-->PPATHOGENS-D-25-02280

BRD4 modulates antimicrobial defense via non-canonical NRF2 activation in macrophages to confer protection against sepsis

PLOS Pathogens

Dear Dr. Hu,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 09 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Vasundhra Bhandari

Academic Editor

PLOS Pathogens

Thomas Guillard

Section Editor

PLOS Pathogens-->--> -->-->Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

-->-->

Michael Malim

Editor-in-Chief

PLOS Pathogens

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This manuscript provides an important conceptual advance by positioning BRD4–NRF2 as a central regulatory axis in innate immunity during sepsis. The integration of mouse genetics, cellular functional assays, and human data is commendable. With additional experiments—particularly verifying NRF2 dependence, endogenous BRD4–NRF2 interaction, and proper statistical control—this work would be substantially strengthened. I recommend major revision, but I am confident that after revision, this study will have high impact and broad relevance.

Reviewer #2: In this manuscript, Hu and colleagues studied the pathogenic role of BRD4, an epigenetic regulator, in sepsis, a life-threatening condition characterized by dysregulated immune

responses and high mortality, with a focus on the relevance of BRD4 in monocytes/macrophages. The claimed myeloid-specific function of BRD4 was supported by the evidence from human patients and murine CLP models. Mechanistically, the interaction between BRD4 and NRF2, which disrupts the NRF2-KEAP1 complex thus enhancing NRF2 stability and nuclear translocation and upregulating scavenger receptors essential for bacterial clearance. The role of BRD4 in monocytes/macrophages and sepsis was explored previously, which dampens the novelty of current study; however, the data presented, particularly those in the mouse CLP model would be of interest to the field.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: This study identifies the BRD4–NRF2 axis as a novel regulatory pathway controlling macrophage antimicrobial defense in sepsis—an insightful connection between epigenetic regulation and host immunity. Using both human and murine data, the authors convincingly show that BRD4 expression decreases in monocytes/macrophages and correlates with disease severity. The use of myeloid-specific Brd4 knockout mice demonstrates that BRD4 loss leads to impaired bacterial clearance and higher mortality. The mechanistic link between BRD4-mediated NRF2 stabilization, scavenger receptor induction, and the rescue effect by sulforaphane (SFN) suggests important therapeutic potential. Overall, the manuscript is solid and of high impact, suitable for PLOS Pathogens, but several aspects require further strengthening.

1. The causal relationship between BRD4 and NRF2 activation needs to be demonstrated more rigorously. The authors conclude that BRD4 stabilizes NRF2, thereby inducing MARCO/MSR1 expression, but they have not performed epistasis analyses in vivo (e.g., double knockout Brd4^f/f; LysM-Cre × Nrf2^f/f, or NRF2 overexpression rescue). To confirm that the proposed BRD4→NRF2 signaling axis is causal, additional experiments are necessary. The authors should test whether the rescue effect of SFN depends on NRF2—using either pharmacologic inhibition (ML385) in vitro or myeloid-specific Nrf2 deletion in vivo. These experiments would establish the directionality and NRF2-dependence of BRD4’s effects.

2. The direct interaction between BRD4 and NRF2 should be more conclusively demonstrated. The current co-immunoprecipitation data rely largely on overexpression in 293T cells. Endogenous BRD4–NRF2 binding should be confirmed in primary macrophages using co-IP or proximity ligation assays (PLA). To support the proposed KEAP1 competition mechanism, in vitro competition assays with recombinant proteins (pull-down or SPR) would be highly informative. In addition, genome-wide analyses (ChIP-seq or CUT&RUN) for NRF2 and BRD4 co-occupancy would strengthen the evidence for cooperative transcriptional regulation.

3. The SFN rescue experiments are compelling but need to rule out off-target effects. Because SFN affects multiple pathways, the authors should clarify whether its protective effects are strictly NRF2-dependent. Combining SFN with NRF2 inhibition, and testing different doses or treatment timing, would improve translational relevance and mechanistic specificity. Although SFN improved survival in both WT and Brd4-CKO mice, this does not by itself prove NRF2 exclusivity—clarifying this would strengthen the conclusion.

4. Concerning the LysM-Cre model, the manuscript should acknowledge its known limitations. LysM-Cre drives recombination in both macrophages and neutrophils; thus, the phenotype might partly reflect altered neutrophil function. Assessing neutrophil phagocytosis, bactericidal activity, or NETosis—or reproducing key experiments using a macrophage-specific driver (e.g., Cx3cr1-CreER)—would help to exclude this confounding factor.

5. The human data are of high translational value but require better statistical control. Multivariate analyses adjusting for age, sex, infection source, comorbidities, and treatment variables should be presented to confirm the independent association between BRD4 (and NRF2) expression and clinical severity. Receiver operating characteristic (ROC) analysis for prognostic performance would also add rigor. Furthermore, sampling timing and disease stage heterogeneity should be clarified, and longitudinal analysis of expression dynamics would be valuable.

