The effect of caloric restriction on the increase in senescence-associated T cells and metabolic disorders in aged mice

Xiaoxiang Yan; Natsumi Imano; Kayoko Tamaki; Motoaki Sano; Ken Shinmura

doi:10.1371/journal.pone.0252547

Peer Review History

Original SubmissionJanuary 23, 2021
18 Feb 2021 Decision Letter - Ichiro Manabe, Editor PONE-D-21-02437 The effect of caloric restriction on the increase in senescence-associated T cells and metabolic disorders in aged mice PLOS ONE Dear Dr. Shinmura, 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 01 2021 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 remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "All authors declare no financial disclosure. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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). 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 ****** 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 ****** 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 general, this research is a good piece of work to expand the knowledge of CR science. The authors showed that SA-T cells are responsible for the development of age-associated metabolic disorders and long-term CR could decrease the number of SA-T cells in mice spleen and adipose tissue. Furthermore, the immunological depletion of PD-1+ T cells improved insulin resistance in aged mice with the reduction of adipose tissue inflammation. Thus, the authors conclude that SA-T cells are attractive therapeutic target for the treatment of age-related chronic inflammation and insulin resistance. Major point To clarify the mechanism of increased insulin sensitivity in aPD-1 treated eWAT, the authors should measure the intracellular insulin signaling molecules and their receptors activation status in the liver and adipose tissue. Furthermore, the measurement of another senescent tissue protein marker would help to understand the pathophysiological roles of PD-1+ cell depletion. At lease, the authors should describe and discuss more these points. (Fig. 6) Also, the authors should discuss the reason why the PD-1＋ T cells increase with age. Minor points p4, line 96 The authors should clarify why they did not measure PD-1+ MP CD4+ and CD8+ cells in the other tissues such as thymus. Also, the authors should clarify that the PD-1+ MP T cells have low level of CD62L or not. (Fig. 5 and 7) p5, line 161, p9 line 283, p10 line 329, Fig. 5, and Fig. 7. Typo? Crown-like structures should be abbreviated CLS. Reviewer #2: The authors evaluated the accumulation of SA-T cells in the visceral adipose tissue and spleen under the caloric restriction (CR) in aged mice. Long-term CR decreased the number of PD-1-positive CD4 or CD8 T cells and decreased M1 macrophage. In addition, the immunological depletion of PD-1 T cells reduced adipose inflammation and improved insulin resistance in aged mice. This study includes an interesting finding and the experiment is well designed. Our concerns are as follows. 1. In Figure 2, the percentage of p21 in eVAT is much higher than that in spleen. Could you evaluate the SA-T cells in eVAT or spleenusing other senescence marker, such as DNA damage marker? 2. In Figure 3 and 5, could you also show the total number of CD4 and CD8 T cells in aged mice or under CR condition? The reviewer also would like to know the percentage of Naïve T cells under CR condition. 3. In Figure 6 and 7, could you show the comparison of each parameter before and after PD-1 depletion antibody? These additional experiments will teach us the potential (prevention or improvement) of senolytic therapy. ****** 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? 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Revision 1
7 May 2021 Author Response Responses to reviewer #1 We would like to thank the reviewer for his/her constructive criticism and helpful suggestions, which have helped us to revise and improve our manuscript. We would also like to thank the reviewer for his/her valuable remarks. In general, this research is a good piece of work to expand the knowledge of CR science. The authors showed that SA-T cells are responsible for the development of age-associated metabolic disorders and long-term CR could decrease the number of SA-T cells in mice spleen and adipose tissue. Furthermore, the immunological depletion of PD-1+ T cells improved insulin resistance in aged mice with the reduction of adipose tissue inflammation. Thus, the authors conclude that SA-T cells are attractive therapeutic target for the treatment of age-related chronic inflammation and insulin resistance.. #1. To clarify the mechanism of increased insulin sensitivity in aPD-1 treated eWAT, the authors should measure the intracellular insulin signaling molecules and their receptors activation status in the liver and adipose tissue. We would like to thank you for your constructive criticism and helpful suggestions, which have helped us revise and improve our manuscript. To examine the effect of anti-PD-1 antibody treatment on intracellular insulin signaling, we evaluated phosphorylation levels of Akt/PKB at the serine 473 residue and those of insulin receptor substrate (IRS) 1 at the serine 307 residue. Phosphorylation levels of IRS1 at baseline were higher in livers obtained from aged mice treated with control IgG than in those from aged mice treated with anti-PD-1 antibody (Fig. 6G). In contrast, phosphorylation levels of Akt at baseline were lower in both the liver and eVAT obtained from aged mice treated with control IgG, compared with those from aged mice treated with anti-PD-1 antibody (Fig. 6G and 6H). In addition, insulin-induced phosphorylation of Akt was significantly improved in eVAT obtained from aged mice treated with anti-PD-1 antibody (Fig. 6H). These results strongly suggest that anti-PD-1 antibody treatment improved insulin signaling, especially in eVAT, by reducing proinflammatory cytokines that could induce insulin resistance. Accordingly, we added each paragraph in the Methods and Results section, and Fig. 6 legend of the revised manuscript (P6, L184-L193; P10, L322-L331; and P10, L344-L348, respectively). #2. Furthermore, the measurement of another senescent tissue protein marker would help to understand the pathophysiological roles of PD-1+ cell depletion. At lease, the authors should describe and discuss more these points. (Fig. 6) In addition to p21, we evaluated the expression levels of p16 and H2AX between PD-1+ and PD-1- CD3+ T cells obtained from the spleen and eVAT. Although there was no difference in the expression levels of p16, we found that the expression levels of H2AX in PD-1+ CD3+ T cells of eVAT were significantly higher than those in PD-1- CD3+ T cells obtained from eVAT, particularly in aged mice (Fig. 2D). Thus, PD-1+ T cells increase with aging and exhibit higher expression levels of p21 and H2AX. These results are consistent with the findings observed in PD-1+ CD4+ T cells obtained from eVAT of high-fat diet-induced obese mice (Shirakawa et al. J Clin Invest 2016;126:4626-39.). Therefore, we speculate that the appearance of PD-1+ T cells in eVAT is closely associated with cellular senescence induced DNA damage. In addition to PD-1+ T cell depletion, the modification of p21/p53 signaling in T cells might have the potential to suppress T-cell immunosenescence. Any interventions for the purpose of reducing DNA damage, such as the suppression of oxidative stress in the circulating environment, might become a different approach to reduce T-cell immunosenescence. Therefore, we added the findings of H2AX analysis in the revised Fig. 2D (Fig. 2 legend, P8, L242-L246) and the results (P7, L228-L232), and this assessment in the Discussion (P12, L394-L407). #3. Also, the authors should discuss the reason why the PD-1＋ T cells increase with age. We discussed this issue as described above and inserted a paragraph in the Discussion section (P12, L394-L407). #4. p4, line 96 The authors should clarify why they did not measure PD-1+ MP CD4+ and CD8+ cells in the other tissues such as thymus. Thank you very much for your constructive advice. In the present study, we focused on the impact of T-cell immunesenescence on glucose intolerance associated with aging. Therefore, we planned to evaluate the increase in PD-1+ T cells in eVAT and the spleen in mice of different ages because we supposed that the T-cell subpopulation in the spleen and eVAT was closely related to chronic inflammation in aged mice. As the reviewer pointed out, T-cell immunesenescence must be observed and might play a role in the thymus and lymph nodes. We would like to evaluate PD-1+ T cells in these tissues in a future study. We briefly describe this as a limitation of the present study (P14, L477-L484). #5. Also, the authors should clarify that the PD-1+ MP T cells have low level of CD62L or not. (Fig. 5 and 7) The PD-1+ T cells exhibited lower levels of CD62L (Fig. 2A). Thus, PD-1+ T cells are classified as memory phenotypes, which are characterized by higher expression of CD44, and lower expression of CD62L. Unfortunately, the number of immune cells obtained from eVAT is relatively low in CR mice and aged mice treated with anti-PD-1 antibody and we divided these immune cells into three tubes for the analysis of macrophages, CD3+ T cells, and regulatory T cells (Treg) (in the present study, we did not show the results of Treg analysis). Therefore, we could not evaluate the expression levels of CD62L in PD-1+ CD44high CD3+ T cells by FACS in CR mice and aged mice treated with anti-PD-1 antibody. However, we believe that it is reasonable to expect that PD-1+ T cells obtained from CR mice and aged mice treated with anti-PD-1 antibody are the same as aged mice (Fig. 2A). #6. p5, line 161, p9 line 283, p10 line 329, Fig. 5, and Fig. 7. Typo? Crown-like structures should be abbreviated CLS. Thank you very much for identifying this issue. We have corrected all the typing errors. Thank you again for your excellent suggestions. Response to reviewer #2 We would like to thank the reviewer for his/her constructive criticism and helpful suggestions, which have helped us to revise and improve our manuscript, as well as for his/her valuable remarks. The authors evaluated the accumulation of SA-T cells in the visceral adipose tissue and spleen under the caloric restriction (CR) in aged mice. Long-term CR decreased the number of PD-1-positive CD4 or CD8 T cells and decreased M1 macrophage. In addition, the immunological depletion of PD-1 T cells reduced adipose inflammation and improved insulin resistance in aged mice. This study includes an interesting finding and the experiment is well designed. Our concerns are as follows. #1. In Figure 2, the percentage of p21 in eVAT is much higher than that in spleen. Could you evaluate the SA-T cells in eVAT or spleen using other senescence marker, such as DNA damage marker? In addition to p21, we evaluated the expression levels of p16 and H2AX between PD-1+ and PD-1- CD3+ T cells obtained from the spleen and eVAT. Although there was no difference in the expression levels of p16, we found that the expression levels of H2AX in PD-1+ CD3+ T cells of eVAT were significantly higher than those in PD-1- CD3+ T cells of eVAT, particularly in aged mice (Fig. 2D). Thus, PD-1+ T cells increase