Dear Dr. Matsuoka,

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Ramada Rateb Khasawneh

Academic Editor

PLOS One

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1)  Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

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Additional Editor Comments:

Overall, this is an interesting and ambitious study with a promising conceptual framework. The approach has clear potential, but several areas would benefit from greater clarity, contextualization within existing literature, and a more cautious interpretation of the findings. Below, I outline the major strengths, followed by key concerns and recommendations for improvement.

1. Immunogenicity and Allogeneic Use — Need for Further Characterization

The manuscript reports “minimal immunogenicity” of cytokine-stimulated PFs based on the absence of HLA-II and co-stimulatory molecules (CD80/86). While this aligns with characteristics observed in MSC-based therapies, such in vitro findings do not fully predict in vivo immune responses. Allogeneic cell transplantation can still lead to immunological events such as allo-antibody formation, innate immune activation, or delayed rejection. A more comprehensive discussion of these potential risks—along with considerations regarding long-term engraftment, persistence, and possible tumorigenicity—would substantially strengthen the manuscript.

Furthermore, clarification is needed regarding the long-term stability and fate of transplanted PFs. It remains unclear whether cytokine-stimulated PFs sustain a lymphangiogenic phenotype over time or undergo phenotypic drift, dedifferentiation, or aberrant contributions to tissue remodeling after transplantation.

2. Specificity of the Observed Effects — Role of PFs vs. Other Cell Populations

The observed increases in lymphatic marker expression (LYVE-1, VEGFR3) and the reduction in fibrosis are attributed to the transplanted PFs. However, existing literature indicates that lymphangiogenesis during fibrosis often involves endogenous mechanisms, including activation of resident lymphatic endothelial cells or macrophage-derived lymphatic-like cells. Alternative explanations—such as host-mediated lymphatic remodeling or macrophage transdifferentiation—should be acknowledged and discussed.

If feasible, lineage tracing or similar techniques would provide stronger evidence regarding whether transplanted PFs directly differentiate into lymphatic endothelial-like cells or contribute structurally to lymphatic remodeling, rather than acting solely through paracrine signaling.

3. Quantitative Strength of Findings and Statistical Interpretation

Several results are described as demonstrating a “greater inhibitory trend,” particularly in the rPF+CKs group compared to rPF alone; however, these differences were not statistically significant. Such findings should be presented explicitly as trends and not interpreted as definitive effects. Similarly, although increases in lymphatic markers and ISH signals are reported, the manuscript acknowledges that the increase in LYVE-1 ISH did not reach statistical significance. These limitations should be more transparently addressed, ideally in both the Results and Discussion sections.

Interpretation of plasma biomarker trends (SP-D, fibrinogen) should also be tempered by the known variability of the bleomycin model and its partial spontaneous resolution. Overstating these findings as clear therapeutic effects may be misleading without stronger statistical support.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Excellent manuscript regarding Cytokine-stimulated pulmonary fibrobsis. Pulmonary fibroblastsmay serve as a novel cell

source for antifibrotic therapy by modulating lymphangiogenesis and tissue

remodeling, providing a potential alternative to conventional stem cell-based

approaches for fibrotic lung diseases as stated by the authors. This may be a new molecular pathway for potential treatment targets in pulmonary hypertension.

Reviewer #2: I would like to thank you and your colleagues for this excellent scientific work and for your valuable contribution to the advancement of science.

Review letter has been uploaded seperately.

Thank you.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Afendoulis Dimitrios

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Peer review-PLOS ONE.docx

Pulmonary fibroblasts activated by the addition of TNF-α and IL-4 enhance lymphangiogenic capacity and ameliorate lung fibrosis in an allogeneic rat model

PONE-D-25-47407R1

Dear Dr. Matsuoka,

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,

Ramada Rateb Khasawneh

Academic Editor

PLOS One

Additional Editor Comments (optional):

Good Luck

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: Interesting manuscript regarding trial of cytokine conditioned fibroblasts, which may be a new pathway

For treatment of pulmonary fibrosis. Most of the reviewers comments have been addressed, enforcing the impact of the data

Presented in the manuscript. My recommendation is to proceed for publication.

Reviewer #2: I would like to thank all the authors for this excellent research. I believe that this study will make a significant contribution to the existing literature and serve as a valuable reference for future research.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes: Afendoulis Dimitrios

Reviewer #2: No

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