PONE-D-20-17668

Association of Serum 25-Hydroxyvitamin D Levels with Severe Necroinflammatory Activity and Inflammatory Cytokine Production in Type I Autoimmune Hepatitis

PLOS ONE

Dear Dr. Abe,

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.

As you can see, both reviewers appreciated your work, both also highlighted several important issues, that need to be addressed.

Please submit your revised manuscript by Aug 14 2020 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Pavel Strnad

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. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed:

(1) https://aasldpubs.onlinelibrary.wiley.com/doi/full/10.1002/hep4.1326 (lines 273-296).

(2) https://www.mdpi.com/2072-6643/6/1/221/htm (lines 250-256)

(3) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808258/ (lines 261-271)

The text that needs to be addressed involves the Discussion section.

In your revision please ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

3. Thank you for stating in your Funding Statement:

"This study was supported in part by AbbVie Inc."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

4. Thank you for stating the following in the Competing Interests section:

"Kazumichi Abe and Hiromasa Ohira has received research funds from AbbVie Inc. All other authors declare no conflict of interest."

We note that you received funding from a commercial source: AbbVie Inc.

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. 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: Partly

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 their study entitled “Association of Serum 25-Hydroxyvitamin D Levels with Severe Necroinflammatory Activity and Inflammatory Cytokine Production” Abe et al. retrospectively analysed a single-centre cohort of AIH patients with regard to their 25-hydroxyvitamin D (25(OH)D) levels. The authors found higher levels of 25(OH)D in AIH patients with acute severe presentation, in line with a positive correlation between 25(OH)D levels and serum albumin and prothrombin time and a negative correlation with serum total bilirubin levels and histological grade of necroinflammatory activity. In addition, the author analysed several pro- and anti-inflammatory cytokines out of the sera of AIH patients and found higher levels of interferon-gamma and IL-33 levels in patients with low levels of 25(OH)D.

I have several major and minor concerns which need to be addressed before this study can be accepted for publication.

Major concerns:

1. My major concern lies in doubts that 25(OH)D plays a role in the pathogenesis of AIH as the authors claim it does. The low levels of 25(OH)D in patients with acute-severe presentation of AIH may be just a result of acute liver damage and of impaired liver function. The low levels of 25(OH)D could depend only on the severity of liver damage, but not on the kind liver disease. 25(OH)D is mainly bound to albumin and vitamin D binding protein in blood, and both transporters are produced in the liver. Due to acute liver injury (of any cause), production of albumin and vitamin D binding protein could be impaired and thereby lower levels of 25(OH)D were measured in peripheral blood of patients with acute presentation of AIH.

a. The authors must add a control groups to their study in order to support that lower levels of 25(OH)D are part of the pathogenesis of AIH. Can the authors perform measurements of 25(OH)D in patients with other causes of acute hepatitis (drug-induced liver injury, acute viral hepatitis)? These analyses should be matched to age, sex and severity of liver injury (e.g. matched to total bilirubin levels)

b. Can the authors also add measurements in age- and sex-matched healthy individuals?

c. What was measured by the 25(OH)D assay used in this study? Protein-bound or free 25(OH)D? Can the authors add measurements of free 25(OH)D, if this has not been performed yet?

2. What were the diagnostic criteria for AIH patients with acute presentation? The authors only mention that for the diagnosis of chronic AIH, the IAIHG and the simplified score were applied and that alcohol consumption and chronic viral hepatitis B and C infection were excluded. Did the authors also apply these diagnostic scores for AIH patients with acute presentation? How were important differential diagnoses of acute hepatitis excluded, such as drug-induced liver injury or acute viral hepatitis? Was acute viral hepatitis E infection excluded by PCR?

3. As I understand it right, 25(OH)D and laboratory values of patients with acute presentation of AIH were analysed at the time point of initial presentation. But at which time point were 25(OH)D and laboratory values analysed in patients with chronic manifestation of AIH? At the time of first diagnosis? Besides, the authors performed analyses at the time of remission. However, the time interval until biochemical remission is achieved, varies from patient to patient. Can the authors give the information what the median time was until remission was achieved in their cohort of AIH patients?

4. Many patients were treated with steroids (prednisolone monotherapy in 89% and combination therapy with azathioprine in 23% of patients). It seems that no patient with chronic AIH was on azathioprine monotherapy. Is this correct? At least those patients with chronic AIH analysed at the time point of remission, being treated for a longer period with steroids, should have been supplemented with vitamin D. How many patients were supplemented with vitamin D when measurements of vitamin D were performed?

5. Throughout the manuscript, the exact median results should be presented and not only p-values (e.g. page 12: “In particular, serum 25(OH)D levels were significantly lower in patients with severe symptoms of AIH than in patients with nonsevere symptoms (P = 0.0018)” – please present the exact levels of 25(OH)D!)

6. Page 12: „…but significantly positively correlated with the necroinflammatory activity grade (r = -0.4039)“ – “r“ is negative, so it is a negative correlation!

Minor concerns:

7. The sex distribution of this study differs from previous international reports, with far more female than male AIH patients in the study by Abe et al. (89% were female) than in previous international studies (about 70% of AIH patients were female). What is the authors’ explanation for this? What is the sex distribution of AIH patients in Japan due to population-based studies?

8. The statement “We investigated the association of serum 25(OH)D levels with pathogenesis” must be avoided: How can laboratory values be associated with pathogenesis itself? In this study, 25(OH)D levels were associated with cytokine levels. Furthermore, pathogenesis of AIH is still unclear and only certain aspects such as proinflammatory mechanisms have been investigated yet.

9. Page 3/4: The incidence of AIH with acute presentation has recently increased, and this form has been established a type I AIH” – this is not correct, paediatric patients with type 2 AIH (anti-LKM+) often manifest with acute and severe hepatitis.

10. In the methods section the authors state that anti-LKM were detected by ELISA. Which test was performed for the detection of ANA? Immunofluorescence or ELISA? What about the other antibodies that should be tested for in a patient suspected to have AIH (Anti-SMA? Anti-SLA/LP? Anti-LC1?)

11. Page 10: “Twenty patients (30%) had severe disease symptoms (TB >5.0 mg/dL and/or PT <40%)” – A laboratory value is not a symptom. Do you mean that 30% presented with icterus?

Reviewer #2: The manuscript reports an association between reduced vitamin D serum levels and necroinflammatory activity / disease severity as well as with some proinflammatory cytokines (INF-gamma and IL-33) in patients with autoimmune Hepatitis. The manuscript is well written and the analyses are thoroughly performed. The novelty of the study is - however - limited, yet the association of Vitamin D deficiency with IL-33 serum levels of some interest. Yet, careful proofreading should be performed in order to eliminate some typos / grammatical issues, and authors should explain or at least discuss why a relevant number of cytokines was not detectable at all.

**********

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

Reviewer #2: 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.

Association of Serum 25-Hydroxyvitamin D Levels with Severe Necroinflammatory Activity and Inflammatory Cytokine Production in Type I Autoimmune Hepatitis

PONE-D-20-17668R1

Dear Dr. Abe,

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,

Pavel Strnad

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 #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 #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

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 #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 #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 #2: The authors have sufficiently responded to my concerns. As mentioned previously, the analysis have been well performed and the manuscript is interesting to read, although the data are rather of confirmatory nature.

**********

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 #2: No