with aging and exhibit higher expression levels of p21 and H2AX. These results are consistent with the findings observed in PD-1+ CD4+ T cells obtained from eVAT of high-fat diet-induced obese mice (Shirakawa et al. J Clin Invest 2016;126:4626-39.). Therefore, we speculate that the appearance of PD-1+ T cells in eVAT is closely associated with cellular senescence induced DNA damage. In addition to PD-1+ T cell depletion, the modification of p21/p53 signaling in T cells might have the potential to suppress T-cell immunosenescence. Any interventions for the purpose of reducing DNA damage, such as the suppression of oxidative stress in the circulating environment, might become a different approach to reduce T-cell immunosenescence. Therefore, we added the findings of H2AX analysis in the revised Fig. 2D (Fig. 2 legend, P8, L242-L246) and the results (P7, L228-L232), and this assessment in the Discussion (P12, L394-L407). #2. In Figure 3 and 5, could you also show the total number of CD4 and CD8 T cells in aged mice or under CR condition? The reviewer also would like to know the percentage of Naïve T cells under CR condition. Thank you very much for your constructive advice. In accordance with the reviewer’s suggestion, we have represented the total number of PD-1+ CD4+ and PD-1+ CD8+ T cells (per g of eVAT) in Figs. 3, 5, and 7. Thus. we show both the total number of PD-1+ CD4+ and PD-1+ CD8+ T cells (per g of eVAT) and the percentage of PD-1+ CD4+, and PD-1+ CD8+ T cells (% of total CD4+ and CD8+ T cells) in Figs. 3, 5 and 7. We considered whether we should also determine the number of CD4+, and CD8+ T cells. The total number of CD4+ and CD8+ T cells is estimated by dividing the total number of PD-1+ CD4+, and PD-1+ CD8 T cells by the ratio of PD-1+ CD4+, and PD-1+ CD8+ T cells, respectively. The change in the number of CD4+ T cells and CD8+ T cells by CR seems to be interesting data, but there may be overlap with the presentation data. In addition, we discussed the changes in CD4+ and CD8+ T cells by CR based on the literature. Therefore, we decided to show both the total number and percentage of PD-1+ CD4+, and PD-1+ CD8+ T cells in Figs. 3, 5, and 7. Unfortunately, the number of immune cells obtained from the eVAT of CR mice was relatively low and the number of CR mice was also limited. We divided these immune cells into three tubes for the analysis of macrophages, CD3+ T cells, and regulatory T cells (Treg) (in the present study, we did not show the results of Treg analysis). Therefore, we could not evaluate the expression levels of CD62L in PD-1+ CD44high CD3+ T cells by FACS in CR mice. Therefore, we did not have data regarding the percentage of naïve T cells in the same series of experiments described in Fig. 5. Because CR mice cannot be obtained easily in Japan, we would like to ask the reviewer to understand that we will show the change in the percentage of naïve T cells by CR at the next opportunity. #3. In Figure 6 and 7, could you show the comparison of each parameter before and after PD-1 depletion antibody? These additional experiments will teach us the potential (prevention or improvement) of senolytic therapy. According to the referee’s suggestion, we compared the changes in each parameter before and after either control IgG or ant-PD-1 antibody treatment in aged mice. Finally, we did not find any changes in BW and food intake before and after treatment (Figs. 6A and 6B). In addition, anti-PD-1 antibody treatment significantly improved glucose intolerance and insulin resistance, although control IgG treatment did not affect the results of the oGTT and ITT (Figs. 6D and 6E). We also confirmed the effective removal of PD-1+ T cells, especially PD-1+ CD4+ T cells, from the eVAT and spleen (Figs. 7C-F). Therefore, we speculate that the effective depletion of senescence-related T cells was responsible for the increase in naïve T cells in eVAT and the spleen of aged mice (Figs. 7A and 7B). These results strongly suggest that senolytic therapy against senescence-related T cells can lead to the rejuvenation of T cells in aged mice. We have added some sentences in the results, Fig. 6 and 7 legends, and the Discussion in the revised manuscript (P9, L316-L318; P10, L339-L343; P10, L353-P11, L358; P11, L372-L375; P12, L414-L415; and P13, L462-L463, respectively). Thank you again for your excellent suggestions. Attachments Attachment Submitted filename: PLOS One response letter final version.docx https://doi.org/10.1371/journal.pone.0252547.r002
18 May 2021 Decision Letter - Ichiro Manabe, Editor The effect of caloric restriction on the increase in senescence-associated T cells and metabolic disorders in aged mice PONE-D-21-02437R1 Dear Dr. Shinmura, 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, Ichiro Manabe 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 #2: 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 #2: 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 #2: This manuscript is well revised, and all comments have been fully addressed.. I have no further comment. ****** 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: Yes: Takuya Chiba Reviewer #2: Yes:** Hironori Nakagami https://doi.org/10.1371/journal.pone.0252547.r003
Formally Accepted
10 Jun 2021 Acceptance Letter - Ichiro Manabe, Editor PONE-D-21-02437R1 The effect of caloric restriction on the increase in senescence-associated T cells and metabolic disorders in aged mice Dear Dr. Shinmura: 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. Ichiro Manabe Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252547.r004

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