Single-Cell RNA-Seq Identifies Immune Remodeling in Lungs of β-Carotene Oxygenase 2 Knockout Mice with Improved Antiviral Response

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have demonstrated that β-Carotene oxygenase-2 knockout results in significantly improved outcomes following intranasal exposure to SARS-CoV-2. The topic and quality of the manuscript are appropriate for publication in Nutrients. However, I have a few questions:

    SARS-CoV-2 infection exhibits significant differences between male and female patients. The authors utilised both male and female mice in the presented study; however, the differences between male and female mice were only illustrated in Figure 4. Why did the authors not present and discuss the differences in the expression profiles of male and female mice?

    The authors obtained lung tissues from the mice, and given that SARS-CoV-2 can have a destructive effect on lung tissue, it would be beneficial to describe the differences in lung tissue concerning sex and BCO2 knockout within the manuscript.

    The introduction comprises only 28 lines, and the manuscript includes merely 31 references, which is rather limited. Furthermore, the differences between male and female responses and the effects of SARS-CoV-2 on lung tissue should be presented in greater detail. 

Author Response

Reviewer#1

Q: The authors have demonstrated that β-Carotene oxygenase-2 knockout results in significantly improved outcomes following intranasal exposure to SARS-CoV-2. The topic and quality of the manuscript are appropriate for publication in Nutrients.

A: Thank you for the positive assessment and constructive review. We have revised the manuscript substantially and addressed each of your questions point-by-point below. In brief, we (i) expanded the Introduction (by ~50%) to frame immune priming/resilience and lung innate signaling (cGAS–STING, RIG-I/MDA5–MAVS, IRF3), (ii) clarified study design and sex usage (male scRNA-seq/IRF3; both sexes for SARS-CoV-2 challenge) and added sex-stratified outcome text, (iii) rewrote the Discussion to emphasize results, mechanisms, and relevance, (iv) added a comprehensive “Scope, limitations, and future work” paragraph, (v) included a concise Conclusions section, and (vi) expanded (from 31 to 43) and reformatted the references to Nutrients style. All changes are highlighted in yellow. We respond to each of your specific questions below and indicate corresponding manuscript changes.

Q: SARS-CoV-2 infection exhibits significant differences between male and female patients. The authors utilised both male and female mice in the presented study; however, the differences between male and female mice were only illustrated in Figure 4. Why did the authors not present and discuss the differences in the expression profiles of male and female mice?

A: We appreciate this point. In our design, discovery assays—single-cell profiling (scRNA-seq/GSVA) and IRF3 immunoblotting—were performed in male mice to control hormonal variability and maximize power under BSL-3/resource constraints, whereas the SARS-CoV-2 challenge cohort included both sexes. Accordingly, we did not generate female scRNA-seq or IRF3 datasets and therefore could not present sex-stratified expression profiles.

To address this limitaiton, we have (i) clarified the sex usage explicitly in Methods and at the start of Results (Study design and sex scope), (ii) added sex-stratified descriptions of outcomes (weight loss and survival) with a note on the stronger effect in females (Fig. 4), and (iii) expanded the Discussion and clarified it in a Scope/Limitations/Future work section stating that female scRNA-seq and sex-specific IRF3 will be prioritized in follow-up studies to test for sex-dependent expression programs and pathway set points. [lines 331-355]

Q:  The authors obtained lung tissues from the mice, and given that SARS-CoV-2 can have a destructive effect on lung tissue, it would be beneficial to describe the differences in lung tissue concerning sex and BCO2 knockout within the manuscript.

A: We agree that tissue-level differences are important in the context of SARS-CoV-2. In the present study, we did not perform lung histopathology or viral-load quantification. Our lung tissues were used for male-only discovery assays (scRNA-seq/GSVA and IRF3 immunoblotting), while challenge outcomes (weight loss, survival) were collected in both sexes under BSL-3 constraints. Consequently, we are not able to report sex- and genotype-specific histologic differences in lung tissue in this dataset.

In response to your suggestion, we have:

• Made this limitation explicit in the Discussion section [lines 345-356].
• Expanded the Discussion with better explanation how these could differ by sex and Bco2
• Outlined planned follow-up studies incorporating sex-aware histopathology, viral titers, and spatial profiling to directly link the cellular programs we report to lung-injury endpoints.

We believe these revisions clarify the current scope and provide a concrete path to the tissue-level analyses you recommended.

Q: The introduction comprises only 28 lines, and the manuscript includes merely 31 references, which is rather limited. Furthermore, the differences between male and female responses and the effects of SARS-CoV-2 on lung tissue should be presented in greater detail. 

A: We have implemented the following revisions:

• Expanded Introduction (~50% increase): The Introduction now opens with the significance of immune priming/resilience, develops BCO2 within carotenoid/mitochondrial/redox biology and the IL18–BCO2 locus, and adds a concise overview of innate antiviral signaling (cGAS–STING; RIG-I/MDA5–MAVS; IRF3).
• Substantially enriched references: The reference list has been expanded to 43 items (from 31 previously), adding recent primary and review articles on epithelial–immune crosstalk and innate signaling (e.g., items 1, 2, 10, 13–16, 16–20, 34 in our numbering).
• Sex differences: We clarify our study design (male scRNA-seq/IRF3; both sexes for challenge) in Methods/Results and add a dedicated Discussion paragraph explaining the outcome differences and outlining planned female scRNA-seq and sex-specific IRF3 analyses. Outcome data are now explicitly sex-stratified (Fig. 4).
• Lung tissue effects: Although histopathology was not performed, we added sentences paragraph in the Discussion interpreting how the single-cell/signaling programs we report (AT2 substrate rerouting, ciliated remodeling, NK/cDC orchestration, IRF3 priming) could map onto tissue injury/repair during infection, and we specify planned histology, viral load, cytokine kinetics, and spatial analyses in the Scope, limitations, and future work paragraph.

We hope these changes address your concerns by providing a fuller conceptual frame, broader sourcing, clearer treatment of sex as a biological variable, and a concrete plan to connect cellular programs to lung tissue outcomes.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper "Single-Cell RNA-Seq Identifies Immune Remodeling in Lungs of β-Carotene Oxygenase 2 Knockout Mice with Improved Antiviral Response" is a well-conceived piece of research with interesting and innovative insights. The methods and results are well described, but the Introduction and Discussion are somewhat lacking and should be expanded to better understand the scope of this research. The Discussion, in particular, should emphasize the results and their relevance to the topic at hand. Conclusions that support the findings in the manuscript are also lacking. 

Methods
Lines 133 – 135 The Authors should specify antibody concentrations and secondary antibodies used in this experiment
Discussion
This section is quite scarce and should highlight the results and their relevance to the topic, novelty and innovation
Lines 254 – 257 Please remove the sentence “Authors should discuss the results and how they can be interpreted from the per- spective of previous studies and of the working hypotheses. The findings and their impli- cations should be discussed in the broadest context possible. Future research directions may also be highlighted”
Conclusions
A concluding section should be added to emphasize the outcome of this research

Author Response

Q: The paper "Single-Cell RNA-Seq Identifies Immune Remodeling in Lungs of β-Carotene Oxygenase 2 Knockout Mice with Improved Antiviral Response" is a well-conceived piece of research with interesting and innovative insights. The methods and results are well described, but the Introduction and Discussion are somewhat lacking and should be expanded to better understand the scope of this research. The Discussion, in particular, should emphasize the results and their relevance to the topic at hand. Conclusions that support the findings in the manuscript are also lacking. 

A: Response to Reviewer 2

Thank you for the thoughtful evaluation and clear guidance. We have substantially revised the manuscript to address each point:

• Expanded Introduction (scope and rationale): We added ~50% new content to frame immune priming/resilience at the respiratory barrier; position BCO2 within carotenoid/mitochondrial/redox biology and the IL18–BCO2 locus; and summarize innate antiviral signaling (cGAS–STING, RIG-I/MDA5–MAVS, IRF3). This establishes the study scope and the a priori mechanistic model that motivates our analyses.
• Rewritten Discussion (emphasis on results and relevance): The Discussion now opens with a system-level synthesis of our single-cell findings (NK/cDC expansion; AT2/ciliated programs; restrained NK effector modules), integrates IRF3 as a basal interferon hub, and explicitly connects BCO2-dependent mitochondrial/redox control to pattern-recognition set points. We added focused paragraphs on challenge outcomes and tissue context and sex as a biological variable, and a concise Scope, limitations, and future work paragraph that maps next steps to the reviewers’ requests.
• Conclusions added and strengthened: A new, one-paragraph Conclusions section distills the major findings and their impact: BCO2 as a nutrient-linked mitochondrial node that calibrates baseline innate set points, the coordinate-and-protect configuration aligned with basal interferon readiness, and improved challenge outcomes (stronger in females). It also articulates the translational significance while clearly noting the mechanistic (non-clinical) scope.
• References expanded: We incorporated additional primary and review sources to support the expanded Introduction and Discussion (e.g., epithelial–immune crosstalk, innate signaling pathways), and formatted them to Nutrients

We hope these revisions meaningfully strengthen the manuscript by clarifying scope, elevating the interpretation of results, and providing conclusions that directly support and contextualize our findings.

 

Q: Methods Lines 133 – 135 The Authors should specify antibody concentrations and secondary antibodies used in this experiment

A: We have added the requested information at Lines 167–170.

Q: Discussion: This section is quite scarce and should highlight the results and their relevance to the topic, novelty and innovation

A: We substantially rewrote and expanded the Discussion to highlight the findings, their relevance, and the manuscript’s novelty [lines 303-356]:

• Results-forward opening synthesis (new first paragraph of Discussion): We now lead with a concise summary of the single-cell shifts (NK/cDC expansion; reduced macrophage/B cells/ECs), epithelial programs (AT2 substrate rerouting; ciliated remodeling), and elevated IRF3, tying these directly to the study aim (baseline interferon readiness and “coordinate-and-protect” bias).
• Mechanistic through-line (added): We explicitly connect BCO2-dependent mitochondrial/redox control to pattern-recognition set-points, clarifying how composition/state changes map to innate signaling thresholds.
• Challenge outcomes in tissue context (added paragraph): We interpret the SARS-CoV-2 weight-loss/survival results in light of our cell-state programs (AT2/cilia resilience, NK/cDC orchestration, IRF3 priming) and specify predicted histologic correlates to guide future tests.
• Sex as a biological variable (added, explicit): We state that discovery assays were male (scRNA-seq/IRF3) and outcomes included both sexes, then discuss the stronger female signal and how it informs hypotheses.
• Scope/limitations/future work (consolidated, sharper): We provide a single, comprehensive paragraph that is concise but actionable.

Novelty and innovation (now stated explicitly in Discussion and Conclusions):

1. We reposition BCO2 from a carotenoid enzyme to a mitochondrial regulator of lung innate set-points, linking nutrition → organelle redox → mucosal immunity.
2. We provide the first single-cell atlas (to our knowledge) of Bco2-deficient lung showing a shift from kill-centric to coordinate-and-protect
3. We connect BCO2 status to IRF3-aligned interferon readiness, articulating a testable model for PRR threshold tuning.
4. We identify a sex-linked outcome signal (stronger benefit in females) that frames sex-aware mechanistic hypotheses for follow-up.

Manuscript changes: These additions appear in the Discussion (first four paragraphs), a strengthened Conclusions paragraph [Lines 572-372], and the expanded Introduction that sets the mechanistic scope. We hope these revisions address your request by making the Discussion more result-centric, clarifying relevance and impact, and stating the novel contributions explicitly.

Q: Lines 254 – 257 Please remove the sentence “Authors should discuss the results and how they can be interpreted from the per- spective of previous studies and of the working hypotheses. The findings and their impli- cations should be discussed in the broadest context possible. Future research directions may also be highlighted”

A: We are sorry about this issue. It has now been removed.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors have addressed all of my concerns with the original manuscript. The revised manuscript is ready for publication