Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption

Hirofumi Fujita; Aoi Ando; Yohei Mizusawa; Mitsuaki Ono; Takako Hattori; Munenori Habuta; Toshitaka Oohashi; Satoshi Kubota; Hideyo Ohuchi

doi:10.1371/journal.pone.0277307

Peer Review History

Original SubmissionJune 24, 2022
25 Aug 2022 Decision Letter - Jung-Eun Kim, Editor PONE-D-22-18067Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorptionPLOS ONE Dear Dr. Fujita, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Oct 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the study by Fujita et al. the authors create a novel global mouse knockout model of the cysteinyl leukotriene receptor 1 (CysTR1). The authors analyze the skeletal phenotype of these mice. Unfortunately, the authors demonstrate that CyslTR1 is not necessary for osteoclast differentiation. The loss of CysTR1 does not lead to changes in the skeleton under pathological conditions. The authors do demonstrate successfully loss of Cysltr1 expression by recombination and that other related genes are not changed in expression due to the knockout of Cysltr1. There are some minor concerns that should be addressed and are listed below. 1. In Figure 4 the images in panel A should be redone as there is no contrast and it is difficult to see the TRAP positive cells. 2. Besides counting number in panel 4D the authors should also count size of the osteoclasts. 3. Should be stated in the text of the manuscript why the authors chose not to analyze the skeleton under physiological conditions. Reviewer #2: This paper is an interesting analysis of the role of cysteinyl leukotriene receptor 1 in osteoclastic bone resorption. Experimental results using cell culture systems and bone resorption-promoting mouse models clearly demonstrate that cysteinyl leukotriene receptor 1 is not directly involved in osteoclast differentiation and bone resorption. Reviewer #3: The authors investigated roles of cysteinyl leukotriene receptor 1 (CysLTR1) in osteoclastogenesis. To clarify the role in vivo they generated two lines of CysLTR1 mutant mice. Macrophages from those mutant mice did not respond to the stimulation of LTD4. However, these mutants failed to impact osteoclastogenesis in in vivo and in vitro studies. The experiments seems to be well organized and performed, and their conclusion is based on solid data. However, there are some concerns in the present manuscript. Major points 1. Figure 4A…To clearly show no difference in osteoclast number between WT and KO, the quantitative data of osteoclast number are needed. In addition, scale bar is missing. 2. The authors only focused on the roles of CysLTR1 in osteoclast differentiation. To confirm whether the deficiency of CysLTR1 impacts on osteoclastic bone resorbing activity, serum bone resorption parameter such as serum CTX should be analyzed. 3. The explanation of roles of CysLT in bone resorption seems to be too short in Introduction. The authors should explain why they focus on CysLT in bone metabolism and roles of CysLT in bone metabolism and bone diseases in more detail. 4. Fig. 6…Scale bars are missing in panels A and D. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0277307.r001
Revision 1
7 Oct 2022 Author Response Answers to reviewer’s comments Reviewer #1: General comment In the study by Fujita et al. the authors create a novel global mouse knockout model of the cysteinyl leukotriene receptor 1 (CysTR1). The authors analyze the skeletal phenotype of these mice. Unfortunately, the authors demonstrate that CyslTR1 is not necessary for osteoclast differentiation. The loss of CysTR1 does not lead to changes in the skeleton under pathological conditions. The authors do demonstrate successfully loss of Cysltr1 expression by recombination and that other related genes are not changed in expression due to the knockout of Cysltr1. There are some minor concerns that should be addressed and are listed below. Comment 1. In Figure 4 the images in panel A should be redone as there is no contrast and it is difficult to see the TRAP positive cells. Answer to comment 1 We thank the reviewer #1 for the kind remarks. According to your advice, we replaced the images to high magnification images in Fig 4A of the Revised Manuscript with Track Changes. Comment 2. Besides counting number in panel 4D the authors should also count size of the osteoclasts. Answer to comment 2 According to your advice, we measured size of osteoclasts using ImageJ software. The data showed that Montelukast decreased osteoclast size in WT and Cystlr1Δ105 BMMs. There was no significance in osteoclast size between WT and Cystlr1Δ105 osteoclasts treated with Montelukast although we observed a tendency of suppression of Montelukast-decreased osteoclast size in Cystlr1Δ105. We added this results and sentences to Revised Manuscript with Track Changes; the Fig 4F; the Results section line 423-427 and line 445-446. Comment 3. Should be stated in the text of the manuscript why the authors chose not to analyze the skeleton under physiological conditions. Answer to Comment 3. In response to the suggestion, we added the data of bone morphological