Porphyromonas gingivalis Vesicles Control Osteoclast–Macrophage Lineage Fate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a significant topic: the impact of P. gingivalis OMVs on the differentiation of osteoclasts versus macrophages using RAW 264.7 cells as a model system.  I suggest that the Introduction and Discussion should more explicitly highlight what is new compared to previous work on Pg-OMVs and osteoclastogenesis (for example, the specific role of timing-dependent effects, OC-STAMP involvement, etc). A clearer statement of the central hypothesis and how these data push the field forward would enhance its impact. I would also suggest a discussion of how these findings and time points can be (and if) correlated with clinical presentations of periodontitis.
I categorize my comments as major and minor concerns, as follows:

Major Comments

1. Improve clarity and visibility of stimulation schedules (S1–S3)
   The three stimulation schedules are central to the study but are described in a way that can be difficult to follow. I strongly recommend improving the layout of the schematic timeline (in Figure 1) to more clearly indicate when RANKL and OMVs are added or removed in S1, S2, and S3 for both RAW264.7 and BMMC experiments. This will greatly aid the interpretation of stage-specific effects throughout the Results. Additionally, explain why the authors used different incubation times with the OMV. Why didn't they incubate with OMV for 7 days in S2 and S3 so that “pro-inflammatory” stimuli would better regulate the timing of the stimuli in S1?
2. Justification and exploration of the OMV dose
   The authors perform a dose–response viability assay and then select 1 µg/mL OMVs for subsequent experiments. However, the functional outcomes (osteoclastogenesis, pit formation, cytokine readouts) are not evaluated across different OMV concentrations. I suggest that the authors provide a stronger rationale for why 1 µg/mL is considered physiologically or pathologically relevant in the context of periodontal lesions. This would help readers understand the significance and relevance of the observed effects.
3.  RAW264.7 vs. primary cell data. Discuss translational relevance
   The study mainly uses RAW264.7 cells, with limited validation in bone marrow–derived mononuclear cells. It would improve the manuscript to expand the primary cell data where possible (e.g., pit formation, OC-STAMP expression, macrophage markers), or at least to more thoroughly discuss the limitations of RAW264.7 as a substitute for primary osteoclast precursors. The Discussion should also more clearly address how these in vitro results can be applied to preclinical in vivo periodontitis research and how they may correlate with clinical disease.
4. Mechanistic depth regarding OMV components and signaling
   This is my primary concern with the manuscript. The authors attribute several effects to LPS/TLR2 signaling and OC-STAMP–mediated fusion, but these mechanistic links remain largely inferred. To substantiate these claims, the authors must include data to demonstrate the presence of LPS in the OMVs. For example, they may consider additional controls such as TLR2/4 blockade or deficiency, comparison with purified LPS, and/or OMVs treated to inactivate specific components (e.g., protease inhibition for gingipains). An ELISA assay could also be used to enhance the strength of the LPS narrative/argument. If new experiments are not feasible at this stage, the authors must soften the causal language and clearly frame these as speculations or hypotheses that may explain the current data.
5. Macrophage polarization readouts and interpretation
   The conclusion that early OMV exposure promotes M1 macrophage differentiation relies on STAT1 phosphorylation and limited surface marker data (F4/80, CD86). The authors should either (a) include expression data for canonical M1/M2 markers (e.g., iNOS, IL-12, TNFα, Arg1, CD206), or (b) soften the language to highlight that OMVs induce an “M1-like” or “pro-inflammatory” phenotype rather than definitive M1 polarization. Clarifying this will strengthen the macrophage findings and make them more appropriately cautious. I also recommend adjusting the gating in Figure 5D. Your control quadrant lines are currently positioned, cutting through the middle of the main population (F4/80 neg, CD86 neg). Alternatively, explain why the quadrants are placed as they are now.

Minor Comments

1. Statistical reporting and sample size information
   For each figure, please clearly indicate the number of biological replicates and the number of independent experiments. It would also be helpful to specify the rationale behind the sample size, the exact statistical tests used, including post-hoc tests following the Kruskal–Wallis test, and how multiple comparisons were addressed. 
2. Clarify the “failed osteoclast” concept
   The link between reduced OMV phagocytosis in certain schedules, enhanced osteoclastogenesis, and the proposed idea of “failed osteoclasts” is intriguing but currently somewhat speculative. I suggest tightening this part of the Discussion to clearly distinguish observed findings (phagocytosis rates, TRAP staining, pit formation) from more speculative models, and outlining what additional experiments would be required to formally demonstrate the existence and function of such “failed osteoclasts.”
3. Language, grammar, and terminology consistency
   The manuscript would benefit from careful editing of the English language. There are multiple typographical and grammatical issues (“linage” vs. “lineage,” “Westren blotting,” misspelled cell line names, double periods, inconsistent spacing). Abbreviations and terms ( “OCgenesis,” “OC/MΦ unit”) should be standardized throughout and defined clearly at first use. A thorough edit will significantly improve readability.
4. Figure and legend clarity
   Please ensure all figures include: (i) clearly labeled axes with units (for example, Fig 2B has no unit), (ii) explicit identification of conditions (Fig 2A shows groups as S1, S1, S3, while on 2B the groups are described as Control, +RANKL, +RANKL+OMV), and (iii) the scale used in the bars for all microscopy images. For flow cytometry data, brief descriptions of gating strategies and representative plots in the supplement would be helpful. Some legends currently repeat text from the Results; these can be streamlined while still providing enough detail for the figures to be interpretable on their own.

Author Response

We appreciate the Reviewers for their thoughtful and constructive feedback on our manuscript. We have carefully addressed each concern and made the necessary revisions to improve the manuscript. Below are our detailed responses to the reviewers’ comments, with their remarks in italics, followed by our replies.

Reviewer 1

This manuscript addresses a significant topic: the impact of P. gingivalis OMVs on the differentiation of osteoclasts versus macrophages using RAW 264.7 cells as a model system. I suggest that the Introduction and Discussion should more explicitly highlight what is new compared to previous work on Pg-OMVs and osteoclastogenesis (for example, the specific role of timing-dependent effects, OC-STAMP involvement, etc). A clearer statement of the central hypothesis and how these data push the field forward would enhance its impact. I would also suggest a discussion of how these findings and time points can be (and if) correlated with clinical presentations of periodontitis.

I categorize my comments as major and minor concerns, as follows:

Thank you for your valuable comments. We agree that the Introduction and Discussion should more explicitly highlight the novelty of this study. In the revised manuscript, we have added the statements to both the Introduction and Discussion sections to emphasize better the new findings and their clinical relevance to periodontitis (Line 96-128, 468-493). In addition, our point-by-point responses addressing your critiques are provided below.

Major comments

1. Improve clarity and visibility of stimulation schedules (S1–S3)

The three stimulation schedules are central to the study but are described in a way that can be difficult to follow. I strongly recommend improving the layout of the schematic timeline (in Figure 1) to more clearly indicate when RANKL and OMVs are added or removed in S1, S2, and S3 for both RAW264.7 and BMMC experiments. This will greatly aid the interpretation of stage-specific effects throughout the Results. Additionally, explain why the authors used different incubation times with the OMV. Why didn't they incubate with OMV for 7 days in S2 and S3 so that “pro-inflammatory” stimuli would better regulate the timing of the stimuli in S1?

We thank the reviewer for this insightful comment regarding the clarity of the stimulation schedules. In response, we have revised the schematic timeline to more clearly illustrate the timing of the M-CSF, RANKL and Pg-OMVs addition for both RAW264.7 and BMM experiments (Fig 1A, Fig S1B, and Fig S4B). Regarding the different OMV incubation times, we intended to evaluate the effects of Pg-OMVs at distinct stages of OC differentiation, reflecting the pathophysiological context of periodontitis, in which osteoclast precursors (monocytes) continuously migrate from the circulation. Unlike the in vitro osteoclastogenesis assay, it is plausible that various differentiation stages of osteoclast precursors coexist within periodontitis lesions. S1 represents Pg-OMVs exposure before RANKL-induced differentiation. S2 corresponds to the early differentiation phase. S3 models the late differentiation and maturation phase. The shorter Pg-OMVs exposure periods used in S2 and S3 were designed to capture stage-specific effects. In particular, in S3, osteoclast maturation becomes pronounced, and prolonged exposure to Pg-OMVs often induces cell death, making accurate assessment difficult. For this reason, the overall experimental period was set to 7 days, with shorter Pg-OMVs’ exposure windows that align with the physiological timing of differentiation and allow reliable evaluation of each stage. We have clarified these points in the revised Discussion sections (Line 354-364).

 

2. Justification and exploration of the OMV dose

The authors perform a dose–response viability assay and then select 1 µg/mL OMVs for subsequent experiments. However, the functional outcomes (osteoclastogenesis, pit formation, cytokine readouts) are not evaluated across different OMV concentrations. I suggest that the authors provide a stronger rationale for why 1 µg/mL is considered physiologically or pathologically relevant in the context of periodontal lesions. This would help readers understand the significance and relevance of the observed effects.

We appreciate the reviewer’s thoughtful suggestions regarding the selection of the Pg-OMVs dose. To determine an appropriate concentration, we first conducted a dose-response viability/growth assay and selected 1 µg/mL Pg-OMVs as the highest dose that promoted cell proliferation without inducing cytotoxicity in RAW264.7 cells (Figure 1 B). In addition, several previous studies analyzing Pg-OMVs-mediated cellular responses have employed 1 µg/mL Pg-OMVs, placing this dose within a widely used and biologically relevant range. We have now added a citation to clarify this rationale (Reference. 59, 60,). We agree that assessing functional outcomes across multiple Pg-OMVs’ concentrations would provide further insight. We acknowledge this as a limitation of the present study and as an important direction for future research. We revised our manuscript in this point in the Discussion section (Line 349-353).

3. RAW264.7 vs. primary cell data. Discuss translational relevance

The study mainly uses RAW264.7 cells, with limited validation in bone marrow–derived mononuclear cells. It would improve the manuscript to expand the primary cell data where possible (e.g., pit formation, OC-STAMP expression, macrophage markers), or at least to more thoroughly discuss the limitations of RAW264.7 as a substitute for primary osteoclast precursors. The Discussion should also more clearly address how these in vitro results can be applied to preclinical in vivo periodontitis research and how they may correlate with clinical disease.

We appreciate the reviewer’s insightful comments regarding the use of RAW264.7 cells and the translational relevance of our findings. RAW264.7 cells are widely used as an osteoclast precursor model because of their reproducibility, homogeneity, and suitability for dissecting molecular mechanisms. However, we fully agree that they do not completely recapitulate the differentiation dynamics or heterogeneity of primary OC precursors. To address this, we included complementary experiments using bone marrow-derived mononuclear cells (BMMCs), which confirmed the stage-dependent effects of Pg-OMVs observed in RAW264.7 cells. Although we were not able to expand all assays in the current study in BMMCs, we have strengthened the Discussion to more explicitly acknowledge these limitations (Line 497-508).

Regarding translational relevance, our findings that the effects of Pg-OMVs vary depending on the differentiation stage of osteoclasts provides an important explanation for the heterogeneity observed in periodontal bone destruction, including differences in the rate, extent, and cellular diversity of bone resorption within periodontal lesions. To clarify this, we revised the Discussion section (Line 468-493, 508-511).

 

4. Mechanistic depth regarding OMV components and signaling

This is my primary concern with the manuscript. The authors attribute several effects to LPS/TLR2 signaling and OC-STAMP–mediated fusion, but these mechanistic links remain largely inferred. To substantiate these claims, the authors must include data to demonstrate the presence of LPS in the OMVs. For example, they may consider additional controls such as TLR2/4 blockade or deficiency, comparison with purified LPS, and/or OMVs treated to inactivate specific components (e.g., protease inhibition for gingipains). An ELISA assay could also be used to enhance the strength of the LPS narrative/argument. If new experiments are not feasible at this stage, the authors must soften the causal language and clearly frame these as speculations or hypotheses that may explain the current data.

We thank the reviewers for their constructive comments. We fully acknowledge that the current data do not allow us to definitively attribute the observed effects to specific Pg-OMVs components—such as LPS, gingipains, or TLR2/4-mediated pathways. We agree that the additional experiments suggested would substantially strengthen the mechanistic interpretation. We suspect that numerous factors contained in Pg-OMVs affect osteoclast differentiation, but identifying specific factors is difficult with the data from this study. Accordingly, we have softened our causal language and revised related statements to clarify that our interpretations represent hypotheses rather than definitive conclusions (Line 404-419).

 

5. Macrophage polarization readouts and interpretation

The conclusion that early OMV exposure promotes M1 macrophage differentiation relies on STAT1 phosphorylation and limited surface marker data (F4/80, CD86). The authors should either (a) include expression data for canonical M1/M2 markers (e.g., iNOS, IL-12, TNFα, Arg1, CD206), or (b) soften the language to highlight that OMVs induce an “M1-like” or “pro-inflammatory” phenotype rather than definitive M1 polarization. Clarifying this will strengthen the macrophage findings and make them more appropriately cautious. I also recommend adjusting the gating in Figure 5D. Your control quadrant lines are currently positioned, cutting through the middle of the main population (F4/80 neg, CD86 neg). Alternatively, explain why the quadrants are placed as they are now.

We thank the reviewers for their valuable comments regarding macrophage polarization and flow cytometry analysis. Regarding macrophage polarization, we agree that the current data, based on STAT1 phosphorylation and limited surface markers, are insufficient to definitively conclude M1 polarization. In the revised manuscript, we have softened the overall terminology to describe the observed phenotype as "M1-like" or "pro-inflammatory" rather than clear-cut M1 polarization. (Line 446-449).

Regarding Figure 5D, we acknowledge that the current quadrant delineation may not be optimal. Originally, quadrants were set based on isotype controls and unstained samples to detect subtle changes in marker expression, but we recognize that their current arrangement may be visually confusing. Therefore, we adjusted the quadrant boundaries to more clearly align with the negative population while remaining consistent with our control-based gating strategy. The revised version of Figure 5D and legend reflect these changes (Fig 5D).

 

Minor comments

1. Statistical reporting and sample size information

For each figure, please clearly indicate the number of biological replicates and the number of independent experiments. It would also be helpful to specify the rationale behind the sample size, the exact statistical tests used, including post-hoc tests following the Kruskal–Wallis test, and how multiple comparisons were addressed.

We thank the reviewers for their important comments regarding statistical reporting and sample size. In the revised manuscript, we have clarified the following points for each figure. For all quantitative data, the number of independent experiments (n) are now explicitly indicated in the figure legends. Sample sizes were determined based on pilot experiments to ensure sufficient statistical power to detect biologically meaningful differences. For comparisons involving more than two groups, we used the Kruskal–Wallis test followed by a Steel-Dwass (multiple comparisons) or Steel (comparison with control) post-hoc test. These clarifications have been incorporated into the Methods section (Line 649-655) and figure legends to ensure full transparency of our statistical analyses.

 

2. Clarify the “failed osteoclast” concept

The link between reduced OMV phagocytosis in certain schedules, enhanced osteoclastogenesis, and the proposed idea of “failed osteoclasts” is intriguing but currently somewhat speculative. I suggest tightening this part of the Discussion to clearly distinguish observed findings (phagocytosis rates, TRAP staining, pit formation) from more speculative models, and outlining what additional experiments would be required to formally demonstrate the existence and function of such “failed osteoclasts.”

We thank the reviewer for this insightful comment regarding the “failed osteoclast” concept.

We agree that the current data, including reduced OMV phagocytosis in certain stimulation schedules, enhanced TRAP-positive osteoclast formation, and pit formation assays, support the presence of stage-dependent effects of Pg-OMVs, but do not conclusively demonstrate the existence of “failed osteoclasts.” In the revised manuscript, we have clarified the Discussion to distinguish clearly between observed findings (e.g., phagocytosis rates, TRAP staining, resorption activity) and speculative interpretations regarding the potential functional consequences of impaired OMV uptake (Line 450-467).

 

3. Language, grammar, and terminology consistency

The manuscript would benefit from careful editing of the English language. There are multiple typographical and grammatical issues (“linage” vs. “lineage,” “Westren blotting,” misspelled cell line names, double periods, inconsistent spacing). Abbreviations and terms ( “OCgenesis,” “OC/MΦ unit”) should be standardized throughout and defined clearly at first use. A thorough edit will significantly improve readability.

We thank the reviewer for highlighting issues related to language, grammar, and terminology.

In the revised manuscript, we have carefully reviewed and corrected typographical errors, grammatical mistakes, and inconsistencies in cell line names. All abbreviations and terms, including “OC-genesis” and “OC/MΦ unit,” have been standardized and are clearly defined at first use.

A thorough language edit has been performed throughout the manuscript to improve readability and ensure consistent terminology.

 

4. Figure and legend clarity

Please ensure all figures include: (i) clearly labeled axes with units (for example, Fig 2B has no unit), (ii) explicit identification of conditions (Fig 2A shows groups as S1, S1, S3, while on 2B the groups are described as Control, +RANKL, +RANKL+OMV), and (iii) the scale used in the bars for all microscopy images. For flow cytometry data, brief descriptions of gating strategies and representative plots in the supplement would be helpful. Some legends currently repeat text from the Results; these can be streamlined while still providing enough detail for the figures to be interpretable on their own.

We thank the reviewer for the detailed suggestions regarding figure presentation.

In the revised manuscript, we have addressed the following points for all figures:

(i): All quantitative figures now include clearly labeled axes with appropriate units.

(ii): Group labels in all figures have been standardized and clearly described. We also apologize for any confusion regarding Fig 2B. The groups “Control”, “+RANKL”, and “+RANKL+OMV” represent the group assignments within each respective experimental schedule. We have revised the figure to improve clarity.

(iii): Scale bars are now included in all microscopy images, and the exact scale is indicated in the figure legends.

 

Flow cytometry figures:

We have added brief descriptions of gating strategies in the figure legends and included representative plots in the Supplementary Information to enhance interpretability (Fig. S8).

 

Figure legends:

Legends have been streamlined to avoid redundancy with the Results section, while still providing sufficient detail for each figure to be interpreted independently.

These revisions improve clarity, consistency, and reproducibility of all figures in the manuscript.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for this nice manuscript. However, I have some small objections almost for each chapter:

1) Introduction. Please, underline the Novelty of the reserahc here in one short paragraph to give better understanding to the reader the result of done huge work!

2) M+M. Please, give the reference to the protocol for each method used in the research starting from the subsection 4.2

-also I would liek to invite you to make flowchart of the research design as so manu methods are used here!

3) Discussion. Please, end this section with the Limitation paragraph.

4) Conclusions earn my main objection. Please, remove the extra words "we revealed..." and shortly add why the Pg-OMVs exert..." and why "they inhibit isteoclastogenesis..."I mean - include very shortly the most important result what indicates this...

Author Response

We appreciate the Reviewers for their thoughtful and constructive feedback on our manuscript. We have carefully addressed each concern and made the necessary revisions to improve the manuscript. Below are our detailed responses to the reviewers’ comments, with their remarks in italics, followed by our replies.

Reviewer 2

1. Please, underline the Novelty of the reserahc here in one short paragraph to give better understanding to the reader the result of done huge work!

Thank you for your valuable comment. In the revised Introduction, we have added a concise paragraph explicitly emphasizing the novelty of our study (Line 111-128). Our study is the first to systematically examine the stage-specific effects of Pg-OMVs on monocyte-derived osteoclast differentiation and to reveal that Pg-OMVs exert bidirectional, differentiation-stage-dependent regulation.

 

2. M+M. Please, give the reference to the protocol for each method used in the research starting from the subsection 4.2

-also I would liek to invite you to make flowchart of the research design as so manu methods are used here!

Thank you for your valuable suggestion. We have added some specific references for each method. In response to your request, we have created a comprehensive flowchart summarizing the overall research design and the sequential workflow. This flowchart has been added as Figure S1 in the revised manuscript.

3. Please, end this section with the Limitation paragraph.

Thank you for this helpful suggestion. We have added a limitation paragraph at the end of the Discussion section to clarify the constraints of our study and to indicate directions for future research (Line 494-511).

 

4. Conclusions earn my main objection. Please, remove the extra words "we revealed..." and shortly add why the Pg-OMVs exert..." and why "they inhibit isteoclastogenesis..."I mean - include very shortly the most important result what indicates this...

Thank you for your insightful comment regarding the Conclusions section. In accordance with your suggestion, we have revised this section by concisely stating why Pg-OMVs exert stage-dependent actions (Line 657-661).

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I have no more remarks or questions.

Thank you and Ill advice to publish the manuscript.

Author Response

Dear Authors, there are six references from the previous century (out of 68) cited in the literature. This is excessive for such a serious journal. Please remove or replace them with more recent sources.

Response to reviewers’ comments

1. We thank the Reviewer for this valuable comment. As suggested in the previous revision, additional references had been added, bringing the total to 81 citations. After carefully re-examining the reference list, in the current revised version, we have removed as older 6 references (published in the previous century) and replaced 2 references with more recent, relevant studies. As a result, the manuscript includes 75 references. In addition, we have revised the reference list to ensure consistency with the main text and reordered the references accordingly. These changes are highlighted in blue in the revised reference list.