The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation

Paris Clarice Papagianis; Siavash Ahmadi-Noorbakhsh; Rebecca Lim; Euan Wallace; Graeme Polglase; J. Jane Pillow; Timothy J. Moss

doi:10.1371/journal.pone.0253456

Peer Review History

Original SubmissionFebruary 9, 2021
24 Mar 2021 Decision Letter - Harald Ehrhardt, Editor PONE-D-21-04396 The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation PLOS ONE Dear Dr. Papagianis, 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. I kindly ask you to address all specific points raised by both reviewers. Their helpful comments can help you to specify some aspects of data presentation within the manuscript. Please submit your revised manuscript by May 08 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. 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PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: No 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: Comments to the Author: The authors hypothesized that human amnion epithelial cells (hAECs) would reduce lung injury in preterm lambs-exposed to antenatal inflammation and investigated the effects of a single dose of intravenous hAECs on lung development and respiratory requirement. The authors found that hAECs administration at birth did not reduce respiratory requirements or lung development in preterm lambs exposed to antenatal inflammation. This study has many data and the results do not confirm the hypothesis and the connection between in vitro and animal study was not evident. General concerns: 1. This study presents many data and most data did not show clear evidence of positive effects. 2. The conclusions in Abstract was not supported by the results: Postnatal administration of a single dose of hAECs activates the pulmonary immune system without changing ventilator requirements in preterm lambs born after intrauterine inflammation. However, activation of pulmonary immune system was not described in the manuscript. 3. Methods: Please describe how the cell numbers of hAECs (30 � 106) was decided in this study. 4. Please describe and discuss the rationale to measure hepatic gene expression in lung injury model. 5. Methods: The animal numbers were not consistent. (LPS/Sal, n=10; or Sal/Sal, n=9) in Abstract. Preterm lamb studies on page 7, Ultrasound-guided intra-amniotic (IA) injection of lipopolysaccharide (LPS;4 mg; 2 mg/mL; Escherichia coli 055:B5; Sigma-Aldrich, NSW, Australia; n=10) or saline (n=10) was performed at 126 days’ GA. 6. Methods: The methods to collect the Physiology variables in Table 2 were not described. 7. Results: Please insert (Table 1) in the third paragraph on page 15. 8. Figure Legend 1: “^Signifies P<0.05 between Sal/Sal and LPS/hAECs” was not shown in the Figure 1. Please describe the meaning of . Reviewer #2: Thank you for submitting the manuscript for review. The study highlights the role of inflammatory axis in the genesis of CLD. By and large the manuscript reads well. I would like to add couple of comments for minor revision 1) Explicitly state the three groups based on the antenatal- post natal interventions - e.g. group A (LPS/S); Group B (S/S) to improve clarity 2) The abstract, discussion, conclusions- explicitly state the effect of hAEC on inflammatory cytokines- while it states hAEC influence pulmonary immunology -- for better clarity state the effect of hAEC on pro and anti-inflammatory cytokine response. This aspect is also discussed appropriately in the limitations. ******** 6. 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Revision 1
12 May 2021 Author Response Dear Editor, Thank you for your consideration of our manuscript “The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation”. We believe we have adjusted this manuscript to meet the journal requirements and comments from the editor and reviewers 1 and 2. Please note that our original blot image is attached as a separate PDF file “S4_raw_image” and as part of supplementary figures (S4 Fig.). Please find our responses to individual reviewers below. We have attached a marked-up version of the manuscript with changes in red text. We have addressed concerns of funding bodies raised by the editor in text on p. 33, line starting 702. Additionally, please note that we inadvertently omitted one author, Dr. Graeme Polglase. We recognise the addition of this author and this author has reviewed the manuscript. Polglase’s contributions to the manuscript are outlined in red on p.ii, line 27-8. All authors support the alterations and submission of this manuscript. We greatly appreciate your consideration of this manuscript. Kind Regards, Paris Papagianis and co-authors. Reviewer #1: The authors hypothesized that human amnion epithelial cells (hAECs) would reduce lung injury in preterm lambs-exposed to antenatal inflammation and investigated the effects of a single dose of intravenous hAECs on lung development and respiratory requirement. The authors found that hAECs administration at birth did not reduce respiratory requirements or lung development in preterm lambs exposed to antenatal inflammation. This study has many data and the results do not confirm the hypothesis and the connection between in vitro and animal study was not evident. General concerns: 1. This study presents many data and most data did not show clear evidence of positive effects. We believe that the data presented provide important insight into the use of translational studies investigating cellular therapies for lung disease. We use a clinically relevant preterm intensive care model which recapitulates human practice to demonstrate that while the impact of hAECs on ventilator requirements is minimal the positive effects on lung parenchyma are apparent. Additionally, we analyse the impact of contemporary NICU care in the context of chorioamnionitis (a common precursor to preterm birth and NICU). The outcomes of these studies are informing the planning and design of clinical trials using hAECs in human infants. 2. The conclusions in Abstract was not supported by the results: Postnatal administration of a single dose of hAECs activates the pulmonary immune system without changing ventilator requirements in preterm lambs born after intrauterine inflammation. However, activation of pulmonary immune system was not described in the manuscript. We describe markers of pulmonary inflammation in Fig. 5. We have removed the word “activation” from the abstract and replaced with “stimulates a pulmonary immune response”. 3. Methods: Please describe how the cell numbers of hAECs (30 � 106) was decided in this study. We chose a hAEC dose of roughly 10 million cells per kilo, based on the average weight of preterm lambs at 128 dGA (~3kg). This dosing approach for hAECs was in line with a planned Phase 2 clinical trial for hAECs in preterm infants with BPD when our study was conducted. 4. Please describe and discuss the rationale to measure hepatic gene expression in lung injury model. Although the focus of this study was on ventilator requirements and lung development/maturation, we opportunistically looked at markers for systemic inflammation. This analysis of hepatic gene expression as an indicator of systemic inflammation informed our conclusion that hAECs were administered safely in our preterm lambs. 5. Methods: The animal numbers were not consistent. (LPS/Sal, n=10; or Sal/Sal, n=9) in Abstract. Preterm lamb studies on page 7, Ultrasound-guided intra-amniotic (IA) injection of lipopolysaccharide (LPS;4 mg; 2 mg/mL; Escherichia coli 055:B5; Sigma-Aldrich, NSW, Australia; n=10) or saline (n=10) was performed at 126 days’ GA. Thank you. Unfortunately, this is typological error in the abstract. Saline/Saline = 9. This group size number has been amended for the Saline/Saline group throughout the manuscript. 6. Methods: The methods to collect the Physiology variables in Table 2 were not described. All the methodology used to collect physiological variables are described in detail in the supplementary methods due to word restrictions in the main paper. A detailed methodology for data collection of physiological variables is cited in the general postnatal management section of our methodology in the main manuscript (line 152 onwards). 7. Results: Please insert (Table 1) in the third paragraph on page 15. Please find Table 1 at line 242 8. Figure Legend 1: “^Signifies P<0.05 between Sal/Sal and LPS/hAECs” was not shown in the Figure 1. Please describe the meaning of *. Figure legends are now adjusted to define all symbols in each figure. ^Signifies P<0.05 between Sal/Sal and LPS/Sal. Signifies P<0.05 between Sal/Sal and LPS/hAECs &Time signifies P<0.05 change over 1-6 days, not between treatment groups.” There is no use of ** in any of the figures, and these symbols are consistent for all subsequent figures. Additionally, we have simplified the description of the colour coding in figure 2 and enhanced the labelling in Figures 1 and 5 to improve readability. Reviewer #2: The study highlights the role of inflammatory axis in the genesis of CLD. By and large the manuscript reads well. I would like to add couple of comments for minor revision 1) Explicitly state the three groups based on the antenatal- post natal interventions - e.g. group A (LPS/S); Group B (S/S) to improve clarity The use of A, B and C for treatment groups may be more confusing as the reader has to remember what each represents. We abbreviate Saline to Sal and human amnion epithelial cells to hAEC, as is standard within the literature. 2) The abstract, discussion, conclusions- explicitly state the effect of hAEC on inflammatory cytokines- while it states hAEC influence pulmonary immunology -- for better clarity state the effect of hAEC on pro and anti-inflammatory cytokine response. This aspect is also discussed appropriately in the limitations We have adjusted the abstract as per reviewer #1 comment which we believe also meets the requirements of this comment. The discussion and conclusion have also been changed accordingly. The discussion: • Line 398 “level of pro- or anti- inflammatory cytokine gene expression in the lungs” • Line 463: “increases the expression of the anti-inflammatory cytokine, IL-10” • Line 471-3: “However, we did not see reduced pro-inflammatory cytokine expression in preterm lambs receiving hAEC after antenatal LPS exposure in this study, despite increased IL-10 compared to LPS/Sal lambs.” The conclusion has been altered: “level of pro- or anti- inflammatory cytokine gene expression in the lungs” Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0253456.r002
7 Jun 2021 Decision Letter - Harald Ehrhardt, Editor The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation PONE-D-21-04396R1 Dear Dr. Papagianis, 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, Harald Ehrhardt 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: The authors have addressed the concerns. I have no further concerns. The authors have addressed the concerns. ****** 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:** Chung-Ming Chen https://doi.org/10.1371/journal.pone.0253456.r003
Formally Accepted
16 Jun 2021 Acceptance Letter - Harald Ehrhardt, Editor PONE-D-21-04396R1 The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation Dear Dr. Papagianis: 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 Prof. Harald Ehrhardt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0253456.r004

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