Immune response against Chlamydia trachomatis via toll-like receptors is negatively regulated by SIGIRR

Mufadhal Al-Kuhlani; Graham Lambert; Sukumar Pal; Luis de la Maza; David M. Ojcius

doi:10.1371/journal.pone.0230718

Peer Review History

Original SubmissionDecember 14, 2019
21 Jan 2020 Decision Letter - Guangming Zhong, Editor PONE-D-19-34581 Immune Response Against Chlamydia trachomatis via Toll-Like Receptors Is Negatively Regulated by SIGIRR PLOS ONE Dear Dr. Ojcius, 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. Reviewer #1 found your study interesting but also raised some concerns: While it may not be necessary to re-run the samples using qRT-PCR for Fig1, please explain why gel separation was used for Fig. 1 while qRT-PCR for Fig.2. It is also important for you to correct and discuss other issues raised by the reviewer. Finally, for Fig. 3, the reviewer requested quantitative data. 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[Note: HTML markup is below. 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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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). 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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: Previous studies have shown that Chlamydia trachomatis infection activates TLR-mediated signaling pathways. Now, Al-Kuhlani et al demonstrated a potential inhibitory role for SIGIRR in TLR signaling in C. trachomatis-infected epithelial cells. Whereas their findings are interesting, the manuscript also has some weaknesses: 1. The expression analyses of SIGIRR in Fig. 1 were done with reverse transcription PCR. The PCR products were analyzed using gel electrophoresis. This is an antiquated technique, and these kinds of results are no longer acceptable to many journals. Given the fact they used to reverse transcription quantitative PCR to generate Fig. 2, they should reanalyze their samples collected for Fig. 1 with quantitative PCR. 2. Given the fact that levels of mRNA and protein are not always in agreement, it would be nice if they perform protein level analysis if antibodies are available. If protein analysis is not feasible, it is important that the authors state mRNA was analyzed in the subheadings of Results and figure titles. The potential discordance between mRNA and protein levels should be stated in the Discussion. 3. For both main text leading to Fig. 1 and the legend, the detection technique should be reverse transcription PCR instead of just PCR. 4. The rationale for selecting the cell lines for SIGIRR expression level analysis in Fig. 1 should be stated. Assuming that it is true that SIGIRR levels in HEK cells are reduced by TLR2, 3, 4 over expression, they should discuss potential implications of the findings and/or underlying mechanisms. 5. In the context of Fig. 2, it is probably more appropriate to change WT to something like NT (non-transformed). 6. Fig. 3 is representative of 3 experiments. They should present quantitative data like in other figures in addition to images. ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0230718.r001
Revision 1
20 Feb 2020 Author Response Reviewer #1: Previous studies have shown that Chlamydia trachomatis infection activates TLR-mediated signaling pathways. Now, Al-Kuhlani et al demonstrated a potential inhibitory role for SIGIRR in TLR signaling in C. trachomatis-infected epithelial cells. Whereas their findings are interesting, the manuscript also has some weaknesses: 1. The expression analyses of SIGIRR in Fig. 1 were done with reverse transcription PCR. The PCR products were analyzed using gel electrophoresis. This is an antiquated technique, and these kinds of results are no longer acceptable to many journals. Given the fact they used to reverse transcription quantitative PCR to generate Fig. 2, they should reanalyze their samples collected for Fig. 1 with quantitative PCR. Authors’ response: The goal for the gel separation was to check for the presence or absence of SIGIRR in these cell lines before we proceeded to introduce the siRNA or vector. Our goal was not to quantify the expression, but simply to determine if these cells were good candidates for the planned experiments (specially the HEK-293-TLRs cells, as they had never been studied before with regards to SIGIRR). Given the large difference between the cell lines, which could be observed even visually, we did not pursue further the quantification of SIGIRR expression. 2. Given the fact that levels of mRNA and protein are not always in agreement, it would be nice if they perform protein level analysis if antibodies are available. If protein analysis is not feasible, it is important that the authors state mRNA was analyzed in the subheadings of Results and figure titles. The potential discordance between mRNA and protein levels should be stated in the Discussion. Authors’ response: In fact, antibodies against SIGIRR are not available. Following the referee’s advice, we therefore state that mRNA was analyzed in the Results and figure legends. 3. For both main text leading to Fig. 1 and the legend, the detection technique should be reverse transcription PCR instead of just PCR. Authors’ response: As mentioned in the response to point 1, the difference in mRNA expression was so dramatically different between the different cell lines (which was clearly obvious to the naked eye), we did not pursue further quantification of SIGIRR expression. 4. The rationale for selecting the cell lines for SIGIRR expression level analysis in Fig. 1 should be stated. Assuming that it is true that SIGIRR levels in HEK cells are reduced by TLR2, 3, 4 over expression, they should discuss potential implications of the findings and/or underlying mechanisms. Authors’ response: Hela cells are the cell of choice for studies of Chlamydia infection, and we therefore verified first that SIGIRR is expressed in HeLa cells. HEK-293 do not express SIGIRR and TLRs, but the stably-transfected HEK cells (HEK-Blue with TLRs) provide a convenient way of measuring NF-�B activation under different conditions. THP-1 served as control for SIGIRR expression since it is known that they express high levels of the protein [Kadota et al, Clin Exp Immunol 2010; 162:348-61]. The HEK cells and the stably-transfected HEK-TLR cells are commercially available. Like the referee, we were intrigued by the observation that the HEK-TLR cells do not express significant amounts of SIGIRR. However, we did not prepare these cells and, unfortunately, do not have an explanation for this result. 5. In the context of Fig. 2, it is probably more appropriate to change WT to something like NT (non-transformed). Authors’ response: We are grateful to the referee for this advice. We therefore made the change in the figure and the text, as recommended. 6. Fig. 3 is representative of 3 experiments. They should present quantitative data like in other figures in addition to images. Authors’ response: Similar to our analysis of Figure 1, the results of the PCR were dramatically different for the different conditions (which could be observed easily by the naked eye). Therefore, we did not pursue further the quantification of this experiment. https://doi.org/10.1371/journal.pone.0230718.r002
9 Mar 2020 Decision Letter - Guangming Zhong, Editor Immune Response Against Chlamydia trachomatis via Toll-Like Receptors Is Negatively Regulated by SIGIRR PONE-D-19-34581R1 Dear Dr. Ojcius, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Guangming Zhong Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have adequately addressed all concerns raised by the reviewers. Reviewers' comments: https://doi.org/10.1371/journal.pone.0230718.r003
Formally Accepted
11 Mar 2020 Acceptance Letter - Guangming Zhong, Editor PONE-D-19-34581R1 Immune Response Against Chlamydia trachomatis via Toll-Like Receptors Is Negatively Regulated by SIGIRR Dear Dr. Ojcius: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Guangming Zhong Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230718.r004

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