analysis under physiological conditions. The results of histomorphometry and CT analysis did not show any noticeable differences between WT and Cystlr1 mutants. We added these results and sentences to Revised Manuscript with Track Changes; the S6 Fig; the Results section line 406 and 409; the Supporting information section line 686-693. Reviewer #2: General comment This paper is an interesting analysis of the role of cysteinyl leukotriene receptor 1 in osteoclastic bone resorption. Experimental results using cell culture systems and bone resorption-promoting mouse models clearly demonstrate that cysteinyl leukotriene receptor 1 is not directly involved in osteoclast differentiation and bone resorption. Answer to comment We thank the peer review of reviewer #2. Reviewer #3: General comment The authors investigated roles of cysteinyl leukotriene receptor 1 (CysLTR1) in osteoclastogenesis. To clarify the role in vivo they generated two lines of CysLTR1 mutant mice. Macrophages from those mutant mice did not respond to the stimulation of LTD4. However, these mutants failed to impact osteoclastogenesis in in vivo and in vitro studies. The experiments seems to be well organized and performed, and their conclusion is based on solid data. However, there are some concerns in the present manuscript. Major points Comment 1. Figure 4A…To clearly show no difference in osteoclast number between WT and KO, the quantitative data of osteoclast number are needed. In addition, scale bar is missing. Answer to Comment 1. We thank the reviewer #3 for the kind remarks. According to your advice, we added quantitative data of osteoclast number of WT, KO and Cysltr1Δ105 femur although the sample size was not sufficient due to dead line of revised manuscript submission. The results of histomorphometry analysis did not show any noticeable differences between WT and Cystlr1 mutants. We added scale bar in Fig 4A. We added these results and sentences to the Revised Manuscript with Track Changes; the S6 Fig A-D; the Materials and methods section line 204-206; the Results section line 432; the Supporting information section line 686-689.　 Comment 2. The authors only focused on the roles of CysLTR1 in osteoclast differentiation. To confirm whether the deficiency of CysLTR1 impacts on osteoclastic bone resorbing activity, serum bone resorption parameter such as serum CTX should be analyzed. Answer to Comment 2. As you pointed out, our study lacked an analysis of the bone resorbing activity of osteoclasts. We performed pit formation assay for bone resorbing activity of osteoclast differentiated from WT and Cysltr1Δ105 BMMs from using osteoassay plate. The data showed that there was no difference in the bone resorption activity in vitro between WT and Cysltr1Δ105 osteoclasts. We added this result and sentences to the Revised Manuscript with Track Changes; the Fig 4D; the Materials and methods section line 213-221; the Results section line 413-417, line 429-430 and line 439-441.　 Comment 3. The explanation of roles of CysLT in bone resorption seems to be too short in Introduction. The authors should explain why they focus on CysLT in bone metabolism and roles of CysLT in bone metabolism and bone diseases in more detail. Answer to Comment 3. Thank you for your valuable suggestions. According to your advice, we added the explanation of CysLTs role in osteoclast differentiation and bone resorption. It explained that CysLTs stimulate isolated osteoclasts to form resorption pits on calcified matrices and stimulate bone resorption in organ culture of mouse calvariae. In addition, LTD4 stimulates osteoclast differentiation from macrophage cell line RAW264.7 in the presence of RANKL. Previous our study showed that the CysLT synthesis substrate, GSH stimulated osteoclast differentiation. We added these sentences to the Revised Manuscript with Track Changes; the Introduction section line 71-77 and line 80-81. We removed sentences from next paragraph line 84. Comment 4. Fig. 6…Scale bars are missing in panels A and D. Answer to Comment 4. We added scale bar in Fig 6A and D. We added a sentence to the Revised Manuscript with Track Changes; the Results section line 481 and line 487. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0277307.r002
24 Oct 2022 Decision Letter - Jung-Eun Kim, Editor Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption PONE-D-22-18067R1 Dear Dr. Fujita, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jung-Eun Kim Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: (No Response) ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #3: The authors have addressed concerns from this reviewer. The manuscript is largely improved. This reviewer has no concerns anymore. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0277307.r003
Formally Accepted
7 Nov 2022 Acceptance Letter - Jung-Eun Kim, Editor PONE-D-22-18067R1 Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption Dear Dr. Fujita: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Jung-Eun Kim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277307.r004

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