High salt diet impairs dermal tissue remodeling in a mouse model of IMQ induced dermatitis

Csenge Pajtók; Apor Veres-Székely; Róbert Agócs; Beáta Szebeni; Péter Dobosy; István Németh; Zoltán Veréb; Lajos Kemény; Attila J. Szabó; Ádám Vannay; Tivadar Tulassay; Domonkos Pap

doi:10.1371/journal.pone.0258502

Peer Review History

Original SubmissionApril 19, 2021
23 Jul 2021 Decision Letter - Minghua Wu, Editor PONE-D-21-11775 High salt intake impairs dermal tissue remodeling PLOS ONE Dear Dr. Pap, 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. 1. High salt did not affect TGF-bata expression even in the IMQ treatment but down stream collagen expression was significantly decreased. Authors may study TGFbeta down stream molecules like smad2/3 expression which transcription factors can directly binding to the promotor region of the target genes like col1a1. 2. Figure legends: presented qPCR result description is not clear. "Values were expressed as mean + SD" but graph presented as a fold changes. Please edit text clearly described. 3. 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Minghua Wu, M.D., Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for including your ethics statement: "All experiments on mice were approved by the Institutional Committee on Animal Welfare (PEI/001/1731-8/2015). Collection of biospecimens for the isolation and culture on human dermal fibroblasts complied with the directive of the Helsinki Declaration and were approved by the local Ethics Committee of the University of Szeged, Hungary (CSR/039/00346-5/2015). Isolation and culture on human PBMCs were approved by Semmelweis University Regional and Institutional Committee of Science and Research Ethics (31224‑5/2017/EKU)." a) Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. 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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: Pajtók et al. examined the effects of high salt intake on skin remodeling in IMQ-treated mice. The manuscript is well written and includes novel findings, however, the authors should address some points as described below. 1. In this reviewer’s experience, when we used an 8% NaCl diet in mice, they don’t eat much maybe because of the taste, therefore we usually use a 4% NaCl diet as a high salt diet. Did the authors measure food intake and body weight in this study? 8% NaCl diet may reduce food intake, which can affect the skin remodeling independently of salt. 2. Based on the authors’ data, the main effect of high salt on cytokines in the IMQ model is IL10 and IL13, not IL17. Thus, figure 6 is confusing. Although the previous studies showed that the clear effect of salt on IL17, it is not clear in the IMQ model. This reviewer suggests that the authors should revise this point in the manuscript and figure 6. ****** 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.] 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.0258502.r001
Revision 1
23 Sep 2021 Author Response Responses to Editor: 1. High salt did not affect TGF-beta expression even in the IMQ treatment but down stream collagen expression was significantly decreased. Authors may study TGFbeta down stream molecules like smad2/3 expression which transcription factors can directly binding to the promotor region of the target genes like col1a1. Thank you for your suggestion. We agree that it is important to better understand the high salt intake induced molecular mechanisms which are responsible for the impaired dermal tissue remodeling. The TGF-ß induced and various SMAD transcription factors mediated signaling pathway is one of the main driver of tissue remodeling process in almost all organ. During its activation the phosphorylated SMAD2/3 molecules translocate to the cell nucleus of fibroblasts and promote gene expression changes that leads to cell differentiation into α-SMA positive myofibroblasts and to production of ECM elements including various collagens and fibronectin [1]. Interestingly, our results showed that while the expression of TGF-ß was increased in the skin of IMQ treated mice on high salt diet (HSD IMQ group) the expression of tissue remodeling alpha smooth muscle actin (ACTA2), fibronectin (FN) and collagen 1a1 (Col1a1) was decreased. In accordance with your suggestion we investigated the potential role of SMAD pathway activation behind this controversy. To this purpose we determined the amount of phosphorylated (p)SMAD2/3 in the skin samples using Western blot analysis. Our results demonstrated that the amount of pSMAD2/3 was significantly decreased in HSD IMQ group compared to NSD IMQ group (Fig 3/f). Numerous previous studies demonstrated that reduced SMAD2/3 expression or pharmacological inhibition of SMAD signaling pathway decreases the expression of these ECM remodeling markers and tissue fibrosis [2]. Therefore, our results showing reduced pSMAD2/3 level in the skin of HSD IMQ mice may explain why the expression of ECM remodeling genes was also reduced in these treatment group compared to that of NSD IMQ controls. Although TGF-ß has been regarded as the main drivers of SMAD signaling, recent studies of ECM