The Neuroimmunological Nexus of Multiple Sclerosis: Deciphering the Microglial Transcriptomic Tapestry

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the manuscript entitled: The Neuroimmunological Nexus of Multiple Sclerosis: Deciphering Microglia Transcriptomic Tapestry. The manuscript covers an important topic the role of microglia in development of multiple sclerosis. Overall the manuscript is interesting but numerous corrections need to be made.

Technical suggestions.

1.      “Running Title” - is unnecessary; similar “Main points” can be deleted.

2.      The reference style is incorrect.

3.      All abbreviations in abstract and in manuscript should be explained e.g. CNS (lines 15 and 34), please check the entire manuscript carefully, many abbreviations are missing (or explanation of abbreviations).

4.      The beginning of the manuscript has no subsection. It should be “1. Introduction” then the numbering of the remaining chapters will change.

Comments about the content.

1.      Line 133 the authors mention fibrin, is there anything known about the influence of elastin derived peptides (EDPs) on microglia? Currently, many manuscripts show the role of EDPs in the development of neurodegenerative diseases. Is there any connection with microglia?

2.      Line 149 - aryl hydrocarbon receptor abbreviation should be added (AhR). In addition, AhR also interacts with EDPs. Line 433 – PPARG also interacts with EDPs. Maybe there is a link with microglia or gila?

3.      The figures are very aesthetic, please increase the resolution if possible.

4.      Line 307 – reference should be added. If you write "many studies", please provide references.

5.      Chapter 5 lines 357 to 366 is very short, maybe expand this chapter or combine it with other.

6.      Add references in lines 413 to 425.

7.      In conclusion, there should be no references. The conclusions are to result from the content of the manuscript.

Author Response

Reviewer 1

Comments and Suggestions for Authors

Review of the manuscript entitled: The Neuroimmunological Nexus of Multiple Sclerosis: Deciphering Microglia Transcriptomic Tapestry. The manuscript covers an important topic the role of microglia in development of multiple sclerosis. Overall the manuscript is interesting but numerous corrections need to be made.

Technical suggestions.

Comment 1_1: “Running Title” - is unnecessary; similar “Main points” can be deleted.

Response 1_1: Thank you for your feedback. We have revised the manuscript accordingly. Specifically, we have removed the "Running Title" and "Main points" sections as per your suggestion.

Comment 1_2: The reference style is incorrect.

Response 1_2: We have changed the reference style as per the MDPI Endnote style.

Comment 1_3: All abbreviations in the abstract and in manuscript should be explained e.g. CNS (lines 15 and 34), please check the entire manuscript carefully, many abbreviations are missing (or explanation of abbreviations).

Response 1_3: Thank you for pointing out this. We have checked and made it uniform wherever the abbreviations are missing we corrected it.

Comment 1_4: The beginning of the manuscript has no subsection. It should be “1. Introduction” then the numbering of the remaining chapters will change.

Response 1_4: We have revised the manuscript to include an “Introduction” section at the beginning and adjusted the numbering of the remaining chapters accordingly.

Comments about the content.

Comment 1_5: Line 133 the authors mention fibrin, is there anything known about the influence of elastin-derived peptides (EDPs) on microglia? Currently, many manuscripts show the role of EDPs in the development of neurodegenerative diseases. Is there any connection with microglia?

Response 1_5: Thank you for your insightful query. Elastin-derived peptides (EDPs) have been well-reported in the context of vascular and age-related vascular diseases, with the main repeating unit being VGVPAG. Although the exact mechanism of action of EDPs is not entirely clear, (Szychowski, K. A., Skóra, B., & Wójtowicz, A. K. (2022). Elastin-derived peptides in the central nervous system: friend or foe. Cellular and Molecular Neurobiology, 42(8), 2473-2487). Research published in 2022 suggests that VGVPAG acts through two receptors: Galectin 3 and the Elastin Receptor Complex (ERC). During our literature survey, we found one paper published in 2024 (Ma, J., Wang, B., Wei, X., Tian, M., Bao, X., Zhang, Y., ... & Hu, M). Accumulation of extracellular elastin‐derived peptides disturbed neuronal morphology and neuron–microglia crosstalk in aged brain. Journal of Neurochemistry). indicating that VGVPAG plays a role in the hyperactivation of microglia both in vitro and in vivo. The study suggests that incubation with EDPs leads neurons to release Galectin 3, which in turn activates microglia. This activation results in increased microglial phagocytosis and synapse loss. While these findings indicate that EDPs may contribute to microglial activation and neuron-microglial crosstalk, more studies are needed to fully understand the influence of EDPs on microglia. We appreciate your suggestion, which has helped us to consider this emerging area for future research.

Comment 1_6: Line 149 - aryl hydrocarbon receptor abbreviation should be added (AhR). In addition, AhR also interacts with EDPs. Line 433 – PPARG also interacts with EDPs. Maybe there is a link with microglia or gila?

