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Dear Dr Ju,
The authors have addressed all of the reviewers' comments. I assessed the revision and am satisfied with the current version, therefore the manuscript is ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
The manuscript is clear, well written, and all previous issues have been corrected.
The design is appropriate, and all requested clarifications and limitations were added.
The conclusions are now properly supported, with overstated claims corrected.
Minor issues were addressed, and the manuscript is consistent and well organized.
The authors have made the requested changes.
The authors have made the requested changes.
The authors have made the requested changes.
Thank you for submitting your manuscript to PeerJ. Your manuscript has been evaluated by two referees and found to be of interest. However, several substantial criticisms were raised that prevent publication of your paper in its current form.
The reviewers' comments are provided below. Please address each critique in detail and resubmit your manuscript.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The manuscript is clear and well-structured, with a solid introduction and a logical discussion. A few points could improve clarity:
1. The abstract still contains editorial instructions, which must be deleted before submission.
2. Figure legends could be more detailed. For example, in Figures 2 and 3, the axes use abbreviations but do not clearly indicate that these are plasma concentrations measured by ELISA. Making this explicit.
3. In the Results section, some passages simply repeat the exact numbers already shown in tables and figures. It would be stronger to focus on interpretation and trends rather than duplication. These data should only be explicitly stated in the text when they are not clearly presented in the figures or tables.
4. The reference list is adequate but could be updated with more recent studies on exercise immunology and macrophage polarization to place this work more firmly in the current context.
The experimental design is appropriate, with a well-established model of diet-induced obesity and a clear exercise protocol. A few areas need clarification or acknowledgment:
1. The conclusions on macrophage polarization rely only on circulating cytokine levels. While valuable, these data would be more convincing if complemented by cellular analyses (e.g., flow cytometry or tissue histology). Since this was not done, the limitation should be highlighted more clearly.
2. Only male mice were used. Given that sex-specific responses in inflammation and exercise are known, this should be noted as a limitation.
3. The treadmill protocol is well described in terms of speed and duration, but the relative intensity (e.g., in relation to VO₂max or maximal running capacity) is not reported.
4. The sample size (n=8 per group) is reasonable, but if possible, display all points in the graphs to represent the animals. Indicate the number of animals in each group in the table header.
The findings are sound and consistent with the literature, showing that exercise reduces pro-inflammatory cytokines and promotes anti-inflammatory markers in obese mice. However, some caution is warranted:
1. The claim that exercise “restores” M2 markers should be phrased more cautiously, as the data are based only on serum assays, not direct cell-level measurements.
2. Without tissue-level analysis of macrophage infiltration (e.g., adipose tissue), the mechanistic interpretation is limited.
3. At times, the discussion presents the study as more novel than it is. It would be better to position the findings within the broader body of evidence, while highlighting what is specifically new in this work.
1. The use of Fisher’s LSD post hoc test is noted. While LSD can be acceptable with only three groups, it is relatively liberal and does not strongly control type I error. A more conservative test, such as Tukey’s, would generally be preferred for multiple group comparisons, or at minimum, the rationale for choosing LSD should be explained. Clarifying this point would strengthen the statistical rigor of the study.
2. Statistical annotations (* vs. chow; # vs. HF-Sed) and tests are appropriate, but they should be clearly explained in each figure legend, not only in the general text.
3. Sometimes the cytokine dosage is described in plasma and sometimes in serum.
4. The conclusion is well stated but should adopt a more cautious tone regarding translational relevance.
Minor comments:
1. Values are means ± SD.; n=8/group.” Correct to: “Values are means ± SD; n = 8 per group.
2. “… p <0.05 vs. Ch-Sed, # p <0.05 vs. HF-Sed.” Standardize spacing: “p < 0.05 vs. Ch-Sed; # p < 0.05 vs. HF-Sed.”
The study is worthwhile and contributes to our understanding of the systemic immunomodulatory effects of exercise in obesity. However, the manuscript requires careful revision of the English, refinement of the methodology, statistical approach, references and a more balanced interpretation of the findings.
1-) The introduction provides useful context, but does not fully justify the knowledge gap. I suggest that the authors emphasize this context more clearly. In addition, it is important that the study have a well-defined hypothesis. Furthermore, more consistent citations should be added to the introduction and throughout the text. The paper as a whole has very few references (16 in total). Even in the methodology, the authors do not cite important references, such as in papers written by the group itself (lines 165-168, for example) or or about the training protocol used.
2-) I would also recommend reviewing the English used throughout the article.
3-) Add a reference for lines 165–168 and add reference for training protocol.
4) Although the exercise protocol is described in detail, the rationale behind twice-daily sessions should be explained, as this differs from the standard approach (also add this information in the abstract ssection). Did the animals undergo a period of adaptation to the equipment?
5) The methodology should also be expanded to include information about the fact that only males were used for the protocol.
6) The experimental design shown in Figure 1A requires improvement.
7) The statistical analysis raises some concerns. A one-way ANOVA followed by a Fisher's LSD test was used; this post hoc test is relatively liberal and increases the probability of a type I error (false positive). More conservative methods (e.g. Tukey's) are recommended.
8) IL-2 is not a canonical M2 marker. IL-2 is classically associated with T-cell proliferation rather than with the M2 status of macrophages. This requires justification or reconsideration.
9) The conclusions are somewhat exaggerated. While exercise clearly reduced pro-inflammatory markers and increased anti-inflammatory markers, claiming that inflammation was 'reversed' is too strong (has not been investigated in specific tissues). A more accurate description would be that exercise 'partially mitigated' or 'attenuated' the pro-inflammatory profile. Similarly, the assertion that the effects are independent of weight loss needs to be nuanced: although body weight remained unchanged, exercise reduced visceral fat and liver weight, which may have contributed to the observed effects. The authors need to discuss the findings in more detail and revise the conclusion based on the data presented, as well as the statistics.
Comments are in Basic reporting.
Comments are in Basic reporting.
Comments are in Basic reporting.
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