Immune Dysregulation and Cytokine Profiling in Acute Mycoplasma pneumoniae Pneumonia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors are requested to address the following points to revise the manuscript:

1. Several sections of the methodology lack appropriate references. Relevant citations should be added to support the described experimental procedures.

2. The authors should clearly describe the environmental or host factors that lead to the upregulation of interleukins and chemokines. This discussion must be supported by appropriate experimental or literature-based evidence.

3. It would be beneficial to provide detailed information on the primer sequences used for RT-PCR, including forward and reverse sequences.

4. The nucleic acid extraction procedure should be described in detail. The authors are expected to clearly outline each methodological step to ensure reproducibility of the experiments.

Author Response

Comments 1: Several sections of the methodology lack appropriate references. Relevant citations should be added to support the described experimental procedures.

Response 1: Thank you for your valuable suggestions. We agree with your comment and have reviewed the methodology sections, adding appropriate references to support the described experimental procedures. The experimental procedures in Sections 2.5, 2.6, and 2.7 now include citations from relevant literature to enhance the credibility and reliability of our methods.

Comments 2: The authors should clearly describe the environmental or host factors that lead to the upregulation of interleukins and chemokines. This discussion must be supported by appropriate experimental or literature-based evidence.

Response 2: We thank the reviewer for this critical suggestion to enhance the mechanistic depth of our discussion. We agree that elucidating the triggers for cytokine upregulation is essential. In direct response to the reviewer's suggestion, we have revised the Discussion section by adding a new paragraph immediately following the description of our key findings. This new paragraph systematically describes the environmental and host factors that lead to the upregulation of interleukins and chemokines, as follows:

Pathogen-derived triggers, known as lipoproteins and TLR2 have been thought to be important for the pathogenesis of M. pneumoniae; host-derived amplification signals, specifically damage-associated molecular patterns (DAMPs) released from tissue damage, activate immune cells via the NLRP3 inflammasome.; and the IL-23/IL-17 axis plays a critical role in specifically driving the neutrophilic chemokine response, consistent with our cellular data.

Each point is supported by appropriate citations from the literature [9,15,34–35,]. This addition provides a clear causal framework that connects our observational findings to established immunological mechanisms, directly addressing the reviewer's comment.

Comments 3: It would be beneficial to provide detailed information on the primer sequences used for RT-PCR, including forward and reverse sequences.

Response 3: We are grateful for the reviewer’s suggestions. But we apologize for that the Mycoplasma pneumoniae (MP) Real-Time PCR Kit was purchased from Shanghai Liferiver Biotechnology Co., Ltd. Regarding your enquiry about providing primer sequences, we have engaged in multiple communications with the reagent supplier to confirm the matter. The current situation is that the supplier's confidentiality agreement restricts them from disclosing specific primer sequences externally. This constitutes a common industry compliance requirement, designed to safeguard their intellectual property and client confidentiality. Nevertheless, we strictly adhered to the reagent usage instructions throughout the experiment to ensure the reliability of the results.

Comments 4: The nucleic acid extraction procedure should be described in detail. The authors are expected to clearly outline each methodological step to ensure reproducibility of the experiments.

Response 4: Thank you for your valuable suggestions. We have added relevant descriptions in section 2.4. of the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract (Lines 10–27)

• Lines 11–13: The background is clear and relevant; however, consider briefly stating the knowledge gap more explicitly to strengthen the rationale.

• Lines 14–15: The animal model and key methods are appropriately summarized. Consider clarifying why a murine model is particularly suitable for studying acute MPP.

• Lines 18–20: The cytokine findings are strong; specifying whether these cytokines were identified as statistically significant compared with controls would improve precision.

• Lines 21–22: Bioinformatics analyses are appropriately mentioned, but the analytical approach (e.g., GO/KEGG) could be briefly specified.

• Lines 23–26: Conclusions are well aligned with the results; consider tempering claims about therapeutic targets by emphasizing the preclinical nature of the findings.

Introduction (Lines 30–79)

• Lines 31–35: The epidemiological and clinical relevance of Mycoplasma pneumoniae is well presented.

• Lines 36–39: The discussion of disease severity and immune dysregulation is appropriate; consider citing one additional recent review to further contextualize immune mechanisms.