remodeling has revealed other pathways that mediates SMAD2/3 activation [3, 4]. Indeed, IL-13 was demonstrated to induce SMAD2/3 phosphorylation in hepatic stellate cells and enhance pSMAD2/3 activation in primary human fibroblasts as well [5, 6]. In addition, IL-13 was demonstrated to facilitate tissue fibrosis in a completely TGF-ß independent manner [7]. Since our results showed decreased IL-13 expression in the skin of HSD IMQ animals compared to NSD IMQ animals, we hypothesize that the reduced IL-13 expression in HSD IMQ animals may contribute to reduced SMAD2/3 activity. Our new results are incorporated into the new version of the manuscript (page 17-18, line 384-408). Changes are highlighted in blue. References: 1. Wynn TA. Cellular and molecular mechanisms of fibrosis. The Journal of Pathology. 2008;214(2):199-210. doi: https://doi.org/10.1002/path.2277. 2. Walton KL, Johnson KE, Harrison CA. Targeting TGF-β Mediated SMAD Signaling for the Prevention of Fibrosis. Frontiers in Pharmacology. 2017;8:461. 3. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature. 2003;425(6958):577-84. doi: 10.1038/nature02006. 4. Nguyen JK, Austin E, Huang A, Mamalis A, Jagdeo J. The IL-4/IL-13 axis in skin fibrosis and scarring: mechanistic concepts and therapeutic targets. Archives of Dermatological Research. 2020;312(2):81-92. doi: 10.1007/s00403-019-01972-3. 5. Liu Y, Meyer C, Müller A, Herweck F, Li Q, Müllenbach R, et al. IL-13 Induces Connective Tissue Growth Factor in Rat Hepatic Stellate Cells via TGF-β–Independent Smad Signaling. The Journal of Immunology. 2011;187(5):2814. doi: 10.4049/jimmunol.1003260. 6. Zhou X, Hu H, Huynh M-LN, Kotaru C, Balzar S, Trudeau JB, et al. Mechanisms of tissue inhibitor of metalloproteinase 1 augmentation by IL-13 on TGF-β1–stimulated primary human fibroblasts. Journal of Allergy and Clinical Immunology. 2007;119(6):1388-97. doi: https://doi.org/10.1016/j.jaci.2007.02.011. 7. Kaviratne M, Hesse M, Leusink M, Cheever AW, Davies SJ, McKerrow JH, et al. IL-13 Activates a Mechanism of Tissue Fibrosis That Is Completely TGF-β Independent. The Journal of Immunology. 2004;173(6):4020. doi: 10.4049/jimmunol.173.6.4020. 2. Figure legends: presented qPCR result description is not clear. "Values were expressed as mean + SD" but graph presented as a fold changes. Please edit text clearly described. Thank you for your comment. In accordance with your suggestion, the texts of the figure legends were revised to be more clear. The mRNA expressions were determined by comparison with the expression of GAPDH or RPLP0 as a housekeeping gene. Data were normalized and presented as the ratio of the mean values of their control groups. Accordingly, we clarified the sections describing the qPCR results in the figure legends. Changes are highlighted in blue. 3. Statistical analysis: please clearly describe in the figure legend which experiment analyzed by t-test or U-test. What is the consideration for using each test in each experiment? In statistical tests, we examined the effect of increased salt intake on gene expression changes under normal and pathological conditions in in vivo and in vitro model systems. In addition, we also examined the changes in gene expression induced by the models itself including in vivo IMQ treatment and in vitro TGF-ß treatment. Therefore, we sought answers to the following three questions: - Does salt loading cause significant changes in inflammatory or profibrotic gene expression in the skin in vivo (NSD↔ HSD) and in dermal fibroblasts and immune cells in vitro (Control↔ NaCl)? - Does salt loading cause a significant change in inflammatory or profibrotic gene expression in IMQ-induced dermatitis in vivo (NSD IMQ↔ HSD IMQ) and in TGFβ-treated dermal fibroblasts in vitro (TGFβ↔ NaCl+TGFβ)? - Do our in vivo (NSD↔ NSD IMQ) and in vitro (Control↔ TGFβ) models cause any inflammatory or profibrotic gene expression changes? In each case, always two groups were compared. First, we determined whether the groups show a normal distribution by Kolmogorov-Smirnov test. Then, depending on whether the group had a normal or non-normal distribution, an unpaired t-test or Mann-Whitney U test was used to determine significance. Multiple comparisons of the raw data derived from migration assays were performed by using multiple t-test and ordinary two-way ANOVA with Dunnett correction. In all cases, p≤0.05 was considered as statistically significant. The figure legends were supplemented with a description of the exact statistical test used in that experiments. In addition, graphs were remade for better interpretation. Dots on the graphs represent individual values in order to show the distribution of the data. Journal Requirements: 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. Both human dermal fibroblasts and human peripheral blood mononuclear cells were obtained from healthy individuals after signing an informed consent. We supplemented both the Methods and the ethics statement with this information. Changes are highlighted in blue. 3. Please consider modifying your title to ensure that it is specific, descriptive, and concise (for example by specifying the animal model used). Thank you for your comment. According to your suggestion, we modified the title of the manuscript to make it more specific. The new title is „High salt intake impairs dermal tissue remodeling in a mouse model of IMQ induced dermatitis”. Changes are highlighted in blue. Responses to Reviewer: 1. In this reviewer’s experience, when we used an 8% NaCl diet in mice, they don’t eat much maybe because of the taste, therefore we usually use a 4% NaCl diet as a highsalt diet. Did the authors measure food intake and body weight in this study? 8% NaCl diet may reduce food intake, which can affect the skin remodeling independently of salt. Thank you for your question. The high salt content of the chow can indeed affect the appetite of the animals and thus their body weight. In our preliminary experiments we investigated whether the high salt diet (HSD) containing 8% NaCl has an effect on the body weight of mice. Our results showed that there was no significant difference in body weight between animals on normal salt diet (NSD) and those on HSD even after two weeks, therefore we terminated the experiment. The graphical representation of this experiment was included in Supporting Information (S2 Fig). Although the duration of our preliminary experiment was shorter than the study design we used in the manuscript (14 days vs. 33 days), our data are in line with recent studies showing no difference in the body weight of mice and rats on HSD containing 8% NaCl even after 10 weeks compared to control animals on NSD [1-4]. These results are incorporated into the new version of the manuscript (page 10, line 220-222). Changes are highlighted in blue. It is also important to note that our results showed that HSD alone had no effect on skin ECM remodeling. Indeed, there was no difference in the expression of ECM remodeling markers, including alpha smooth muscle actin (ACTA2), fibronectin (FN) and collagen1a1 (Col1a1) between mice kept on NSD and HSD alone. However, under inflammatory conditions induced by the imiquimod (IMQ) treatment the high salt intake resulted in decreased ECM production in HSD IMQ group compared to NSD IMQ group (Fig. 3/a-c). Therefore, our results suggest that high salt consumption exerts inhibitory effect on skin ECM remodeling under inflammatory conditions. References: 1. Faraco G, Brea D, Garcia-Bonilla L, Wang G, Racchumi G, Chang H, et al. Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response. Nature Neuroscience. 2018;21(2):240-9. doi: 10.1038/s41593-017-0059-z. 2. Ferguson JF, Aden LA, Barbaro NR, Van Beusecum JP, Xiao L, Simmons AJ, et al. High dietary salt–induced DC activation underlies microbial dysbiosis-associated hypertension. JCI Insight. 2019;4(13). doi: 10.1172/jci.insight.126241. 3. Guo C-P, Wei Z, Huang F, Qin M, Li X, Wang Y-M, et al. High salt induced hypertension leads to cognitive defect. Oncotarget. 2017;8(56):95780-90. doi: 10.18632/oncotarget.21326. PubMed PMID: 29221166. 4. Ogihara T, Asano T, Ando K, Chiba Y, Sekine N, Sakoda H, et al. Insulin Resistance With Enhanced Insulin Signaling in High-Salt Diet–Fed Rats. Diabetes. 2001;50(3):573. doi: 10.2337/diabetes.50.3.573. 2. Based on the authors’ data, the main effect of high salt on cytokines in the IMQ model is IL10 and IL13, not IL17. Thus, figure 6 is confusing. Although the previous studies showed that the clear effect of salt on IL17, it is not clear in the IMQ model. This reviewer suggests that the authors should revise this point in the manuscript and figure 6. Thank you for your remark. Although the inducing effect of increased salt intake on dermal proinflammatory IL-17 cytokine expression is one of our novel findings, the high salt diet (HSD) indeed did not affect its expression in mice receiving IMQ treatment. Therefore, in accordance with your suggestion we removed the IL-17 related parts of Figure 6. In accordance with this, we also removed the parts of Figure 6 showing the inhibitory effect of high salt environment on motility of dermal fibroblast since these in vitro results were not investigated in our in vivo model. We also revised the figure legend of Figure 6. Please find the revised caption of Figure 6 on page 20, line 457-461. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0258502.r002
29 Sep 2021 Decision Letter - Minghua Wu, Editor High salt diet impairs dermal tissue remodeling in a mouse model of IMQ induced dermatitis PONE-D-21-11775R1 Dear Dr. Pap, 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, Minghua Wu, M.D., Ph.D. 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ****** 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) ****** 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 https://doi.org/10.1371/journal.pone.0258502.r003
Formally Accepted
15 Oct 2021 Acceptance Letter - Minghua Wu, Editor PONE-D-21-11775R1 High salt diet impairs dermal tissue remodeling in a mouse model of IMQ induced dermatitis Dear Dr. Pap: 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. Minghua Wu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258502.r004

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