6. Mechanistically, the authors could explore how BRD4 inhibits NRF2 ubiquitination beyond KEAP1 competition—whether it recruits deubiquitinases or influences Cullin3 E3 ligase activity.

7. In the discussion, the authors should elaborate on the dual nature of BRD4—as both an enhancer of inflammatory gene expression and a protector of antimicrobial function. Presenting a phase-specific therapeutic framework (inhibiting BRD4 in early hyperinflammation, activating it during the immunosuppressive phase) would increase clinical relevance. Additionally, potential risks of sustained NRF2 activation (tumor promotion, metabolic effects) should be briefly mentioned.

Reviewer #2: Figure 3A, it is not clear the bacterial burden was measured under anaerobic or aerobic culture condition? If the colony-formed Gram-negative and -positive bacteria could be isolated and identified, would they be more relevant species than E. coli and S. aureus to assess the myeloid-specific impact of BRD4 in bacterial clearance and sepsis, as in Figure 3?

Myeloid-specific Brd4 knockout mice was previously shown resistant to LPS-induced sepsis, while the current study demonstrated that Brd4 conditional knockout mice were more susceptible to CLP-induced polymicrobial sepsis (ref 21). Such phenotype difference should be discussed in depth, in particular, the identification of the recruitment of Brd4 to the promoters of Marco and Msr1 was based on the LPS treatment data (Supplementary Figure 5 and Figure 5A).

What were the transcription levels of Brd4 in Figure 1 J and K? It would be necessary to ascertain whether the decreased levels of BRD4 in later time points could result from the protein degradation.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: Yes: Fengyi Wan

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PPATHOGENS-D-25-02280R1

BRD4 modulates antimicrobial defense via non-canonical NRF2 activation in macrophages to confer protection against sepsis

PLOS Pathogens

Dear Dr. Hu,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 17 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Vasundhra Bhandari

Academic Editor

PLOS Pathogens

Thomas Guillard

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have carefully addressed the major concerns raised in the previous round of review and have performed several important additional experiments that substantially strengthen the manuscript. In particular, the newly added NRF2 reconstitution experiments in Brd4 conditional knockout mice, the endogenous BRD4–NRF2 interaction analysis in primary macrophages, and the NRF2 inhibitor (ML385) experiments confirming the specificity of SFN-mediated rescue significantly improve the mechanistic rigor of the study. These new data provide convincing support for the proposed BRD4–NRF2 signaling axis in regulating macrophage antimicrobial function during sepsis. The authors have also made reasonable efforts to address the concerns regarding the LysM-Cre model by examining neutrophil antimicrobial functions, which adds valuable insight into the broader role of BRD4 in myeloid innate immunity.

Reviewer #2: The authors have addressed the concerns and critiques in a satisfying manner. The revised verson of the manuscript with newly added experimental data and extensive revisions is suitable for publication.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: Not applicable.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Overall, the revised manuscript represents a substantial improvement and now provides a coherent mechanistic framework linking BRD4 to NRF2-dependent antimicrobial responses.

However, a few minor issues remain that should be clarified before publication.

1. The ChIP-seq analysis supporting BRD4–NRF2 co-occupancy relies on publicly available datasets. While this analysis provides useful bioinformatic support, the authors may clarify this limitation in the manuscript and discuss potential context-dependent differences between experimental systems.

2. In the KEAP1 competition assays, a BRD4 deletion construct (BRD4-PID) was used instead of full-length BRD4. The authors may briefly discuss whether additional domains of BRD4 could influence NRF2 binding in vivo.

3. The discussion of the LysM-Cre model would benefit from a brief statement acknowledging that both macrophages and neutrophils contribute to the observed phenotype, as suggested by the newly added neutrophil functional assays.

Reviewer #2: Not applicable.

**********

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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: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

Figure resubmission:

-->While revising your submission, we strongly recommend that you use PLOS’s NAAS tool (https://ngplosjournals.pagemajik.ai/artanalysis) to test your figure files. NAAS can convert your figure files to the TIFF file type and meet basic requirements (such as print size, resolution), or provide you with a report on issues that do not meet our requirements and that NAAS cannot fix.-->-->

After uploading your figures to PLOS’s NAAS tool - https://ngplosjournals.pagemajik.ai/artanalysis, NAAS will process the files provided and display the results in the "Uploaded Files" section of the page as the processing is complete. If the uploaded figures meet our requirements (or NAAS is able to fix the files to meet our requirements), the figure will be marked as "fixed" above. If NAAS is unable to fix the files, a red "failed" label will appear above. When NAAS has confirmed that the figure files meet our requirements, please download the file via the download option, and include these NAAS processed figure files when submitting your revised manuscript.-->

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Prof. Hu,

We are pleased to inform you that your manuscript 'BRD4 modulates antimicrobial defense via non-canonical NRF2 activation in macrophages to confer protection against sepsis' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Vasundhra Bhandari

Academic Editor

PLOS Pathogens

Thomas Guillard

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (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: Shigeki Nakamura