• Lines 42–56: The literature review is thorough and balanced. Some sentences are lengthy and could be streamlined for clarity.

• Lines 58–65: The discussion of macrolide resistance is relevant; however, its connection to the current experimental design could be more explicitly stated.

• Lines 67–76: Study objectives are clearly defined. Consider separating aims into numbered points for improved readability.

Materials and Methods (Lines 80–168)

• Lines 81–89: Strain selection and culture conditions are clearly described; include a brief justification for using the FH strain as the primary model.

• Lines 91–101: The animal model is well detailed and ethically sound. Consider adding a rationale for the chosen inoculation dose.

• Lines 103–109: Histopathology methods are standard and adequately described.

• Lines 110–118: qPCR methodology is appropriate; primer sequences or reference to prior validation could enhance reproducibility.

• Lines 119–124: The Ella™ system is clearly described; consider stating the sensitivity or detection limits.

• Lines 125–136: Cytokine multiplex analysis is robust; clarify criteria for defining “significantly upregulated” proteins.

• Lines 137–161: Mass cytometry methods are comprehensive; however, antibody panels could be summarized in a supplementary table.

• Lines 162–167: Statistical analysis is appropriate, though justification for sample size and use of only t-tests could be added.

Results (Lines 169–310)

• Lines 170–175: MP characterization is clear and well supported by Figure 1.

• Lines 181–198: Disease phenotype description is strong; avoid referencing unpublished data within the main Results section.

• Lines 206–219: Immune cell dynamics are well presented; consider reporting absolute cell numbers in addition to percentages.

• Lines 225–264: UMAP and FlowSOM analyses are a major strength. Some interpretations could be more conservative given the small sample size (n = 3).

• Lines 284–299: Cytokine profiling is comprehensive; highlighting the top 5 most biologically relevant cytokines may improve focus.

Discussion (Lines 311–380)

• Lines 312–320: The clinical relevance is clearly articulated and well referenced.

• Lines 331–343: Interpretation of myeloid dominance is convincing; consider acknowledging alternative explanations such as trafficking versus expansion.

• Lines 344–353: The link between cytokine signaling and immune remodeling is insightful; however, mechanistic causality should be stated cautiously.

• Lines 355–364: Therapeutic implications are interesting but somewhat speculative; emphasize the need for functional validation.

• Lines 370–379: Limitations are appropriately acknowledged and strengthen the manuscript’s credibility.

Conclusions (Lines 381–393)

• Lines 382–387: Conclusions accurately reflect the results.

• Lines 388–392: Future directions are appropriate; consider specifying which cytokine pathways are the most promising targets.

References

• The references are current, relevant, and well integrated. Minor formatting consistency checks are recommended.

Author Response

Comments 1: Abstract (Lines 10–27) Lines 11–13: The background is clear and relevant; however, consider briefly stating the knowledge gap more explicitly to strengthen the rationale.

Response 1: Thank you for your valuable and detailed feedback. We have revised this point as required in the revised manuscript.

Comments 2: Lines 14–15: The animal model and key methods are appropriately summarized. Consider clarifying why a murine model is particularly suitable for studying acute MPP.

Response 2: Thank you for this valuable suggestion. To clarify the rationale for our model selection, we have revised the Abstract. Specifically, we have added a brief justification indicating that the murine model recapitulates key pathophysiological features of acute human MPP, such as airway inflammation and cytokine release. This makes it a well-established and suitable system for studying the immunological mechanisms in question.

Comments 3: Lines 18–20: The cytokine findings are strong; specifying whether these cytokines were identified as statistically significant compared with controls would improve precision.

Response 3: Thank you for your valuable and detailed feedback. We have revised this point as required in the revised manuscript.

Comments 4: Lines 21–22: Bioinformatics analyses are appropriately mentioned, but the analytical approach (e.g., GO/KEGG) could be briefly specified.

Response 4: Thank you for your valuable and detailed feedback. We have revised this point as required in the revised manuscript.

Comments 5: Lines 23–26: Conclusions are well aligned with the results; consider tempering claims about therapeutic targets by emphasizing the preclinical nature of the findings.

