation of CD4+ T-cells, which produce high levels of IFNγ, IL-17 and IL-6. Alternatively, a western blot analysis of spinal cords or brain homogenates showing reduction of specific bands intensity (18.8, 17, or 14kDa) could support the PCR amplification analysis.

6. Why using a cuprizone-induced demyelination instead the experimental autoimmune encephalomyelitis (EAE) model, which is the commonly used? A rationale for this should be added and in the discussion pros and contrast of the cuprizone-intoxication model.

7. Some statements in the discussion seems to be inaccurate or overinterpreted. For example: lines 382-386, you state that in vivo effect of Fh ESP on remyelination are in contrast with the Dowling et al studies [28]. The meaning of this apparent discrepancy is not clear to me.

8. Lines 386-391: It is not clear to me relation that you make between the results of Baska et al [29] obtained with THP1 macrophages, and the miRNA expression with the data presented herein.

Minor comments

1. All scientific names should be in italic: i.e. Fasciola hepatica, please correct it throughout the text and references list.

2. Please, provide the Ethical approval number of protocol in the Material and Methods section

3. Please, revise introduction and correct or add adequate citations when necessary. For example, lines 70-73, mention diverse mechanisms used by ESPs (downregulation of pro-inflammatory pathways, inhibition of inflammasome activation, suppression of T and B cell responses, T-cell expansion and activation of M2-macrophages. However, only is cited the work of Valdes-Fernandez et al 2024, which was exclusively about GST and M2-type induced macrophages.

Lines 73-75: The publication of Cengiz ZT et al 2023, which is not available in PubMed, is about antioxidant enzymes and oxidative stress in human fascioliasis and it is not related to multiple sclerosis or autoimmune diseases

Lines 75-78: The publications of Musah-Eroje M 2018 and Ali SBG, 2021 are about F. hepatica immunomodulation and do not mention neuroprotective microglia phenotype or myelin repair.

Lines 82-83: You state that cuprizone is a no toxic model, which is incorrect. It is considered a toxic model. Cuprizone causes toxicity by triggering mitochondrial stress, resulting in severe oxidative stress.

Lines 83-84: Administered ESPs were evaluated….., seems has no sense. Please check syntaxis.

4. Please, also revise the references cited in the discussion, which seems to be wrongly cited. E.g., in line 375: The study by Maria E. et al [26] demonstrated that…… The reference #26 in the reference list corresponds to: Lund ME et al 2016.

5. Please add the Doi and PMCID/PMID number to all references listed.

6. Please revise grammatical and syntaxis throughout the entire text

7. Please correct the graphical abstract. After centrifugation, the arrow wrongly mark the pellet that contains all debris of FES instead the supernatant. The mouse seems to be drinking instead eating solid food (rodent chow).

Reviewer #2: This manuscript describes an investigation into the capacity for the excretory/secretory products of Fasciola hepatica to prevent progression of neurodegenerative disease in a murine model of cuprizone induced demyelination. This study builds on the body of previous literature showing the potent beneficial effect of helminth infection and their secreted products to slow/stop the progression of immune mediated disease. The study follows a standard approach of treatment and assessment of outcomes using acceptable technical approaches.

I would suggest the conclusions and title of the paper are a little exaggerated based on the actual experimental data presented. While there is clearly an effect of the injection of FhES, there is no robust measure of remyelination per se. Accordingly, I think the conclusion should be tempered and the title edited to better reflect the experimental design and actual outcomes. In the Cuprizone model, while demyelination is certainly underway by week 2 (when the FhES is administered) this continues over the subsequent days if cuprizone is continually administered in the food up to 5 weeks before remyelination begins. In this study, the mice were on Cuprizone for during the same period as they received FhES. It is therefore more likely, that the FhES slowed down demyelination rather than induced remyelination – the increased expression of Olig2 being the only possible biomarker of oligodendrocyte activation, is not sufficient to conclude the induction of remyelination.

Additional comments:

• Line 65 – at the very least the seminal and foundational paper in this area by Correlae et al should be included here.

• Fasciola hepatica (and all other parasite names) should always be in italics.

• Line 66 – the work of Mills et al which explored the effect of ES in murine models of EAE should be included here

Methods:

• The reason for the sole choice of male mice should be included here

• What source was utilized for the protocol to harvest the Fasciola ES (which should be correctly named FhES)? 24h is a long time to culture these worms, with optimal secretion and work health being around 8h – after that the parasites being to break down. Giiven this, a protein gel of the harvested ES could be included in the paper as supplementary data.

