Exploring the effects of IFN-τ on LPS-induced endometritis in cows based on transcriptomics

PLOS One

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PLOS One

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Additional Editor Comments:

The manuscript "Exploring the effects of IFN-τ on LPS-induced endometritis in cows based on transcriptomics" is an interesting investigation by the authors, conducted systematically with valuable findings. However, there are some points to be addressed before acceptance for publication as also pointed by the reviewers. So, it is recommended for a minor revision. In addition to the reviewers' comments, address the following points: 1. Correct the short title, 2. Insert literature about the clinical application of IFN-tau in endometritis (if available or justify your point), 3. Reduce the number of references, limiting it to 40 (delete less relevant citations), 4. Improve English grammar for better readability.

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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

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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: Manuscript ID: PONE-D-25-59265

Title: Exploring the effects of IFN-τ on LPS-induced endometritis in cows based on transcriptomics

The manuscript entitled “Exploring the effects of IFN-τ on LPS-induced endometritis in cows based on transcriptomics” describes the transcriptomic profile in bovine endometrial cell culture simulating LPS induced endometritis. Further, the effect of IFN-� on the modulation of transcript profile in LPS induced endometritis in bovine endometrial epithelial cell culture model. The manuscript is well designed and describes the experimental methodology and illustrated the significant findings in terms of differentially expressed genes and their associated biological pathway.

However, the rationale behind the use of IFN� as possible therapeutic target in endometritis is not clear. It is a proven fact that IFN� is a key signal molecule for maternal recognition of pregnancy in ruminants. It is produced by the mononuclear trophoblast cells during peri implantation period and suppress PGF2� release from the endometrium by modulating expression of oxytocin receptor (OTR) and Estradiol receptor. Thus, IFN� prevent luteolysis to maintain progesterone secretion from the corpus luteum required for establishment and maintenance of pregnancy. Progesterone has immunosuppressive effect on endometrium and aggravates the endometritis further. on the other hand, in bovine endometritis, PGF2� is considered as a drug of choice as it causes luteolysis and induces estrus, thus helps in reversal of progesterone effect.

In present study, the role of IFN� as a possible therapeutic option in bovine endometritis emphasizing only the anti-inflammatory role overlooking its role in modulating key steroidogenic pathway and their receptor expression limits its relevance as therapeutics in clinical use. It would be better to relook the transcriptomic profile considering the biological relevance of IFN-� in LPS induced bovine endometritis model and to explore the risk of MRP and increase chances of embryonic mortality in endometritis cows.

The rationale of the study needs to be focused considering these facts and the manuscript need to be revised thoroughly. Following points are to be considered for the revision.

1. Many abbreviations were used throughout the text without mentioning their expanded form. Always use the expanded form of the abbreviations when used for the first time.

2. Line 40: microorganisms invading the uterus through the open cervix during pregnancy. Check this statement. During pregnancy cervix remains closed and it prevents entry of pathogens into the uterus. While pathogen may get entry into the uterus through open cervix at estrus or during parturition.

3. The rationale behind use of IFN-� and LPS in the transcriptomic profiling in bovine endometrial epithelial cell culture model is not clear.

It is well established fact that in endometritis animal chances of embryonic survival is low due to inflammatory reactions in the endometrium, production of inflammatory mediators including PGF2� and failure of MRP due to sub-optimal production of IFN�. As IFN� is key signaling molecule for MRP, It would be better to highlight the importance of IFN� induced transcriptomic profile in LPS treated bovine endometrial epithelial cell culture model to understand the pathophysiology of early embryonic mortality in bovine endometritis.

4. In the experimental design three groups, Control (C), LPS (L) and IFN� + LPS (F) groups were included. To understand the effect of IFN� on the transcriptomic profile an additional group of IFN� alone is required.

5. Line #198-202: The Q20 base distribution ranged from 98.95% to 99.57% (Q20 > 90% was considered acceptable), Q30 bases ranged from 96.45% to 97.98% (Q30 > 80% was considered acceptable), and GC content ranged from 41.99% to 56.48%.

However, the values mentioned in Suppl. Table 1. does not match with the values mentioned in the text. Please check and clarify.

6. In the introduction and discussion, include relevant literature supporting the role of IFN � in modulating transcriptomic profile in MRP and during embryonic mortality in endometritis cows.

7. Conclusion: Concise the conclusion focusing the most significant findings and possible clinical applications in bovine endometritis.

Reviewer #2: The manuscript presents a well-designed transcriptomic study investigating the anti-inflammatory role of IFN-τ in LPS-induced bovine endometritis. The research question is clear, the methodology is robust, and the findings are supported by appropriate bioinformatic analyses. The study contributes meaningfully to understanding the molecular mechanisms of endometritis and potential therapeutic applications of IFN-τ. However, several editorial and formatting issues must be addressed to meet PLOS ONE.

Major Concerns

1. Figures and tables are referenced in the text (e.g., Fig. 1, Table 1), but the actual figures are not embedded in the submitted PDF (only file names are listed). Ensure all figures and tables are included in the manuscript file or as separate high-resolution files.

2. The statement is appropriate and includes an accession number (PRJNA1337137). Ensure the link is functional and that data will be publicly available upon publication.

3. The manuscript uses a commercial bovine endometrial epithelial cell line (BENDs). While this does not require IACUC approval, a clear statement confirming the cell line’s ethical sourcing and absence of animal experimentation should be provided in the Methods and the Ethics Statement section (currently “N/A”).

Minor Concerns

Short Title: Incorrectly lists section headers. Replace with a short, descriptive title.

Keywords: Should be listed as: endometritis; IFN-τ; transcriptomics; LPS (currently included in abstract).

Abbreviations: Define all abbreviations at first use (e.g., BENDs, LPS, IFN-τ, DEmRNAs, GO, KEGG).

Inconsistent Symbols: Use “IFN-τ” consistently (not “IFN-r”).

Page 10, Line 10: “Glycosphingolipid biosynthesis-lacto and neolacto series” – ensure spelling consistency (“lacto” vs. “lacta”).

Cell Culture: Specify the source and passage number of BEND cells.

RNA-seq Analysis: Mention software versions and parameters used for differential expression analysis (e.g., DESeq2, threshold: |log2FC| > 1, padj ≤ 0.05).

Ethical Compliance: Add a sentence confirming that no live animals were used and that the cell line was obtained commercially.

Clarity: Some descriptions are dense. Consider breaking into subsections (e.g., 3.1. RNA Quality Control, 3.2. Differential Expression, 3.3. Enrichment Analysis).

Supplementary Material: Ensure all supplementary figures/tables are cited in the text (e.g., Supplementary Fig. 1, Supplementary Table 1).

The discussion is comprehensive but could be more concise. Focus on integrating findings with existing literature and highlighting novel insights from this study.

Format references according to PLOS ONE style (e.g., Vancouver style). Ensure all in-text citations match the reference list.

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Reviewer #1: Yes: Manas Kumar Patra

Reviewer #2: No

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Exploring the effects of IFN-τ on LPS-induced endometritis in cows based on transcriptomics

PONE-D-25-59265R1

Dear Dr. Liu,

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,

Vikash Chandra, PhD

Academic Editor

PLOS One

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

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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

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Reviewer #1: Authors have addressed most of the comments raised by me. The revised manuscript is now suitable for acceptance.

Reviewer #2: Unresolved Minor Concern:

1. Corrected the conceptual error about cervical status (revised "during pregnancy" to "during estrus" for pathogen entry in endometritis).

2. Clarified the rationale for IFN-τ/LPS use (LPS as a key endometritis pathogen component; IFN-τ as anti-inflammatory/maternal recognition signal) and added relevant literature on IFN-τ’s role in embryo tolerance.

3. Resolved data inconsistency (Q20/Q30/GC content values now match Suppl. Table 1).

4. Streamlined the conclusion to focus on core findings (IFN-τ’s multi-pathway anti-inflammatory effects) and clinical applications.

5. Improved English grammar via native speaker revision

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