Response 1_6: Thank you for your comments. We have addressed them as follows:

• We have added the abbreviation for aryl hydrocarbon receptor (AhR).
• EDPs do interact with glial cells, mainly astrocytes. There are reports suggesting their interaction with astrocytes. For example, in an in vitro study (Szychowski, K. A., & Gmiński, J. (2020). The elastin-derived peptide VGVAPG does not activate the inflammatory process in mouse cortical astrocytes in vitro. Neurotoxicity Research, 37(1), 136-145.), VGVPAG increased the activity of caspase-1 in mouse primary astrocytes. Caspase-1 plays a dual role in both the inflammatory process and cytoprotective effects.
• VGVPAG acts as a Peroxisome Proliferator-Activated Receptor Gamma (PPAR-ϒ) agonist. The PPAR-ϒ agonist disrupts the main inflammatory pathway, Nuclear Factor kappa-light-chain-enhancer of activated B cells, which is the primary mechanism by which PPARG agonists reduce inflammation. Therefore, VGVPAG acts as a PPAR-ϒ agonist and decreases markers of inflammation in mouse astrocytes. Further research is needed before reaching firm conclusions (Szychowski, K. A., & Gmiński, J. (2020). Elastin-derived peptide VGVAPG affects the proliferation of mouse cortical astrocytes with the involvement of aryl hydrocarbon receptor (Ahr), peroxisome proliferator-activated receptor gamma (Pparγ), and elastin-binding protein (EBP). Cytokine, 126, 154930)
• Moreover, both receptors, PPAR-ϒ and AhR, are present in microglia. AhR is well expressed in microglia involved in inflammatory disorders. AhR and PPAR-ϒ are involved in the mechanism of action of VGVPAG: AhR silencing results in an increase in S100B protein and Ki67. The S100B protein is involved in the initiation of inflammation in macrophages and microglial cells. For PPAR-ϒ, the effect of its silencing depends on the concentration of the medium. The role of EDPs in microglial cells is not well-documented, and more research is needed in this area. Both reports confirm the beneficial role of VGVPAG on astrocytes.

Comment 1_7: The figures are very aesthetic, please increase the resolution if possible.

Response 1_7: Thank you for your kind words. We appreciate your positive feedback. The diagrams were created using the paid version of BioRender.com. Previously, the resolution was set at 300 DPI. We have now increased the resolution to 600 DPI to enhance the clarity and quality of the figures.

Comment 1_8: Line 307 – reference should be added. If you write "many studies", please provide references.

Response 1_8: Thank you for your comment. We have added references where many studies are added.  All newly added references are highlighted in yellow

Comment 1_9: Chapter 5 lines 357 to 366 is very short, maybe expand this chapter or combine it with others

Response 1_9: Thank you for the suggestion. Yes, we have combined this part in the previous section which is section in section 4.

Comment 1_10: Add references in lines 413 to 425.

Response 1_10: Thank you for your Comment. We have included references for the lines that discuss the morphological aspects and classification of different microglia.

      Comment 1_11: In conclusion, there should be no references. The conclusions are to result from the content of the manuscript.

Response 1_11: Thank you for your insightful comment. We have revised the conclusion section to ensure that it is based solely on the content presented within the manuscript. All references have been removed from the conclusion. We appreciate your guidance in improving the clarity and coherence of our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

First of all, I would like to congratulate the authors for this very exhaustive review. It has been very interesting to read. I think that it is a very good job, and I have minor suggestions, and maybe the most relevant one is to explain better the potential relevance of the information provided for the authors for current and future clinical practice, add a more translational focus to make it more interesting for clinicians as well.

 

Here are the other minor concerns: 

 

1) Abstract: please avoid to use acronyms without mention its significance first: for istance CNS or MS (in this case you could add the acronym after the first mention). Correct in line 17: "an d" to "and".

2) Introduction: 

I believe that the paragraph between line 43 to 84 Is excessive. Evaluate the possibility to create a summary table with critical information.

Explain better the necessity of this study including clinical perspective. Remove the ideas that you are not going to explore, if it is clear what you are going to do is somewhat repetitive. 

I would suggest to remove the sentence between 95-97. This should be explained at each section that evidence is presented. 

Consider to refer better how MS is diagnosed, MS types,... it is critical to have a short mention to it. 

Create subsections for point 2. For instance, divide phagocytosis related section to those related with the complement, it could increase the capacity to properly distinguish the relevance of each section. 

Section 6 Title: MS-associated microglia vs Neurodegenerative disorders associated microglia.

3) Divide conclusions section in discussion (most of it indeed is discussion) and Crete a subsection about limitations/research gaps and proposals (critical lecture and creative one) by the authors on how to respond to this gaps. This could be done with a table; and conclusion. This last one should be the take home message section, with the main 2 or 3 ideas summarized. 

4) After the introduction: add a section of research methodology. Explain how the research was performed, which databases were used, paper selection criteria... A flowchart could be helpful.

