PONE-D-24-47001Synovial Gene Expression after Hemarthrosis Differs Between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion ProteinPLOS ONE

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Reviewers' comments:

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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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 study examines at day 3 and day 14, the effects of murine FVIII-Fc fusion protein (mFcFVIII) in comparison to recombinant human FVIII (rhFVIII) on synovial molecular processes in a context of hemarthrosis. Hemarthrosis were induced in FVII-KO mice by sub-patellar needle puncture and an approach bases on RNAseq and differential gene expression analysis complimented by KEGG and Reactome pathway analysis were applied to synovial tissue. The authors observe a small proportion of genes affected by FVIII treatment and distinct differences between both FVIII concentrates, both related and unrelated to injury/bleed. At day 3, mFcVIII had effects on immune and inflammatory processes whereas rhFVIII seems more modulated mRNA and protein processes. At day 14, molecular profiling revealed expression changes in favour of a transition to synovial tissue repair and remodelling and the authors argue that mFcFVIII compared to rhFVIII facilitated the M1 to M2 macrophage transition. The study, only based on transcriptomic analysis, seems methodologically well conducted, but some issues need to be addressed:

Major remarks:

- The limitations of studies based solely on transcriptomic approaches are not indicated in discussion. These limitations must be added.

- In results section, the authors written at the beginning of the section “injured mice treated with rhFVII vs mFcFVII revealing similar expression profiles without statistical difference” that is in contradiction with the results that follow. Can the authors revise the wording of this sentence to be more explicit?

- The classification of genes/pathways as on-target (related to injury/bleed) and off-target (unrelated to injury/bleed) is not very clear. For example, in results, cytokine-cytokine receptor interaction is on-target and perturbation of immune system is off-target and focal adhesion is on-target and cell adhesion is off-target. This point needs to be clarified.

- In results section, the second paragraph focus on used methodology and should be moved in material and methods section.

- Did the authors apply the cell-type deconvolution from bulk RNASeq using immune cell references at day 3. It would be interesting to present this data.

- Concerning the differential expression levels of macrophage subtype-specific markers M1 (figure 5B), the authors indicated in results section “among the 5 M1 markers, only il6 was mildly increased after saline treatment compared to baseline and decreased with both FVIII-preparations, indicating overall abating inflammation”. The effect observed is very modest and the authors seem to have overinterpreted their results.

- It is the same remark for the discussion about these results (“mFcFVIII facilitate a polarization to M2, not observed to the same extent with rhFVIII)”. Concerning the M2 markers, the difference between rhFVIII and mFcFVIII for lrf4 seems minimal. The last sentence of the results needs to be modified. What are the results for the anti-inflammatory M2 macrophages markers?

Minor remarks:

- The use of abbreviations needs to be reviewed. There should be no abbreviation in the abstract and once an abbreviation is defined in the text it should then be systematically used.

- The title of the Table I is cut.

- In supplementary figures, the figure 1 is illegible for the part on the right.

Reviewer #2: The authors used next-generation RNA sequencing (RNA-Seq) to analyse the temporal evolution of molecular processes in synovial tissue after haemarthrosis in a mouse model of haemophilia A. They studied changes in gene expression and disruption of molecular pathways with or without treatment with factor VIII (FVIII) on days 3 and 14 after haemarthrosis. In addition, they compared the effects of murine FVIII fused to an Fc portion (mFcFVIII) with those of recombinant human FVIII (rhFVIII, without Fc portion) to determine whether this Fc portion influences processes linked to inflammation, tissue repair or immune responses. The results show that in the acute phase (D3), mFcFVIII had unique on-target effects related to immune and inflammatory regulation, whereas rhFVIII primarily affected mRNA and protein processing. At day 14, macrophage profiling indicated a transition from M1 to M2, and only mFcFVIII uniquely influenced pathways and genes associated with tissue remodelling and repair.

This study, conducted by a team with internationally recognized expertise in the field and supported by prior publications on the models used, is robustly designed and executed. The limitations of the work are clearly and transparently presented. The most innovative findings stem from the use of mFcFVIII, which, after 14 days, demonstrated a unique influence on molecular pathways and genes associated with tissue remodeling and repair. The authors attribute this beneficial effect to the mFc portion of the molecule.

However, this conclusion could be further strengthened by including one or two control groups of hemophilia A mice treated either with isolated mFc or with free rhFVIII combined with mFc. Additionally, the study would benefit from experimental evidence, such as histological observations, explicitly demonstrating the beneficial effects of mFcFVIII on vascular tissues in hemophilic mice.

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Reviewer #1: No

Reviewer #2: No

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Synovial Gene Expression after Hemarthrosis Differs Between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion Protein

PONE-D-24-47001R1

Dear Dr. Joseph,

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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 #1: All comments have been addressed

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: Yes

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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 #1: (No Response)

Reviewer #2: All my comments have been thoroughly addressed by the authors. The additional experiments have been properly conducted, and the text has been revised accordingly.

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Reviewer #1: Yes:  Annabelle Dupont, Université de Lille, CHU de Lille service d'hémostase et transfusion, UMR Inserm 1011

Reviewer #2: No

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