PONE-D-20-38304

Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: down-modulation by H4 binding to C-reactive protein and Surfactant protein D.

PLOS ONE

Dear Dr. Hartshorn,

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 recommend addressing the points highlighted by the referees.

Please submit your revised manuscript by Feb 19 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.

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,

Nades Palaniyar, MSc., PhD.

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 note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

3.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

[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: No

**********

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: The manuscript attempts to unravel the impact of H4 histone on the activation of neutrophils in the context of IAV infection. Importantly, authors reveal the molecular interaction between H4 and CRP as well as H4 and SP-D and their inhibitory effect on H4 induced neutrophil activation and oxidative burst.

The authors did not comparatively evaluate CRP or SP-D binding to H4 and whether they compete for the same binding site on H4.

Also, authors have not discussed the impact of these molecular interactions together on the infectious foci of IAV in vitro and neutrophil uptake of IAV.

Discussion needs to mention the physiological concentrations of these ligands and H4 in BAL of IAV infected individuals and how is it likely to affect the IAV replication. Most of the discussion is focused on the ability of SP-D and CRP on the H4 induced neutrophil activation. Both H4 and SP-D have antiviral effects. Once they bind to each other, it was expected to see an increase in the viral foci. Authors may offer an explanation in this regard. Is it likely that H4 bound to SP-D may still show antiviral effects? or SP-D bound to H4 may still show antiviral effects?

In the Fluorescent focus assay of IAV infectivity, the authors only mention MOI. It would be relevant to mention the number of viral units and the number of cells. Importantly, also indicate how the viral units were enumerated.

Ethics statement may mention the study approval date and duration.

Reviewer #2: The authors show the effect of histone H4 on the activation of neutrophils and promoting neutrophils’ responses that lead to a respiratory burst, and how H4 can exacerbate the inflammatory response of neutrophils to influenza A virus (IAV). Interestingly, they demonstrate that SP-D or CRP can inhibit the pro-inflammatory effect of H4 in neutrophils.

The results and data provided by the authors are interesting and novel, besides the demonstrated role of CRP and SP-D to reduce the H4 pro-inflammatory response in neutrophils in-vitro is relevant and the first step to perform future in vivo experiments. However, some additional details of the experiments or information should be provided, as well as explanation or comments in some results.

Specific comments:

1. The methods section should be carefully reviewed. More details should be provided, as well as the method details in the figure legends. Authors should provide buffer protein compositions (e.g. for all the recombinant SP-Ds used and commercial H4 and CRP). What is control buffer? and if it is PBS++, it should be stated it in methods, sometimes PBS++ is also indicated in the figure legend and others isn’t – revise figure legends. Concentrations of reagents should be provided: e.g. how much calcium was added to PBS? Incubations of peptides with the neutrophils were performed at 37 ˚C or at 37 ˚C in an incubator with 5% (v/v) CO2? Same comment applies for conditions when “treatments” are performed e.g. in the measurement of caspase 3 activity it says “human neutrophils were treated with indicated proteins for 5 hours” (where? Which temperature?).

2. Production of recombinant human SP-D in CHO cells yield a combination of different oligomeric forms of SP-D. Therefore, it should be specified if rhSP-D dodecamers were isolated from the mixture of oligomers obtained and purified from the CHO cells. Otherwise, it should be indicated as rhSP-D without specifying the oligomeric form or emphasizing that is the most abundant form but not 100%.

3. In Figure 2, the graphs for A-B seem identical to the graphs for C-D but for the label “with BAPTA”. Authors should double check if they copied the right graphs to figure 2. It seems that C-D might be the wrong ones and A-B were taken again by mistake.

4. The authors showed binding of CRP and SP-D to H4 with two different methods. There is no question that binding takes place between CRP and H4 and also between SP-D and H4. However, the second method applied, the co-precipitation by centrifugation is somehow intriguing. The gels show that the method is working and reporting binding like the ELISAs. It is very surprising that the bound complex of SP-D (especially the NCRD) and H4 (a 12 kDa protein) precipitates with a centrifugation at 1,200 x g for 5 min. Ultracentrifugation at 100,000 x g is performed to pellet pulmonary surfactant from bronchoalveolar lavages and SP-D is recovered in the supernatant instead of the pellet (Taeusch, H. W., J. Bernardino de la Serna, et al. 2005. Biophys. J.), in addition, in SP-D purification from BAL or amniotic fluid a centrifugation step at 2,000 to 10,000 x g depending on the paper is performed (e.g. Leth-Larsen, R., et al., 1999. Biochem J; Strong P., et al., 1998. J of Immunological Methods). