The ubiquitin-like modifier FAT10 does not affect IL-12 expression and signaling

Jinjing Cao; Gerardo Omar Alvarez Salinas; Gunter Schmidtke; Michael Basler

doi:10.1371/journal.pone.0323005

Peer Review History

Original SubmissionJanuary 10, 2025
10 Jan 2025 Author Response https://doi.org/10.1371/journal.pone.0323005.r001
24 Feb 2025 Decision Letter - Mariola Ferraro, Editor PONE-D-25-01617The ubiquitin-like modifier FAT10 does not affect IL-12 expression and signalingPLOS ONE Dear Dr. Basler, 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 Apr 10 2025 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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: 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: The study investigates the impact of FAT10 on IL-12 expression and signaling, which has not been extensively studied before. They do this in two very specific models of immune cells. There is no indication of prior publication of these specific results. But this may be a follow up to previous results. The experiments are well-described, but some analyses (e.g., small sample sizes in Fig. 4, lack of flow cytometry gating strategy in figure 3) could be improved for better rigor. Increasing sample size or better more obvious normalization will improve figures like figure one where you see a separation of points which may be a reasoning for expression to not be different. Evaluating fat10 gene expression in C57/B6 mice in figure 4 would help the reader believe that fat10 expression is still up during this window of time. Did the authors do a time course experiment to show why they chose 24 hours of stimulation? It is also not scientifically explained why the authors would shift away from the TNF/IFNg model of expression seen in figure 1A. Yes, conclusions are presented in an appropriate fashion but with limitations. The data show makes assumptions about expression which would support the conclusion that FAT10 does not affect IL-12 expression or signaling. However, variability in some experiments and small sample sizes in signaling studies limit the strength of the conclusions. Including more controls and sometimes replicates would improve the results presented in this study to ensure that the study is properly powered and controlled. Major Comments 1. Clarity in Experimental Design In Th1 differentiation assays, I do not think they included control. it would be beneficial to include a control condition without IL-12 to demonstrate the necessity of IL-12 in this system. 2. STAT Signaling Pathway Investigation (line 208) The authors analyze STAT1 and STAT4 phosphorylation, but IL-12 is also known to activate STAT3 to a certain extent. They briefly stated this without clearly justifying why they did not look at STAT3 3. IL-12 Expression and Secretion (line 244) Figure 2C shows that IL-12 secretion is not altered in FAT10 knockout cells. Since IL-12 has two subunits (p35 and p40), the authors did not measure p35 expression separately, and did not create a limitation section to acknowledged this as a limitation. 4. Potential Redundancy in Text The authors repeatedly emphasize that FAT10 does not alter IL-12 expression across different sections (Results, Discussion). Conclusion The manuscript is well-conceived and presents valuable findings in immunology and cytokine signaling. With minor revisions to improve clarity, correct typographical errors, and address the misplaced reference, the manuscript will be well-suited for publication. Key Recommendations Address the STAT3 signaling question. Consider IL-12 p35 measurement. Remove redundant statements in Discussion. not should be no in line 214. Reviewer #2: While the introduction is technically sound, I feel a re-write may be in order to emphasize the importance of this research and how it's applications may benefit immunological research. That issue aside, there were several factors that warranted my recommendation for major revision in Results section of this manuscript. On line 169 under the "FAT10 does not influence IL-12 expression in DC2.4 cells" segment, there didn't appear to be any mention of validating successful lentiviral transfection. In the next segment "FAT10 has no significant effect on IL-12 expression in bone marrow-derived dendritic cells", BMDCs were stimulated with IFN-γ and LPS to induce IL-12 expression. However, in the previous segment a combination IFNγ/TNF was used to stimulate DC2.4s. The rationale behind using only IFN-γ as opposed to the combination IFNγ/TNF wasn't immediately clear upon review. Lastly, in "FAT10 does not affect Th1 cell differentiation", the rationale as to why IFN-γ was selected as the sole cytokine of focus was unclear. TNF is also considered an important signature cytokine for Th1 differentiation, why was that not selected in addition to IFN-γ to validate results? Overall, while I do partly agree with the overall conclusions of this paper that FAT10 does not appear to affect IL-12 signaling, I feel more validatory work is necessary to confirm many of these results. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). 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Revision 1