• Why was this particular regime of FhES administration chosen? A reference is given, but this is from a very different murine model which runs over a much longer time frame. In addition, given the neurotropic disease under examination here, why was IV not chosen as an administration route?

• Why was the 10ug dose of ES chosen, and why was it diluted in PBS rather than saline?

• Was the stability of the housekeeping gene expression measured. This is critical if it is to be used as a relative marker to quantify the expression of other genes. This analysis needs to be completed and included in the paper.

• The quantification of myelination mentions choosing regions of interest – this suggests preferred regions of tissue were assessed. Ideally the same regions of cerebellar sections in all mice should be chosen and quantified blind.

Results

• TNF is no longer called ‘alpha’ due to the recharacterization of TNFbeta as LT – please correct this terminology throughout

• There is no explanation here or in the methods of the statistical analyses that were used to calculate the significance of any data – as such this makes it very difficult to properly interpret the outcomes.

• I would not use the term ‘partial suppression’ – either there was a reduction or not of cytokines in response to the FhES – the extent of this suppression can then be determined by statistical analyses.

• Why is the PCR gene expression data presented as a ratio in Figure 5 when in the methods it describes the quantification as calculated using house keeping gene?

• Can the authors clarify how many animals were used to calculate the data shown in Figs 4 and 5. Although the methods describe the use of 7 mice per group there appears to only be 3-4 represented in these data sets?

• Line 310 – to claim the ES restores MBP and Olig2 expression is a very exaggerated and wholly inaccurate when looking at the actual data that is presented. Levels of neither gene returns to the expression seen in the control mice which would represent a restoration. The language and conclusion from this data set needs to be tempered.

Discussion

• The discussion is far too long and takes too may exaggerated explorations into likely mechanisms given the small set of basic data that is presented here. It is best to edit this to a consideration of this study of ES efficacy compares to others (models, dose, read outs etc) – then make a unifies proposal for mechanism based on the evidence from this and the existing body of literature. (something like that presented in lines 382-412.

• The content of lines 425-454 is too hypothetical and makes assumptions incorrectly based on an exaggerated interpretation of the MBP and Olig2 expression data. This type of claim is not acceptable.

• Line 375 – the author of this citations [26] is Lund et al

• Line 377 – there is no basis for this mechanistic connection between ref #27 and the data that is shown here

• Lines 455 – this section is predominantly a reiteration of what was mentioned before – although recognizing some of the limitations is a positive move.

• The conclusion incorrectly states the induction of remyelination (please see my previous comments) there is just not enough actual evidence to make this claim.

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Reviewer #1: No

Reviewer #2: No

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-->PONE-D-26-04172R1-->-->Fasciola hepatica Excretory-Secretory Products Attenuate Demyelination and Reduce Neuroinflammation in the Cuprizone –Induced Multiple Sclerosis Model-->-->PLOS One

Dear Dr. Shabani,

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.

Please submit your revised manuscript by Jun 14 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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-->

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Tanja Grubić Kezele, Ph.D., M.D.

Academic Editor

PLOS One

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Additional Editor Comments:

Your manuscript, entitled "Fasciola hepatica Excretory-Secretory Products Attenuate Demyelination and Reduce Neuroinflammation in the Cuprizone – Induced Multiple Sclerosis Model", has been reviewed. Your efforts to revise the manuscript are appreciated. However, the peer review process continues because Reviewer 2 requests further revisions on certain issues the author should address. Please find the reviewer's commentary below.

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Reviewers' comments:

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Reviewer #2: (No Response)

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Reviewer #2: Yes

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

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: The revised manuscript addresses all my comments raised in the review. However, there are two minor issues remaining:

1. Line 75 - Fasciola hepatica should be italicized

2. Line 156 - please provide the references to support this statement

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Reviewer #2: No

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Fasciola hepatica Excretory-Secretory Products Attenuate Demyelination and Reduce Neuroinflammation in the Cuprizone –Induced Multiple Sclerosis Model

PONE-D-26-04172R2

Dear Dr. Shabani,

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,

Tanja Grubić Kezele, Ph.D., M.D.

Academic Editor

PLOS One

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