PONE-D-23-02446Induction of Pro-Inflammatory Genes by Fibronectin DAMPS in Three Fibroblast Cell Lines:  Role of TAK1 and MAP kinasesPLOS ONE

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Reviewers' comments:

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Comments to the Author

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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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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?

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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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 is a rather straightforward study by experienced groups that address the remodeling of the extracellular matrix (ECM) and the Toll- Like Receptor-4 (TLR4) complexes and downstream signaling pathways in chronic inflammation. The authors previously showed that fibronectin’s extra domain A (FnEDA) and the partially unfolded first Type III domain (FnIII-1c) function as Damage Associated Molecular Pattern (DAMP) molecules to stimulate the induction of inflammatory cytokines by serving as agonists for TLR4. In this manuscript, they investigated the molecular steps regulating the fibronectin driven induction of inflammatory genes in three human fibroblast cell lines (human foreskin fibroblasts: A1F, adult human dermal fibroblasts: HDF, and Human Kidney fibroblasts: HKF).

Using the inhibitor and siRNA-mediated gene silencing of TAK1 and the automated capillary-based western system (WES), they showed that

1) Fibronectin DAMP mediates induction of IL-8 in fibroblasts (figure 1).

2) Fibronectin DAMPs, FnEDA and FnIII-1c, phosphorylate TAK1 at Thr184/187 in fibroblasts (figure 2).

3) TAK1 kinase activity regulating the fibronectin DAMP mediates IL-8 synthesis in both dermal and kidney fibroblasts (figure 3 and 4).

4) Fibronectin DAMP induced activation of the NF-κB and MAPK pathways requires TAK1 in all three cell lines (figure 5-7).

Using RT² Profiler PCR Array, they further demonstrated that

5) that differential expression of pro-inflammatory genes in fibroblast cell lines in response to fibronectin DAMPS (figure 8).

6) JNK inhibition reduced gene expression induced by fibronectin DAMPs/TAK1 to a higher degree than either p38 or ERK inhibition (figure 9).

Based on the above results they propose that FnEDA and FnIII-1c induce several pro-inflammatory cytokines whose expression is dependent on both TAK1 and JNK MAPK and highlight cell-type specific differences in the gene-expression profiles of the fibroblast cell-lines. I think this work is well organized and a nice contribution to the field of the remodeling of the ECM. This work could serve as a basis for further consideration of signaling molecules downstream of fibronectin DAMPs as a potential therapeutic target for hypertrophic skin disease.

I have only a concern to address as below.

There are no blot data in figure 6F, 6G, and 7F.

Reviewer #2: In this study by Maddali et al., the authors explored the role of two fibronectin DAMPs (FnEDA and FnIII-1c) in the activation of TGFb activated kinase 1 (TAK1) in fibroblasts derived from the kidney, foreskin or dermis. The authors show that both FnEDA and FnIII-1c are capable of activating the TAK1 and MAPK pathways in all three cell types, but that interestingly there appears to be organ specific differences in the inflammatory response. In particular they showed that with the exception of IL-8 different pro-inflammatory cytokines are upregulated in the different cell types. The authors also show that activation of TAK1 leads to activation of the IKK/NF-kB and MAPK pathways. This is a well written and very interesting study.

One point that should be addressed, however, is the observation that FnEDA and FnIII-1c appear to vary in their ability to IKK/NF-kB and MAPK pathways (Figure). FnIII-1c seems to be a weaker DAMP then FnEDA and did not show a very robust response. Do the authors have any explanation why this would be the case?

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Reviewer #1: No

Reviewer #2: No

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Induction of Pro-Inflammatory Genes by Fibronectin DAMPS in Three Fibroblast Cell Lines:  Role of TAK1 and MAP kinases

PONE-D-23-02446R1

Dear Dr. McKeown-Longo,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

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: In my opinion, in this revised manuscript, the responses to the major points raised previously were adequate and now this is acceptable for publication in PLOS ONE.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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