 

Author Response

Reviewer 2

Comments and Suggestions for Authors

First of all, I would like to congratulate the authors for this very exhaustive review. It has been very interesting to read. I think that it is a very good job, and I have minor suggestions, and maybe the most relevant one is to explain better the potential relevance of the information provided for the authors for current and future clinical practice, add a more translational focus to make it more interesting for clinicians as well.

Here are the other minor concerns: 

Comment 2_1: Abstract: please avoid to use acronyms without mention its significance first: for istance CNS or MS (in this case you could add the acronym after the first mention). Correct in line 17: "an d" to "and".

Response 2_1: Thank you for your valuable feedback. We have made the following corrections to the abstract: All acronyms are now defined upon their first mention. For example, "central nervous system (CNS)" and "multiple sclerosis (MS)". The typographical error in line 17 has been corrected from "an d" to "and". We appreciate your thorough review and believe these changes have improved the clarity and readability of the abstract.

Comment 2_2: Introduction: I believe that the paragraph between line 43 to 84 Is excessive. Evaluate the possibility to create a summary table with critical information.

Response 2_2: Thank you for your suggestion regarding the introduction. We have shorten it and made it a concise way. While we understand the potential benefit of a summary table, we believe it may not be necessary as we have thoroughly explained the relevant information in the different subsections. Each subtype of microglia has been detailed with disease-specific information, which may overlap if summarized again in a table. However, if you still strongly feel that a summary table would enhance clarity and organization, we are more than willing to create one. Your feedback is valuable to us, and we aim to ensure the manuscript is as clear and concise as possible.

Comment 2_3: Explain better the necessity of this study including clinical perspective. Remove the ideas that you are not going to explore if it is clear what you are going to do is somewhat repetitive. 

Response 2_3: Thank you for your Suggestion. In the Introduction as per your suggestion, the clinical perspective has been integrated as updated Text. We acknowledge the importance of clarifying the necessity of this study from a clinical perspective. In conclusion, already 2-3 lines were related to this” Thus, these discoveries offer hope for developing transformative treatments involving microglial-mediated therapies, specifically targeting proteins, with a concept of immunomodulation compared to global immunosuppressive therapies, which have detrimental side effects for MS Regarding the redundancy of certain ideas, we have ensured to focus specifically on microglia and MS, avoiding unnecessary repetition of concepts that have been adequately covered in previous sections.

Comment 2_4: I would suggest to remove the sentence between 95-97. This should be explained in each section that evidence is presented. 

Response 2_4: Thank you for your suggestion. We have removed the sentences between 95-97 (as per the line number of previous manuscript version) We have included only the hallmarks that are directly relevant to this article and are commonly reported as significant to the disease discussed. While we understand the value of providing evidence in relation to hallmarks pertinent to each subsection, doing so without specific references to transcription factors, as the original research articles (and other sources) did not describe, could lead to inaccuracies and speculation. Expanding on these sections might result in a much longer version, which could deviate from the intended focus of our article. Nonetheless, we genuinely appreciate your insightful suggestion. If further details (in form of different subsections already described in the manuscript) on these points is deemed essential, we are more than willing to incorporate additional details to enhance the quality of our work.

Comment 2_5: Consider to refer better how MS is diagnosed, MS types,... it is critical to have a short mention to it. 

Response 2_5: Thank you for your suggestion. Yes, we have included a very short description as many papers have already described it in detail and excellent versions. The updated Text is (Page number 1 and Line number 35-41).

Comment 2_6: Create subsections for point 2. For instance, divide phagocytosis related section to those related with the complement, it could increase the capacity to properly distinguish the relevance of each section

Response 2_6: Thank you for your suggestion. We have divided section 3 (updated in our manuscript,previously was section 2) in subsection-

• Unsettled role of microglial phagocytosis-Phagoptosis and Trogocytosis
• Involvement of indirect pathways in microglial’s cascading effects

Comment 2_7: Section 6 Title: MS-associated microglia vs Neurodegenerative disorders associated microglia.

Response 2_7: Thank you for the suggestion. This we have incorporated in the heading.

Comment 2_8: 3) Divide conclusions section in discussion (most of it indeed is discussion) and Crete a subsection about limitations/research gaps and proposals (critical lecture and creative one) by the authors on how to respond to this gaps. This could be done with a table; and conclusion. This last one should be the take home message section, with the main 2 or 3 ideas summarized. 

Response 2_8: Thank you for your comment. Yes, we have divided the discussion and conclusion parts. The discussion has been designated as section 9. Conclusion has been designated as section 10. In conclusion, no references are there.

Comment 2_9: After the introduction: add a section of research methodology. Explain how the research was performed, which databases were used, paper selection criteria... A flowchart could be helpful.

Response 2_9: Thank you for your comment. This we have added after the introduction as Updated Text” For this review, we have searched the databases Scopus and Pubmed with a keyword like Single-cell-RNA-sequencing, microglia, Transcriptomic profile of microglia, microglia cells in Multiple Sclerosis”.

Author Response File: Author Response.pdf