Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major Comments

1. The mechanistic evidence remains limited. Most conclusions rely solely on mRNA expression analysis without protein-level validation (ELISA, Western blot, or flow cytometry). Additional functional validation would substantially strengthen the mechanistic claims.
2. The identification and characterization of macrophage populations are insufficient. Bone marrow chimera experiments are mainly supported by immunofluorescence, whereas flow cytometric quantification using CD45.1/F4/80/CD11b/Ly6C/Ly6G markers would provide more convincing evidence for infiltrating macrophage origin.
3. Several figures require substantial improvement. The immunofluorescence images are of relatively low quality and resolution, representative images are limited, statistical annotations are difficult to interpret, and the bar graphs should ideally include individual biological data points. Representative images of postoperative adhesions would also improve data presentation.

Minor Comments

1. Clarify randomization and blinded assessment of adhesion scores.
2. Provide detailed statistical methods, including multiple-comparison corrections.
3. Improve figure resolution and enlarge labels for readability.
4. Standardize graph colors, axis scales, and statistical annotation across all figures.
5. Include more recent literature (2024–2026) on macrophage heterogeneity and peritoneal adhesion biology.
6. Replace "Opposing roles" with a more cautious description if not directly demonstrated by the experimental evidence.

Author Response

Major Comments

1. The mechanistic evidence remains limited. Most conclusions rely solely on mRNA expression analysis without protein-level validation (ELISA, Western blot, or flow cytometry). Additional functional validation would substantially strengthen the mechanistic claims.

 

Response:

We agree that the study is primarily transcriptional and that mRNA expression does not necessarily predict protein abundance or biological activity. We have revised the manuscript to describe the qPCR results as changes in gene expression rather than direct evidence of cytokine or macrophage function. We have removed language implying protein-level validation.

No additional ELISA, Western blot, or flow-cytometry data were available for the current revision. We now explicitly identify the absence of protein-level validation as a limitation.

 

 

1. The identification and characterization of macrophage populations are insufficient. Bone marrow chimera experiments are mainly supported by immunofluorescence, whereas flow cytometric quantification using CD45.1/F4/80/CD11b/Ly6C/Ly6G markers would provide more convincing evidence for infiltrating macrophage origin.

 

Response:

We agree. We have clarified that the current immunofluorescence approach identifies F4/80-positive cells and, in the chimera experiments, donor-marker-positive cells, but does not fully characterize macrophage subsets. We have consequently moderated the terminology throughout the manuscript and now state that the chimera data support recruitment of donor-derived hematopoietic cells with a macrophage-like phenotype into ischemic buttons.

A definitive distinction between resident macrophages, recruited monocytes, monocyte-derived macrophages, and other myeloid cells would require multiparameter flow cytometry using at least CD45, CD11b, F4/80, Ly6C, Ly6G, and MHC-II, with additional markers such as TIM-4 or CD102 where appropriate. These limitations and future experiments are now discussed.

 

 

1. Several figures require substantial improvement. The immunofluorescence images are of relatively low quality and resolution, representative images are limited, statistical annotations are difficult to interpret, and the bar graphs should ideally include individual biological data points. Representative images of postoperative adhesions would also improve data presentation.

 

Response:

We agree and revised the figures accordingly.

 

 

Minor Comments

1. Clarify randomization and blinded assessment of adhesion scores.

 

Response: Randomization and blinded assessment was clarified.

 

1. Provide detailed statistical methods, including multiple-comparison corrections.

 

Response: We now specify the statistical tests, exact comparisons, correction method, and the unit of analysis.

 

1. Improve figure resolution and enlarge labels for readability.

 

Response: We revised the figures accordingly.

 

1. Standardize graph colors, axis scales, and statistical annotation across all figures.

 

Response: We revised the figures accordingly.

 

1. Include more recent literature (2024–2026) on macrophage heterogeneity and peritoneal adhesion biology.

 

Response: We inculded the following more recent literature:

• Henlon Y, Panir K, McIntyre I, Hogg C, Dhami P, Cuff AO, Senior A, Moolchandani-Adwani N, Courtois ET, Horne AW, Rosser M, Ott S, Greaves E. Single-cell analysis identifies distinct macrophage phenotypes associated with prodisease and proresolving functions in the endometriotic niche. Proc Natl Acad Sci U S A. 2024
• Li L, Hirao H, Honda M, Adawy A, Pan C, Fujiwara Y, Yoshii D, Xue W, Tomita M, Su J, Hibi T, Komohara Y. Preventive role of CD163-positive macrophages in postoperative peritoneal adhesions. Inflamm Regen. 2025 Aug 15;45(1):26.
• Liu Z-Y, Li R-F, Qin H-Y, Ma P-F. Postoperative adhesion formation: the role of peritoneal macrophages and targeting therapy. Frontiers in Immunology. 2025;16:1601642. doi:10.3389/fimmu.2025.1601642.