21 Mar 2025 Author Response Response to the reviewer comments you will find in the uploaded "Response to reviewers" file. 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: The study investigates the impact of FAT10 on IL-12 expression and signaling, which has not been extensively studied before. They do this in two very specific models of immune cells. There is no indication of prior publication of these specific results. But this may be a follow up to previous results. The experiments are welldescribed, but some analyses (e.g., small sample sizes in Fig. 4, lack of flow cytometry gating strategy in figure 3) could be improved for better rigor. Reply: We agree that sample size in Fig. 4 is rather low. Therefore, we increased sample size in Fig. 4. Furthermore, we included the gating strategy of Figure 3 (in supplementary Fig. S1). Increasing sample size or better more obvious normalization will improve figures like figure one where you see a separation of points which may be a reasoning for expression to not be different. Reply: Real-time RT-PCR experiments were performed in Figure 1. Each data point represents the average of duplicates. The cycle threshold (Ct) value was obtained from the experiment. Gene expression was calculated using the 2−ΔΔCt method, where ΔCt = Ct (target gene) - Ct (reference gene), and ΔΔCt = ΔCt (treated sample) - ΔCt (control sample). All data were already normalized to untreated DC2.4 cells. Evaluating fat10 gene expression in C57BL/6 mice in figure 4 would help the reader believe that fat10 expression is still up during this window of time. Reply: We included the FAT10 expression data of CD4+ cells from C57BL/6 mice stimulated for 1 day with IFNγ/TNF in the revised manuscript (supplementary Fig. S2). Furthermore, we have repeated the Western blot experiments for Figure 4 to increase sample size. Did the authors do a time course experiment to show why they chose 24 hours of stimulation? Reply: Initially, when setting up the experiments, we tried different time points and compared gene expression levels with STAT activation. If the incubation time was too short, FAT10 gene expression was difficult to detect. If the incubation time was too long, the culture medium turned yellow, indicating that culture conditions were suboptimal. Finally, we chose 24 hours, a time point FAT10 expression was readily detected and STAT activation with IL-12 could be observed. It is also not scientifically explained why the authors would shift away from the TNF/IFNg model of expression seen in figure 1A. Reply: In Figure 3, we only used IFNγ stimulation without TNF stimulation for the following reason: To investigate whether FAT10 plays a role in IL-12 secretion in BMDCs, we need FAT10 expression. Therefore, cells were stimulated with IFNγ for 24 h. After the induction of FAT10, IL-12 is induced with LPS. If we do the initial FAT10 induction in the presence of TNF, TNF will mature BMDCs and therefore will induce the expression of IL-12. However, to investigate a contribution of FAT10 inIL-12 induction, we first need a FAT10 expression. As seen in Fig. 2A, FAT10 expression in BMDCs is also strongly induced in the absence of TNF. We explained this in the revised manuscript Yes, conclusions are presented in an appropriate fashion but with limitations. The data show makes assumptions about expression which would support the conclusion that FAT10 does not affect IL-12 expression or signaling. However, variability in some experiments and small sample sizes in signaling studies limit the strength of the conclusions. Including more controls and sometimes replicates would improve the results presented in this study to ensure that the study is properly powered and controlled. Reply: We increased the sample size in Figure 4 to ensure the experiment is properly powered. In our opinion, the other experiments with n≥5 are properly powered. Major Comments 1. Clarity in Experimental Design In Th1 differentiation assays, I do not think they included control. it would be beneficial to include a control condition without IL-12 to demonstrate the necessity of IL-12 in this system. Reply: It is well established that IL-12 is required for Th1 differentiation (f.i. Trinchieri G. Annu Rev Immunol. 1995;13:251-76, Trinchieri G. Blood. 1994;84(12):4008-27, Neurath et al., J Exp Med. 1999;182(5):1281-90, Nakamura et al., J Immunol. 1997;158(3):1085-94, etc.). Therefore, we did not include this control. Nevertheless, we discuss this as a limitation of our study and mainly conclude that FAT10 does not influence Th1 differentiation. 2. STAT Signaling Pathway Investigation (line 208) The authors analyze STAT1 and STAT4 phosphorylation, but IL-12 is also known to activate STAT3 to a certain extent. They briefly stated this without clearly justifying why they did not look at STAT3. Reply: See below. 3. IL-12 Expression and Secretion (line 244) Figure 2C shows that IL-12 secretion is not altered in FAT10 knockout cells. Since IL-12 has two subunits (p35 and p40), the authors did not measure p35 expression separately, and did not create a limitation section to acknowledged this as a limitation. Reply: See below. 4. Potential Redundancy in Text The authors repeatedly emphasize that FAT10 does not alter IL-12 expression across different sections (Results, Discussion). Reply: See below. Conclusion The manuscript is well-conceived and presents valuable findings in immunology and cytokine signaling. With minor revisions to improve clarity, correct typographical errors, and address the misplaced reference, the manuscript will be well-suited for publication. Reply: We have updated the references in our manuscript.Key Recommendations 1. Address the STAT3 signaling question. Reply: The study focuses on the relationship between FAT10 and IL-12 in Th1 cells. The signaling pathway related to Th1 cells are mainly the STAT4 and STAT1 signaling pathways (Trinchiere, Nat Rev Immunol. 2003 Feb;3(2):133-46). STAT3 plays a minor role. Therefore, we only investigated STAT1 and STAT4 signaling. We indicate this in the revised manuscript. 2. Consider IL-12 p35 measurement. Reply: We performed real-time RT-PCR to detect the expression levels of IL-12p40 and IL-12p35 in DC2.4 cells. However, the cycle threshold (Ct) value for IL-12p35 could not be reliably measured in our hands. Therefore, we only measured IL- 12p40. We discuss this as a limitation in the revised manuscript. 3. Remove redundant statements in Discussion. Reply: We tried to remove redundant statements in the discussion of the revised manuscript. 4. not should be no in line 214. Reply: We changed this. Reviewer #2: While the introduction is technically sound, I feel a re-write may be in order to emphasize the importance of this research and how it's applications may benefit immunological research. That issue aside, there were several factors that warranted my recommendation for major revision in Results section of this manuscript. On line 169 under the "FAT10 does not influence IL-12 expression in DC2.4 cells" segment, there didn't appear to be any mention of validating successful lentiviral transfection. Reply: When the DC2.4 cells were successfully infected with lentivirus, they overexpressed FAT10, and were named DC2.4-mFAT10. In Figure 1C one can see that DC2.4-mFAT10 cells, in contrast to not-infected DC2.4 cells (Fig. 1A, C), strongly express FAT10.This shows that DC2.4 cells were successfully infected with lentiviruses. In the next segment "FAT10 has no significant effect on IL-12 expression in bone marrow-derived dendritic cells", BMDCs were stimulated with IFN-γ and LPS to induce IL-12 expression. However, in the previous segment a combination IFNγ/TNF was used to stimulate DC2.4s. The rationale behind using only IFN-γ as opposed to the combination IFNγ/TNF wasn't immediately clear upon review. Reply: In Figure 3, we only used IFNγ stimulation without TNF stimulation for the following reason: To investigate whether FAT10 plays a role in IL-12 secretion in BMDCs, we need FAT10 expression. Therefore, cells were stimulated with IFNγ for 24 h. After the induction of FAT10, IL-12 is induced with LPS. If we do the initial FAT10 induction in the presence of TNF, TNF will mature BMDCs and therefore will induce the expression of IL-12. However, to investigate a contribution of FAT10 in IL-12 induction, we first need a FAT10 expression. As seen in Fig. 2A, FAT10expression in BMDCs is also strongly induced in the absence of TNF. We explained this in the revised manuscript. Lastly, in "FAT10 does not affect Th1 cell differentiation", the rationale as to why IFN-γ was selected as the sole cytokine of focus was unclear. TNF is also considered an important signature cytokine for Th1 differentiation, why was that not selected in addition to IFN-γ to validate results? Reply: IFN-γ is “the” signature cytokine to describe Th1 cells. For sure, TNF can additionally be used to describe Th1 cells. However, expression of TNF correlates with the expression of IFNγ. In our opinion, no substantial gain of information can be obtained by including TNF in the analysis. Therefore, in our opinion it is not justified to redo the experiments to include TNF. Overall, while I do partly agree with the overall conclusions of this paper that FAT10 does not appear to affect IL-12 signaling, I feel more validatory work is necessary to confirm many of these results. Reply: In our opinion, the experiments are very well controlled and solid. Furthermore, we addressed the question whether FAT10 is involved in Il-12 signaling from many different aspects. Finally, all of the different setups provided the same conclusive result. Namely, that FAT10 does not play a role in Il-12 signaling. Therefore, we strongly feel that our results are highly valid and conclusive https://doi.org/10.1371/journal.pone.0323005.r003
1 Apr 2025 Decision Letter - Mariola Ferraro, Editor The ubiquitin-like modifier FAT10 does not affect IL-12 expression and signaling PONE-D-25-01617R1 Dear Dr. Basler, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Mariola J Ferraro, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0323005.r004
Formally Accepted
Acceptance Letter - Mariola Ferraro, Editor PONE-D-25-01617R1 PLOS ONE Dear Dr. Basler, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Mariola J Ferraro Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0323005.r005

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