Response 5: We sincerely thank the reviewer for this insightful comment. We agree that it is important to clearly frame the preclinical nature of our findings. In response, we have revised the conclusion in the Abstract.

Comments 6: Introduction (Lines 30–79)

Lines 31–35: The epidemiological and clinical relevance of Mycoplasma pneumoniae is well presented.

Response 6: Thank you for your comment.

Comments 7: Lines 36–39: The discussion of disease severity and immune dysregulation is appropriate; consider citing one additional recent review to further contextualize immune mechanisms.

Response 7: We thank the reviewer for this positive and constructive suggestion. We agree that citing

review number 6 would help to better contextualize immune mechanisms.

Comments 8: Lines 42–56: The literature review is thorough and balanced. Some sentences are lengthy and could be streamlined for clarity.

Response 8: We appreciate your feedback regarding the clarity of our writing. In response, we have carefully reviewed the manuscript and identified lengthy sentences. We have revised these sentences to make them more concise and clearer, ensuring that the overall readability of the literature review is improved while maintaining the integrity of the content.

Comments 9: Lines 58–65: The discussion of macrolide resistance is relevant; however, its connection to the current experimental design could be more explicitly stated.

Response 9:  We thank the reviewer for this insightful comment, which helps to sharpen the focus of our introduction. We agree that the connection between macrolide resistance and our experimental focus on immunopathology could be made more explicit. We have revised this point as required in the revised manuscript.

Comments 10: Lines 67–76: Study objectives are clearly defined. Consider separating aims into numbered points for improved readability.

Response 10: We thank the reviewer for the positive feedback and the practical suggestion to improve readability. As suggested, we have revised the ‘Study objectives’ section in the Introduction by presenting the aims as a numbered list. This modification clearly delineates the primary goals of our study, ensuring they are immediately accessible to the reader.

Comments 11: Materials and Methods (Lines 80–168)

Lines 81–89: Strain selection and culture conditions are clearly described; include a brief justification for using the FH strain as the primary model.

Response 11: We thank the reviewer for the constructive comment. As suggested, we have revised the ‘Materials and Methods’ 2.1. section to include a brief justification for selecting the FH strain as our primary infection model.

Comments 12: Lines 91–101: The animal model is well detailed and ethically sound. Consider adding a rationale for the chosen inoculation dose.

Response 12: We appreciate your positive feedback on the animal model. In our preliminary experiments to determine the inoculation dose for infecting mice, we selected doses ranging from 10⁷ to 10⁹ CCU/mL of Mycoplasma pneumoniae. The experimental results revealed that 10⁹ CCU/mL was sufficient to induce a severe inflammatory response and a robust immune response in the mice. This selection aligns with the goals of our study. This rationale was also explained in reference 25 of 2.1. section in our revised manuscript.

Comments 13: Lines 103–109: Histopathology methods are standard and adequately described.

Response 13: Thank you for your comment.

Comments 14: Lines 110–118: qPCR methodology is appropriate; primer sequences or reference to prior validation could enhance reproducibility.

Response 14: We are grateful for the reviewer’s suggestions. But we apologize for that the Mycoplasma pneumoniae (MP) Real-Time PCR Kit was purchased from Shanghai Liferiver Biotechnology Co., Ltd. Regarding your enquiry about providing primer sequences, we have engaged in multiple communications with the reagent supplier to confirm the matter. The current situation is that the supplier's confidentiality agreement restricts them from disclosing specific primer sequences externally. This constitutes a common industry compliance requirement, designed to safeguard their intellectual property and client confidentiality. Nevertheless, we strictly adhered to the reagent usage instructions throughout the experiment to ensure the reliability of the results.

Comments 15: Lines 119–124: The Ella™ system is clearly described; consider stating the sensitivity or detection limits.

Response 15: Thank you for your suggestion. We have added the sensitivity of the Ella™ System into the revised manuscript.

Comments 16: Lines 125–136: Cytokine multiplex analysis is robust; clarify criteria for defining “significantly upregulated” proteins.

Response 16: We thank the reviewer for this important comment, which enhances the rigor and clarity of our methodology. In direct response, we have revised the 2.6. section to explicitly state the quantitative criteria used to define “significantly upregulated” cytokines/chemokines.