 

 

1. Replace "Opposing roles" with a more cautious description if not directly demonstrated by the experimental evidence.

 

Response: We agree. The title and conclusion have been changed to describe “distinct and potentially divergent contributions” rather than definitively asserting opposing roles. This wording better reflects the observational and intervention-based evidence while preserving the main biological message.

Reviewer 2 Report

Comments and Suggestions for Authors

Please refer to the attached file.

Comments for author File: Comments.pdf

Author Response

This manuscript addresses a clinically important question that is the relative contributions of resident versus infiltrating macrophages to postoperative adhesion formation using a well- established ischemic button model and a reasonable combination of depletion, chimera, and knockout approaches. The central finding that clodronate depletion reduces adhesions while CCR2 deficiency increases them is potentially interesting, but several methodological details are missing or internally inconsistent, and a few technical issues need to be resolved before the conclusions can be considered well-supported. I outline these below, with line references to the submitted text where possible.

 

1. Central logical tension between the depletion and chimera results is not resolved (lines 171–174, 201–214, 296–314). The authors report that clodronate achieved >95–98% depletion efficiency in mesentery and peritoneal lavage, yet F4/80+ cell counts within the ischemic button itself at day 3 were not significantly different between clodronate- and vehicle-treated animals. If the lesional macrophage pool is unaffected by clodronate, it is difficult to attribute the reduced adhesion score at day 7 specifically to “resident macrophages,” since the lesion's own macrophage content was not reduced. The authors acknowledge this discrepancy in the Discussion (lines 306–314) but offer three competing explanations (early contribution, incomplete drug penetration, replacement by infiltrating cells) without any data to adjudicate between them. This is the single most important gap in the paper and should be addressed either with additional time-course depletion/reconstitution data or with substantially more cautious causal language throughout the abstract, results, and conclusions.

 

Response:

We agree. This is an important limitation of the original interpretation. The clodronate experiment demonstrates that depletion of macrophage-rich peritoneal and mesenteric compartments is associated with reduced adhesion formation, but it does not prove that resident macrophages within the ischemic button at day 3 are the direct cellular mediators of this effect. The persistence of F4/80-positive cells in the ischemic button may reflect incomplete penetration of clodronate into the lesion, early effects of depletion before the day-3 assessment, recruitment or replacement by other phagocytic cells, or limitations of F4/80-based enumeration. The present experiments cannot distinguish these possibilities.

We have therefore replaced categorical statements such as “identifies resident macrophages as net pro-adhesive effectors” with the more cautious formulation that depletion of macrophage-rich resident compartments was associated with reduced adhesion formation. We have also removed the assertion that the data directly demonstrate temporally separated functions. The Discussion now emphasizes that the findings are consistent with, but do not definitively establish, an early contribution of resident macrophage-rich compartments.

 

 

1. Chimera generation details are incomplete (lines 108–113). The Methods do not state: (a) the reconstitution period between bone marrow transplantation and ischemic button surgery (standard practice is 6–8 weeks to allow full hematopoietic reconstitution and to allow radioresistant tissue-resident populations to normalize); (b) whether chimerism was confirmed by blood flow cytometry prior to surgery, and to what percentage; (c) whether lethal (9 Gy as a single dose, or split-dose) irradiation was combined with antibiotic prophylaxis; (d) genetic background matching between CD45.1 donors and C57BL/6J hosts. These are essential for readers to judge whether the chimera model is valid, particularly given that resident peritoneal macrophages are known to be radioresistant and only slowly replaced by bone-marrow-derived cells which is directly relevant to the paper's central question.

 

Response: We agree that these details are essential. We have expanded the Methods section to report the interval between transplantation and surgery, preoperative assessment of peripheral blood chimerism, irradiation protocol, antibiotic prophylaxis, and donor/recipient genetic background. We also now explicitly acknowledge that radioresistant resident macrophages may persist after irradiation and that CD45.1 positivity is evidence of donor hematopoietic origin, but does not by itself establish a complete phenotypic distinction between resident and infiltrating macrophage subsets.

 

 

 

1. F4/80 alone is an incomplete marker for resident vs. infiltrating peritoneal macrophage subsets. It is well established that the peritoneal cavity contains at least two functionally distinct F4/80+ populations; large peritoneal macrophages (F4/80^hi, resident) and small peritoneal macrophages/recruited monocyte-derived cells (F4/80^lo, MHCII^hi) and that F4/80 intensity or additional markers (CD11b, Ly6C, MHCII, CD102/ICAM2, Tim4) are typically needed to separate these populations. Given that the paper's core thesis rests on distinguishing resident from infiltrating macrophage identity, relying on F4/80 positivity alone (plus CD45.1 in the chimera) is a significant limitation that should be explicitly discussed, and ideally supplemented with additional markers if data exist.

 

Response:

We agree. F4/80 staining alone does not resolve the relevant peritoneal macrophage subsets. In the revised manuscript, we no longer describe all F4/80-positive cells as definitively resident or infiltrating macrophages. We clarify that CD45.1 labeling in the chimera experiments supports hematopoietic donor origin, whereas phenotypic discrimination of large resident macrophages, small recruited macrophages, monocytes, and other myeloid populations would require multiparameter flow cytometry or fate-mapping approaches.

No additional CD11b, Ly6C, Ly6G, MHC-II, CD102/ICAM2, or TIM-4 data were available in the submitted dataset. We therefore present this as a methodological limitation and identify multiparameter flow cytometry as an important direction for future work. If such data are available, they should be incorporated rather than using the limitation-only wording below.

 

 

1. No statement regarding blinding or randomization of adhesion scoring. The adhesion score (0–4) is a semi-subjective visual scale. Please state explicitly whether the surgeon/scorer was blinded to treatment group when assigning adhesion scores, and how animals were randomized to treatment arms. This is standard and expected for this type of outcome measure.

 

Response:

We agree that the original description was insufficient. All items of the ARRIVE checklist are now mentioned in the manuscript.

 

 

1. Single time-point analysis of lesional macrophage content limits claims of “temporally separated roles” (line 28, 173). Macrophage numbers in the ischemic button were assessed only at day 3 for the depletion experiments (and largely day 1/day 3 for cytokines). Without a time course (e.g., days 1, 3, 5, 7), the claim that resident and infiltrating macrophages act in “temporally separated” fashion (abstract, line 28) is not directly demonstrated. Please either soften this claim or provide additional time points.

 

Response:

We agree. The available data do not establish a complete temporal sequence or temporally separated roles. We now state that the current study samples selected postoperative time points rather than providing a complete time course. We have also revised the Discussion to make clear that the proposed early resident-compartment contribution is an interpretation of the depletion phenotype, not a directly demonstrated time-resolved mechanism.

 

 

1. CCR2 global knockout is not macrophage-specific. CCR2 is expressed on additional leukocyte subsets (e.g., certain NK cell, dendritic cell, and lymphocyte populations), so the CCR2−/− phenotype cannot be attributed to monocyte/macrophage recruitment alone. This should be acknowledged as a limitation (it is only partially addressed in the Limitations paragraph, lines 372–377) and, ideally, discussed alongside literature using more specific tools (e.g., LysM-Cre or CX3CR1-based fate-mapping) for corroboration.

 

Response:

We agree. CCR2 is expressed by and regulates the trafficking of multiple immune-cell populations. Thus, the phenotype of CCR2-deficient mice cannot be attributed exclusively to altered monocyte/macrophage recruitment. We have expanded the limitations paragraph and now describe the CCR2-deficient model as testing the contribution of CCR2-dependent hematopoietic recruitment more broadly. We also mention that macrophage-specific genetic approaches, inducible fate mapping, and multiparameter flow cytometry would be required to establish macrophage-specific causality.

 

 

1. Off-target effects of clodronate liposomes. Clodronate liposomes deplete phagocytic cells generally (including some dendritic cells and monocytes) rather than being exclusively “resident macrophage”-specific and can have systemic as well as local effects. This is acknowledged briefly (lines 372–374) but should be integrated earlier into the Results interpretation rather than left solely to the Limitations paragraph.

 

 

Response:

We agree and have moved this limitation from the final paragraph of the Discussion into the interpretation of the depletion experiment. Clodronate liposomes target phagocytic cells and are not exclusively specific for resident macrophages; systemic and local effects, including effects on monocytes and selected dendritic-cell populations, cannot be excluded. The revised Results and Discussion therefore refer to “macrophage-rich resident compartments” rather than claiming selective resident macrophage depletion.

 

 

 

1. Sex of animals. Only male mice were used (line 100). Given known sex-based differences in peritoneal immune cell composition and fibrinolytic activity, this should be noted as a limitation.

 

Response:

We have added a limitation stating that only male mice were studied. This restricts generalizability because sex may influence peritoneal immune-cell composition, inflammatory responses, fibrinolysis, and adhesion formation.

 

 

1. Sample sizes and exact test/comparison mapping. "n = 5–8 for all groups" is stated generically for each figure; please report the exact n for each specific panel/comparison and clarify precisely which pairwise comparisons within each ANOVA were tested (all pairwise, or pre-specified contrasts only), since Bonferroni correction depends on the number of comparisons made.

 

Response:

We agree. The generic statement “n = 5–8 for all groups” was insufficient. We have revised the figure legends and statistical section to report the exact number of biological replicates for each panel and group. We also specify whether comparisons were prespecified pairwise contrasts or all pairwise comparisons within an analysis of variance.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript falls under Biomedicines scope and presents findings of research entitled “Opposing Roles of Resident and Infiltrating Macrophages during Adhesion Formation After Abdominal Surgery”. The manuscript consists of 14 pages, 5 figures, S1 table and the list of total 29 literature references. The paper presents interesting results as well as an inquisitive and reliable interpretation of the research results. The topic original and relevant in the field of study. The Abstract provides the highlights of the key contents of the main text. The Introduction provides enough background information to justify the study. The Results are consistent with the declared methodology, presented clearly enough, supported by the figures and table. Researchers aimed distinguishing the contributions of resident peritoneal macrophages and infiltrating macrophages, and assess how CCR2-dependent recruitment influences inflammatory and reparative responses during adhesion formation after abdominal surgery, and concluded that resident macrophages contribute to early adhesion formation, whereas CCR2-dependent bone marrow–derived macrophages support inflammatory control and limit adhesion severity. The references are appropriate and relevant to the research. However, minor typographical and grammatical errors need addressing.

Comments on the Quality of English Language

Minor typographical and grammatical errors need addressing.     

Author Response

This manuscript falls under Biomedicines scope and presents findings of research entitled “Opposing Roles of Resident and Infiltrating Macrophages during Adhesion Formation After Abdominal Surgery”. The manuscript consists of 14 pages, 5 figures, S1 table and the list of total 29 literature references. The paper presents interesting results as well as an inquisitive and reliable interpretation of the research results. The topic original and relevant in the field of study. The Abstract provides the highlights of the key contents of the main text. The Introduction provides enough background information to justify the study. The Results are consistent with the declared methodology, presented clearly enough, supported by the figures and table. Researchers aimed distinguishing the contributions of resident peritoneal macrophages and infiltrating macrophages, and assess how CCR2-dependent recruitment influences inflammatory and reparative responses during adhesion formation after abdominal surgery, and concluded that resident macrophages contribute to early adhesion formation, whereas CCR2-dependent bone marrow–derived macrophages support inflammatory control and limit adhesion severity. The references are appropriate and relevant to the research. However, minor typographical and grammatical errors need addressing.

 

Response: We thank the Reviewer. The manuscript has undergone language revision. We corrected spelling, grammar, punctuation, terminology, and inconsistent use of hyphens and abbreviations. We also standardized “postoperative,” “bone marrow-derived,” “anti-inflammatory,” “F4/80-positive,” and the nomenclature for cytokines and genes. The title and interpretive statements were revised to ensure that the conclusions match the strength of the evidence.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. The current evidence is still insufficient to clearly define resident versus infiltrating macrophages and to assign distinct biological functions to these two populations.
2. Without functional or cell-specific validation, the conclusion that CCR2-dependent infiltrating macrophages acquire a reparative, anti-inflammatory, or pro-resolving phenotype remains stronger than what the current data directly support.
3. The figure resolution is now acceptable. However, the overall figure organization and presentation would still benefit from further polishing.

Author Response

We thank the reviewer for the careful reassessment and constructive comments. We have revised the manuscript to more clearly separate direct experimental observations from cell-subset-specific or functional interpretations. In particular, we have removed statements that implied definitive identification of resident versus infiltrating macrophages or acquisition of a reparative/pro-resolving phenotype by CCR2-dependent recruited macrophages. Changes are highlighted in yellow in the revised manuscript.

Comment 1: The current evidence is still insufficient to clearly define resident versus infiltrating macrophages and to assign distinct biological functions to these two populations.

 

Response: We agree. The previous wording still implied a sharper resident-versus-infiltrating distinction than can be supported by clodronate depletion, F4/80 staining, bone marrow chimerism, and global CCR2 deficiency. We therefore revised the manuscript throughout so that these experimental approaches are described operationally rather than as definitive macrophage-subset identifiers.

Specifically, the title has been changed from “Distinct and Potentially Divergent Contributions of Resident and Infiltrating Macrophages…” to “Macrophage-Rich Peritoneal Compartments and CCR2-Dependent Hematopoietic Recruitment Are Differentially Associated with Adhesion Formation After Abdominal Surgery.” In the Abstract, Introduction, Methods, Results, Discussion, Conclusions, and figure legends, we now preferentially use “macrophage-rich peritoneal and mesenteric compartments,” “F4/80-positive cells,” “donor-derived hematopoietic cells,” and “CCR2-dependent hematopoietic recruitment.”

We also added explicit text noting that F4/80/CD45.1 staining does not fully resolve resident macrophages, recruited monocytes, monocyte-derived macrophages, or other myeloid populations; irradiation may leave radioresistant resident cells; clodronate is not selective for resident macrophages; and global CCR2 deficiency is not macrophage specific. The Discussion and Conclusions now state that the study identifies divergent associations between experimentally defined perturbations, not definitive opposing functions of phenotypically defined resident and infiltrating macrophage subsets.

Comment 2: Without functional or cell-specific validation, the conclusion that CCR2-dependent infiltrating macrophages acquire a reparative, anti-inflammatory, or pro-resolving phenotype remains stronger than what the current data directly support.

 

Response: We agree and have removed this conclusion. The Results no longer state that “CCR2-dependent recruited macrophages promote anti-inflammatory/reparative programs.” Instead, we report the direct observation: global CCR2 deficiency was associated with lower day-3 whole-tissue expression of MR1, IL10, and Arg1 in ischemic buttons. We now explicitly state that these data do not identify the cellular source of these transcripts and do not demonstrate acquisition of a reparative, anti-inflammatory, or pro-resolving macrophage phenotype.

The Discussion has been revised in the same way. The previous sentence stating that CCR2-dependent infiltrating macrophages “acquire a reparative, anti-inflammatory, or pro-resolving phenotype” has been replaced by association-level language. We further emphasize that transcript measurements were performed in whole tissue, that protein-level activity was not validated, and that Arg1/MRC1/IL10 expression alone is not proof of pro-resolving cellular function.

We also removed the stronger claim that CCR2-recruited infiltrating macrophages are necessary for full inflammatory resolution. The revised interpretation is that intact CCR2-dependent recruitment is associated with lower adhesion severity and with a distinct day-3 transcriptional pattern, while the responsible cell type and mechanism remain unresolved and require lineage-resolved, cell-specific functional studies.

Comment 3: The figure resolution is now acceptable. However, the overall figure organization and presentation would still benefit from further polishing.

 

Response: Thank you. We have further polished the figure presentation at the manuscript level by standardizing all figure legends, panel descriptions, terminology, postoperative time-point notation, sample-size reporting, and statistical-test descriptions. Where cell identity is not definitively established, the legends now use “F4/80-positive cells” or “donor-derived hematopoietic cells” rather than “macrophages.”

We also clarified the panel sequence in Figures 2–5 so that inflammatory transcripts at postoperative day 1 and wound-healing-associated transcripts at postoperative day 3 are presented consistently, and we corrected inconsistencies in panel labels and spacing. No underlying data or statistical results were changed.

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The interpretation of the macrophage subsets and their functions is now more appropriate. The figures have been improved。 But the overall organization of several figures could still be further polished, particularly the panel size, spacing, and layout.

Author Response

Thank you for your constructive and helpful comments. We are pleased that the revised interpretation of the macrophage subsets and their functions is now considered more appropriate, and that the improvements to the figures are recognized. Following your suggestion, we have further polished the overall organization of the figures, with particular attention to panel size, spacing, alignment, and layout, to improve their consistency, clarity, and visual presentation. We believe these additional revisions have improved the overall quality and readability of the figures.