Abstract

Filifactor alocis, an asaccharolytic anaerobic Gram-positive rod (AAGPR), is an emerging marker of periodontitis. Severe periodontitis causes destruction of the alveolar bone that supports teeth and can even lead to tooth loss. Based on our previous report thatF. alocis-derived extracellular vesicles (FA EVs) contain various effector molecules and have immunostimulatory activity, we investigated the effect of FA EVs on osteogenesis using mouse bone-derived mesenchymal stromal cells (BMSCs). FA EVs dramatically inhibited bone mineralization similar to whole bacteria and reduced the expression levels of osteogenic marker genes. The osteogenic differentiation of TLR2-deficient BMSCs was not inhibited by FA EVs, suggesting that their inhibitory effect on osteogenesis is dependent on TLR2 signaling. FA EVs effectively activated TLR2 downstream signaling of the MAPK and NF-kappa B pathways. In addition, FA EVs regulated RANKL and OPG gene expression, increasing the RANKL/OPG ratio in BMSCs in a TLR2-dependent manner. Our study suggests thatF. alocis-derived EVs interfere with bone metabolism via TLR2 activation, providing insight into the pathogenesis of bone loss associated with periodontitis.


Authors

Song, Min-Kyoung;  Kim, Hyun Young;  Choi, Bong-Kyu;  Kim, Hong-Hee

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  • 1 reviewer
  • pre-publication peer review (FINAL ROUND)
    Decision Letter
    2020/07/16

    16-Jul-2020

    Dear Ms Song:

    It is a pleasure to accept your manuscript entitled "Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling" in its current form for publication in Molecular Oral Microbiology.

    Please note although the manuscript is accepted the files will now be checked to ensure that everything is ready for publication, and you may be contacted if final versions of files for publication are required.

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    Thank you for your excellent contribution. On behalf of the Editors of Molecular Oral Microbiology, we look forward to your continued contributions to the Journal.

    Sincerely,
    Dr Richard Lamont
    Editor in Chief, Molecular Oral Microbiology

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    Author Response
    2020/07/10

    In this 2nd revision, we amended the title as the Editor-in-Chief recommended and modified the text of manuscript to accommodate suggestions provided by the Reviewer 1.

    Reviewers' Comments to Author:

    Reviewer: 1

    Comments to the Author
    The authors have adequately answered to most of the reviewer’s comments and suggestions. I have 4 minor comments/suggestions for this revised manuscript.

    1. 4th query from reviewer 2: A justification of why 7 or 4 days were used to expose the BMSCs with either the Fa-EVs or the whole bacterium is needed.

    The authors have answered this query satisfactorily but this explanation should also be included in the revised manuscript (wherever they think it fits best; materials and methods or discussion).

    We included the answers to the 4th question from Reviewer 2 in Material and Method section of the re-revised manuscript (page 6, line 141-143).

    1. 5th query from reviewer 2: In Fig 1, the authors state that the BMSCs were cultured with different MOI of F. alocis during 4 days. How was this experiment done, using a tissue culture incubator or in the anaerobic chamber? Four days in an aerobic environment, such as the tissue culture incubator, will be deleterious to F. alocis. If the incubation was done in aerobic conditions, could it be that dead F. alocis also impacts on bone mineralization? The authors should clarify the culture conditions. Determining if the phenotype is similar between live vs. dead F. alocis will be a relevant point.

    The authors have stated that F. alocis was cultured aerobically for 4 days. Definitely bacteria will be fully dead in 4 days. This is also confirmed by supplementary figure 1. Heat killed bacteria gave similar results as whole bacteria after 4 days. This concludes that result shown in figure 1B is not due to live whole F. alocis but due to the dead bacteria. Therefore authors should change this result (page 9, line 197-198) in the manuscript accordingly.

    As suggested by the reviewer, we stated that the results in Figure 1B may have been attributed to dead bacteria, not live F. alocis, in the manuscript (page 8, pages 198-202).

    1. Page 7, line 140-42: Materials and Methods section for osteogenic differentiation should clearly specify that F. alocis was maintained under aerobic conditions.

    We revised the manuscript reflecting this point as suggested (page 6, line 139-141)

    1. Page 19, line 497-98: BMSCs were cultured in osteogenic medium with F. alocis at the indicated MOI for four days. The authors should add “in the CO2 incubator” at the end of the sentence after four days.

    We revised the manuscript as suggested (page 18, line 498-499)



    Cite this author response
  • pre-publication peer review (ROUND 2)
    Decision Letter
    2020/07/07

    We recognize that the impact of the COVID-19 pandemic may affect your ability to return your revised manuscript to us within the requested timeframe. If this is the case, please let us know.

    07-Jul-2020

    Dear Ms Song:

    Manuscript ID MOM-04-20-1156.R1 entitled "Periodontitis pathogen Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling".

    Thank you for submitting a revised version of your manuscript to Molecular Oral Microbiology. This has now been evaluated and the reviewers are generally satisfied with most of your responses. Reviewer 1, however, has highlighted a few remaining issues that require further clarification. In addition to these points, please provide the figures as eps or tif files at 300 dpi or higher. Please also consider amending the title to 'Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling'.

    For these minor revisions, please highlight the changes to your manuscript within the document by using the track changes mode in MS Word or by using bold or colored text. To access your manuscript, log into https://mc.manuscriptcentral.com/mom and enter your Author Center, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been amended to denote a revision. Please DO NOT upload your revised manuscript as a new submission’

    You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript using a word processing program and save it on your computer. Once the revised manuscript is prepared, you can upload it and submit it through your Author Center.

    When submitting your revised manuscript, you will be able to respond to the comments made by the reviewers in the space provided. You can use this space to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the reviewers. If referring to page or line numbers please use those in the revised version.

    IMPORTANT: Your original files are available to you when you upload your revised manuscript. Please delete any redundant files before completing the submission.

    Because we are trying to facilitate timely publication of manuscripts submitted to Molecular Oral Microbiology, your revised manuscript should be uploaded as soon as possible. If it is not possible for you to submit your revision within 30 days then please contact me to discuss the possibility of extending the revision time.

    Once again, thank you for submitting your manuscript to Molecular Oral Microbiology and we look forward to receiving your revision.

    Sincerely,
    Dr Richard Lamont
    Editor in Chief, Molecular Oral Microbiology

    Reviewers' Comments to Author:

    Reviewer: 1

    Comments to the Author
    The authors have adequately answered to most of the reviewer’s comments and suggestions. I have 4 minor comments/suggestions for this revised manuscript.

    1. 4th query from reviewer 2: A justification of why 7 or 4 days were used to expose the BMSCs with either the Fa-EVs or the whole bacterium is needed.

    The authors have answered this query satisfactorily but this explanation should also be included in the revised manuscript (wherever they think it fits best; materials and methods or discussion).

    1. 5th query from reviewer 2: In Fig 1, the authors state that the BMSCs were cultured with different MOI of F. alocis during 4 days. How was this experiment done, using a tissue culture incubator or in the anaerobic chamber? Four days in an aerobic environment, such as the tissue culture incubator, will be deleterious to F. alocis. If the incubation was done in aerobic conditions, could it be that dead F. alocis also impacts on bone mineralization? The authors should clarify the culture conditions. Determining if the phenotype is similar between live vs. dead F. alocis will be a relevant point.

    The authors have stated that F. alocis was cultured aerobically for 4 days. Definitely bacteria will be fully dead in 4 days. This is also confirmed by supplementary figure 1. Heat killed bacteria gave similar results as whole bacteria after 4 days. This concludes that result shown in figure 1B is not due to live whole F. alocis but due to the dead bacteria. Therefore authors should change this result (page 9, line 197-198) in the manuscript accordingly.

    1. Page 7, line 140-42: Materials and Methods section for osteogenic differentiation should clearly specify that F. alocis was maintained under aerobic conditions.

    2. Page 19, line 497-98: BMSCs were cultured in osteogenic medium with F. alocis at the indicated MOI for four days. The authors should add “in the CO2 incubator” at the end of the sentence after four days.

    Decision letter by
    Cite this decision letter
    Reviewer report
    2020/07/06

    The authors addressed all of my queries. I don't have any additional questions.

    Reviewed by
    Cite this review
    Reviewer report
    2020/07/02

    The authors have adequately answered to most of the reviewer’s comments and suggestions. I have 4 minor comments/suggestions for this revised manuscript.

    1. 4th query from reviewer 2: A justification of why 7 or 4 days were used to expose the BMSCs with either the Fa-EVs or the whole bacterium is needed.

    The authors have answered this query satisfactorily but this explanation should also be included in the revised manuscript (wherever they think it fits best; materials and methods or discussion).

    1. 5th query from reviewer 2: In Fig 1, the authors state that the BMSCs were cultured with different MOI of F. alocis during 4 days. How was this experiment done, using a tissue culture incubator or in the anaerobic chamber? Four days in an aerobic environment, such as the tissue culture incubator, will be deleterious to F. alocis. If the incubation was done in aerobic conditions, could it be that dead F. alocis also impacts on bone mineralization? The authors should clarify the culture conditions. Determining if the phenotype is similar between live vs. dead F. alocis will be a relevant point.

    The authors have stated that F. alocis was cultured aerobically for 4 days. Definitely bacteria will be fully dead in 4 days. This is also confirmed by supplementary figure 1. Heat killed bacteria gave similar results as whole bacteria after 4 days. This concludes that result shown in figure 1B is not due to live whole F. alocis but due to the dead bacteria. Therefore authors should change this result (page 9, line 197-198) in the manuscript accordingly.

    1. Page 7, line 140-42: Materials and Methods section for osteogenic differentiation should clearly specify that F. alocis was maintained under aerobic conditions.

    2. Page 19, line 497-98: BMSCs were cultured in osteogenic medium with F. alocis at the indicated MOI for four days. The authors should add “in the CO2 incubator” at the end of the sentence after four days.

    Reviewed by
    Cite this review
    Author Response
    2020/06/17

    Response to Comments from the reviewers:

    Reviewers' Comments to Author:

    Reviewer: 1

    In the manuscript entitled “Periodontitis pathogen Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling” submitted by Song et al., the authors have shown that extracellular vesicles from F. alocis interfere with bone metabolism via activating TLR2 receptors which may explain the pathogenesis of bone loss during severe periodontitis. In their earlier manuscript, they have isolated the extracellular vesicles from F. alocis and showed their immunostimulatory activity. This is an interesting follow up manuscript as this study show, for the first time, the role of vesicles from oral Gram-positive bacteria in bone metabolism.

    The results presented will contribute to our understanding in this field as there is no information in connection with F. alocis extracellular vesicles and bone homeostasis.

    The results are clear and I do have any major comments.

    Minor comments:

    1. Page 3, line 82: “Bacterial extracellular vesicles (BEVs) contain various cargo” instead of “Bacteria extracellular vesicles (BEVs) contain various cargo”.

    2. Page 4, line 103: “and increase RANKL/OPG gene expression ratio for osteoclastogenesis” rather than “and increase RANKL/OPG for osteoclastogenesis”.

    3. Page 5, line 108: “InvivoGen” instead of “Invivogen”.

    4. Page 6, line 137: “assessed by Alizarin and/or von Kossa staining” because von Kossa stain was used in figure 1 to assess bone mineralization.

    5. Page 6, line 145: Please put period (.) at the end of the sentence.

    6. Page 8, line 177: F. alocis instead of Filifactor alocis.

    7. Page 9, line 213: “These results indicate that FA EVs inhibited….” rather than “This result indicates that FA EVs inhibited….”

    8. Page 10, lines 243-245: “In contrast, in the absence of TLR2, expression changes in RANKL and OPG after FA EV treatment were moderated compared to WT” is not clearly stated. Consider revising the sentence.

    9. Page 11, line 271: “For example, P. gingivalis 16S rRNAs were detected in…..” Here the authors are referring that P. gingivalis is seen at other sites (other than periodontal sites). Two very recent reports have identified F. alocis at extra-oral sites, in patients with lung cancer and chronic dysphagia [1, 2]. The authors should refer these two articles also.

    10. Gray, R. M. & Vidwans, M. Mixed anaerobic thoracic empyema: the first report of Filifactor alocis causing extra-oral disease. New Microbes New Infect. 29:100528 (2019).

    11. Wang, K. et al. A Preliminary Study of Microbiota Diversity in Saliva and Bronchoalveolar Lavage Fluid from Patients with Primary Bronchogenic Carcinoma. Med Sci Monit. 25, 2819–2834 (2019).

    12. Page 18, line 465: Please put period (.) at the end of the sentence.

    13. Please provide the full name of the gene hprt which was used as control in figures 1 and 2.

    We revised the manuscript reflecting all the 11 points as suggested.

    Reviewer: 2

    The same group published an article in 2019 describing that the emerging oral pathogen Filifactor alocis produced extracellular vesicles that had the ability to induce an array of cytokines and chemokines from THP-1 cell and human keratinocyte cell line. This manuscript by Song et al, further characterizes the role that F. alocis-derived extracellular vesicles now in terms of osteoclastogenesis. This is an interesting work and provides valuable information about the pathogenic potential of F. alocis.

    1) More detail is required to understand how the infection with BMSCs was done, what was the cell density used?

    We provide the cell number for infection and culture conditions in the revised manuscript (page 5~6).

    2) None of the 4 figure legends specify how many biological replicates were done. Even when using the CHO-TLR2 or TLR4 cell line, are the data shown an average of triplicate sample replicates, or biological replicates?

    The Statistical Analysis section in the Materials and Methods has been revised to make this point clear (page 7). All experiments were performed at least three times and representative results from one experiment were presented. All histograms, except those for RT-qPCR and ELISA, are from three separate wells of cultures. The data point of RT-qPCR and ELISA analyses is average of triplicates of a sample generated by pooling four and two wells of cultures, respectively. The histogram at the bottom of Figure 2A (experiment using the CHO-TLR2 or TLR4 cell line) are also from three separate wells of cultures, and the top of Figure 2A (FACS graph) shows only one representative well.

    3) For the WT or TLR2KO BMSCs how many mice were used? Are these BMSCs obtained from pooling cells from different mice? Are these cells coming from male or female mice?

    WT or TLR2KO BMSCs were obtained by pooling cells from 4 different male mice each. This is stated in the revised manuscript (page 5).

    4) A justification of why 7 or 4 days were used to expose the BMSCs with either the Fa-EVs or the whole bacterium is needed.

    The treatment time was based on the time required for osteogenic differentiation of BMSCs in the absence of EVs or bacterium. Empirically, our BMSCs require about 4~7 days to generate mineralization nodules depending on the BMSCs preps. We determine culture period each time by visually checking cell conditions and mineralization nodules under a microscope.

    5) In Fig 1, the authors state that the BMSCs were cultured with different MOI of F. alocis during 4 days. How was this experiment done, using a tissue culture incubator or in the anaerobic chamber? Four days in an aerobic environment, such as the tissue culture incubator, will be deleterious to F. alocis. If the incubation was done in aerobic conditions, could it be that dead F. alocis also impacts on bone mineralization? The authors should clarify the culture conditions. Determining if the phenotype is similar between live vs. dead F. alocis will be a relevant point.

    (The entire response including figure is attached as a file.)
    F. alocis was cultured under anaerobic condition, but during the co-culture with BMSCs, it was incubated under an aerobic condition in a cell culture incubator. As the reviewer pointed out, culturing in aerobic conditions for several days can be detrimental to F.alocis.
    To examine whether the reduced mineralization shown in Figure 1B is also caused by dead F.alocis, we co-cultured heat-killed F.alocis with BMSCs. Heat-killed F.alocis was obtained by heating F.alocis at 70 °C for an hour. As shown in the figure below, both F.alocis and heat-killed F.alocis seem to have similar inhibitory effects on mineralization. This result has been added to the revised manuscript as Supplementary Figure 1.

    6) Measuring RANKL, OPG and M-CSF levels in the supernatants collected from the Fa-EV treated BMSCs will be of value to determine if the increases in mRNA levels also translate into an increase in protein release. Ultimately the release of high levels of RANKL and M-CSF combined with low levels of OPG will be relevant to make the case that Fa-EVs promote osteoclast formation.

    (The entire response including figure is attached as a file.)
    As suggested by the reviewer, we measured the amount of RANKL, OPG and M-CSF in the supernatants of BMSCs treated with FA EVs. The experimental scheme is as above.
    Consistent with mRNA results, the amount of secreted RANKL was increased but OPG was decreased over time by FA EVs (Figure 4B, revised manuscript). The ratio of RANKL to OPG is more obvious. FA EVs also increased M-CSF significantly. However, all these changes were not observed in the supernatants of TLR2 KO BMSCs. Collectively, FA EVs promote osteoclast formation by regulating the gene expression and secretion of RANKL, OPG, and M-CSF in a manner dependent on TLR2.



    Cite this author response
  • pre-publication peer review (ROUND 1)
    Decision Letter
    2020/05/06

    We recognize that the impact of the COVID-19 pandemic may affect your ability to return your revised manuscript to us within the requested timeframe. If this is the case, please let us know.

    06-May-2020

    Dear Ms Song:

    Manuscript ID MOM-04-20-1156 entitled "Periodontitis pathogen Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling".

    Thank you for submitting your paper to Molecular Oral Microbiology. The manuscript has now been evaluated and the comments of the reviewers are included at the bottom of this letter. As you will see, the reviewers believe this to be an interesting and timely study. Nonetheless, the reviewers have raised a number of issues that should be addressed before the manuscript is considered further. If you would like to modify your paper according to the suggestions of the reviewers, it will be reconsidered for publication.

    In your revision, please highlight the changes to your manuscript within the document by using the track changes mode in MS Word or by using bold or colored text. To access your manuscript, log into https://mc.manuscriptcentral.com/mom and enter your Author Center, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been amended to denote a revision. Please DO NOT upload your revised manuscript as a new submission.

    You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript using a word processing program and save it on your computer. Once the revised manuscript is prepared, you can upload it and submit it through your Author Center.

    When submitting your revised manuscript, you will be able to respond to the comments made by the reviewers in the space provided. You can use this space to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the reviewers. If referring to page or line numbers please use those in the revised version.

    If you would like help with English language editing, or other article preparation support, Wiley Editing Services offers expert help with English Language Editing, as well as translation, manuscript formatting, and figure formatting at www.wileyauthors.com/eeo/preparation. You can also check out our resources for Preparing Your Article for general guidance about writing and preparing your manuscript at www.wileyauthors.com/eeo/prepresources.

    IMPORTANT: Your original files are available to you when you upload your revised manuscript. Please delete any redundant files before completing the submission.

    Because we are trying to facilitate timely publication of manuscripts submitted to Molecular Oral Microbiology, your revised manuscript should be uploaded as soon as possible. If it is not possible for you to submit your revision within 60 days, we may have to consider your paper as a new submission. If you feel that you will be unable to submit your revision within this time please contact me to discuss the possibility of extending the revision time.

    Once again, thank you for submitting your manuscript to Molecular Oral Microbiology and we look forward to receiving your revision.

    Sincerely,
    Dr Richard Lamont
    Editor in Chief, Molecular Oral Microbiology

    Reviewers' Comments to Author:

    Reviewer: 1

    In the manuscript entitled “Periodontitis pathogen Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling” submitted by Song et al., the authors have shown that extracellular vesicles from F. alocis interfere with bone metabolism via activating TLR2 receptors which may explain the pathogenesis of bone loss during severe periodontitis. In their earlier manuscript, they have isolated the extracellular vesicles from F. alocis and showed their immunostimulatory activity. This is an interesting follow up manuscript as this study show, for the first time, the role of vesicles from oral Gram-positive bacteria in bone metabolism.

    The results presented will contribute to our understanding in this field as there is no information in connection with F. alocis extracellular vesicles and bone homeostasis.

    The results are clear and I do have any major comments.

    Minor comments:

    1. Page 3, line 82: “Bacterial extracellular vesicles (BEVs) contain various cargo” instead of “Bacteria extracellular vesicles (BEVs) contain various cargo”.

    2. Page 4, line 103: “and increase RANKL/OPG gene expression ratio for osteoclastogenesis” rather than “and increase RANKL/OPG for osteoclastogenesis”.

    3. Page 5, line 108: “InvivoGen” instead of “Invivogen”.

    4. Page 6, line 137: “assessed by Alizarin and/or von Kossa staining” because von Kossa stain was used in figure 1 to assess bone mineralization.

    5. Page 6, line 145: Please put period (.) at the end of the sentence.

    6. Page 8, line 177: F. alocis instead of Filifactor alocis.

    7. Page 9, line 213: “These results indicate that FA EVs inhibited….” rather than “This result indicates that FA EVs inhibited….”

    8. Page 10, lines 243-245: “In contrast, in the absence of TLR2, expression changes in RANKL and OPG after FA EV treatment were moderated compared to WT” is not clearly stated. Consider revising the sentence.

    9. Page 11, line 271: “For example, P. gingivalis 16S rRNAs were detected in…..” Here the authors are referring that P. gingivalis is seen at other sites (other than periodontal sites). Two very recent reports have identified F. alocis at extra-oral sites, in patients with lung cancer and chronic dysphagia [1, 2]. The authors should refer these two articles also.

    10. Gray, R. M. & Vidwans, M. Mixed anaerobic thoracic empyema: the first report of Filifactor alocis causing extra-oral disease. New Microbes New Infect. 29:100528 (2019).

    11. Wang, K. et al. A Preliminary Study of Microbiota Diversity in Saliva and Bronchoalveolar Lavage Fluid from Patients with Primary Bronchogenic Carcinoma. Med Sci Monit. 25, 2819–2834 (2019).

    12. Page 18, line 465: Please put period (.) at the end of the sentence.

    13. Please provide the full name of the gene hprt which was used as control in figures 1 and 2.

    Reviewer: 2

    The same group published an article in 2019 describing that the emerging oral pathogen Filifactor alocis produced extracellular vesicles that had the ability to induce an array of cytokines and chemokines from THP-1 cell and human keratinocyte cell line. This manuscript by Song et al, further characterizes the role that F. alocis-derived extracellular vesicles now in terms of osteoclastogenesis. This is an interesting work and provides valuable information about the pathogenic potential of F. alocis.

    1) More detail is required to understand how the infection with BMSCs was done, what was the cell density used?

    2) None of the 4 figure legends specify how many biological replicates were done. Even when using the CHO-TLR2 or TLR4 cell line, are the data shown an average of triplicate sample replicates, or biological replicates?

    3) For the WT or TLR2KO BMSCs how many mice were used? Are these BMSCs obtained from pooling cells from different mice? Are these cells coming from male or female mice?

    4) A justification of why 7 or 4 days were used to expose the BMSCs with either the Fa-EVs or the whole bacterium is needed.

    5) In Fig 1, the authors state that the BMSCs were cultured with different MOI of F. alocis during 4 days. How was this experiment done, using a tissue culture incubator or in the anaerobic chamber? Four days in an aerobic environment, such as the tissue culture incubator, will be deleterious to F. alocis. If the incubation was done in aerobic conditions, could it be that dead F. alocis also impacts on bone mineralization? The authors should clarify the culture conditions. Determining if the phenotype is similar between live vs. dead F. alocis will be a relevant point.

    6) Measuring RANKL, OPG and M-CSF levels in the supernatants collected from the Fa-EV treated BMSCs will be of value to determine if the increases in mRNA levels also translate into an increase in protein release. Ultimately the release of high levels of RANKL and M-CSF combined with low levels of OPG will be relevant to make the case that Fa-EVs promote osteoclast formation.

    Decision letter by
    Cite this decision letter
    Reviewer report
    2020/05/05

    Comments
    The same group published an article in 2019 describing that the emerging oral pathogen Filifactor alocis produced extracellular vesicles that had the ability to induce an array of cytokines and chemokines from THP-1 cell and human keratinocyte cell line. This manuscript by Song et al, further characterizes the role that F. alocis-derived extracellular vesicles now in terms of osteoclastogenesis. This is an interesting work and provides valuable information about the pathogenic potential of F. alocis.
    Comments:
    1) More detail is required to understand how the infection with BMSCs was done, what was the cell density used?
    2) None of the 4 figure legends specify how many biological replicates were done. Even when using the CHO-TLR2 or TLR4 cell line, are the data shown an average of triplicate sample replicates, or biological replicates?
    3) For the WT or TLR2KO BMSCs how many mice were used? Are these BMSCs obtained from pooling cells from different mice? Are these cells coming from male or female mice?
    4) A justification of why 7 or 4 days were used to expose the BMSCs with either the Fa-EVs or the whole bacterium is needed.

    5) In Fig 1, the authors state that the BMSCs were cultured with different MOI of F. alocis during 4 days. How was this experiment done, using a tissue culture incubator or in the anaerobic chamber? Four days in an aerobic environment, such as the tissue culture incubator, will be deleterious to F. alocis. If the incubation was done in aerobic conditions, could it be that dead F. alocis also impacts on bone mineralization? The authors should clarify the culture conditions. Determining if the phenotype is similar between live vs. dead F. alocis will be a relevant point.

    6) Measuring RANKL, OPG and M-CSF levels in the supernatants collected from the Fa-EV treated BMSCs will be of value to determine if the increases in mRNA levels also translate into an increase in protein release. Ultimately the release of high levels of RANKL and M-CSF combined with low levels of OPG will be relevant to make the case that Fa-EVs promote osteoclast formation.

    Reviewed by
    Cite this review
    Reviewer report
    2020/04/29

    In the manuscript entitled “Periodontitis pathogen Filifactor alocis-derived extracellular vesicles inhibit osteogenesis through TLR2 signaling” submitted by Song et al., the authors have shown that extracellular vesicles from F. alocis interfere with bone metabolism via activating TLR2 receptors which may explain the pathogenesis of bone loss during severe periodontitis. In their earlier manuscript, they have isolated the extracellular vesicles from F. alocis and showed their immunostimulatory activity. This is an interesting follow up manuscript as this study show, for the first time, the role of vesicles from oral Gram-positive bacteria in bone metabolism.

    The results presented will contribute to our understanding in this field as there is no information in connection with F. alocis extracellular vesicles and bone homeostasis.

    The results are clear and I do have any major comments.

    Minor comments:

    1. Page 3, line 82: “Bacterial extracellular vesicles (BEVs) contain various cargo” instead of “Bacteria extracellular vesicles (BEVs) contain various cargo”.

    2. Page 4, line 103: “and increase RANKL/OPG gene expression ratio for osteoclastogenesis” rather than “and increase RANKL/OPG for osteoclastogenesis”.

    3. Page 5, line 108: “InvivoGen” instead of “Invivogen”.

    4. Page 6, line 137: “assessed by Alizarin and/or von Kossa staining” because von Kossa stain was used in figure 1 to assess bone mineralization.

    5. Page 6, line 145: Please put period (.) at the end of the sentence.

    6. Page 8, line 177: F. alocis instead of Filifactor alocis.

    7. Page 9, line 213: “These results indicate that FA EVs inhibited….” rather than “This result indicates that FA EVs inhibited….”

    8. Page 10, lines 243-245: “In contrast, in the absence of TLR2, expression changes in RANKL and OPG after FA EV treatment were moderated compared to WT” is not clearly stated. Consider revising the sentence.

    9. Page 11, line 271: “For example, P. gingivalis 16S rRNAs were detected in…..” Here the authors are referring that P. gingivalis is seen at other sites (other than periodontal sites). Two very recent reports have identified F. alocis at extra-oral sites, in patients with lung cancer and chronic dysphagia [1, 2]. The authors should refer these two articles also.

    10. Gray, R. M. & Vidwans, M. Mixed anaerobic thoracic empyema: the first report of Filifactor alocis causing extra-oral disease. New Microbes New Infect. 29:100528 (2019).

    11. Wang, K. et al. A Preliminary Study of Microbiota Diversity in Saliva and Bronchoalveolar Lavage Fluid from Patients with Primary Bronchogenic Carcinoma. Med Sci Monit. 25, 2819–2834 (2019).

    12. Page 18, line 465: Please put period (.) at the end of the sentence.

    13. Please provide the full name of the gene hprt which was used as control in figures 1 and 2.

    Reviewed by
    Cite this review
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