Comments 17: Lines 137–161: Mass cytometry methods are comprehensive; however, antibody panels could be summarized in a supplementary table.

Response 17: We are grateful for the reviewer’s suggestions. We have created a supplementary table that summarizes the antibody panels used in our mass cytometry methods. This table includes detailed information on each antibody to provide clarity and enhance the comprehensibility of our methods section.

Comments 18: Lines 162–167: Statistical analysis is appropriate, though justification for sample size and use of only t-tests could be added.

Response 18: Thank you for your suggestion. We have added the sample size information and supplemented the manuscript with statistical methods.

Comments 19: Results (Lines 169–310)

Lines 170–175: MP characterization is clear and well supported by Figure 1.

Response 19: Thank you for your comment.

Comments 20: Lines 181–198: Disease phenotype description is strong; avoid referencing unpublished data within the main Results section.

Response 20: Thank you for your suggestions. We have carefully reviewed the Results section and removed all references to unpublished data. The revised manuscript now only includes published data to ensure clarity and adherence to publication standards. Thank you for highlighting this important point.

Comments 21: Lines 206–219: Immune cell dynamics are well presented; consider reporting absolute cell numbers in addition to percentages.

Response 21: We are grateful for the reviewer’s suggestions. We have revised the manuscript to include absolute cell numbers alongside the percentages in the relevant sections. This addition provides a clearer understanding of the immune cell dynamics presented in our study.

Comments 22: Lines 225–264: UMAP and FlowSOM analyses are a major strength. Some interpretations could be more conservative given the small sample size (n = 3).

Response 22: We appreciate your feedback regarding the interpretation of our results. We have revised this point as required in the revised manuscript.

Comments 23: Lines 284–299: Cytokine profiling is comprehensive; highlighting the top 5 most biologically relevant cytokines may improve focus.

Response 23: We appreciate the suggestion to enhance the focus of our cytokine profiling. In response, we have revised the manuscript to include a section that highlights the top 5 most biologically relevant cytokines based on their significance in the revised manuscript.

Comments 24: Discussion (Lines 311–380)

Lines 312–320: The clinical relevance is clearly articulated and well referenced.

Response 24: Thank you for your comment.

Comments 25: Lines 331–343: Interpretation of myeloid dominance is convincing; consider acknowledging alternative explanations such as trafficking versus expansion.

Response 25: We appreciate the suggestion and have revised the manuscript to acknowledge alternative explanations for myeloid dominance.

Comments 26: Lines 344–353: The link between cytokine signaling and immune remodeling is insightful; however, mechanistic causality should be stated cautiously.

Response 26: We thank the reviewer for raising this important point regarding mechanistic interpretation. We agree that caution is necessary when inferring causality from correlative data.

We have revised this point as required in the revised manuscript.

Comments 27: Lines 355–364: Therapeutic implications are interesting but somewhat speculative; emphasize the need for functional validation.

Response 27: We thank the reviewer for this constructive comment. We agree that while our findings point to interesting therapeutic directions, these implications are indeed preliminary and require experimental validation.

Comments 28: Lines 370–379: Limitations are appropriately acknowledged and strengthen the manuscript’s credibility.

Response 28: Thank you for your comment.

Comments 29: Conclusions (Lines 381–393)

Lines 382–387: Conclusions accurately reflect the results.

Response 29: Thank you for your comment.

Comments 30: Lines 388–392: Future directions are appropriate; consider specifying which cytokine pathways are the most promising targets.

Response 30: We thank the reviewer for this excellent suggestion to sharpen the focus of our conclusion. Among these, the CCL5 pathway emerges as a promising target given its role in neutrophile recruitment and macrophage infiltration. Furthermore, targeting the upstream master regulators, such as IL-6 and TNF-α, that drive the cytokine storm could provide an extended therapeutic window. We have revised this point as required in the revised manuscript.

Comments 31: References

The references are current, relevant, and well integrated. Minor formatting consistency checks are recommended.

Response 31: We have thoroughly reviewed the references and made necessary adjustments to ensure consistency in formatting throughout the paper. All references now adhere to the required style guidelines.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept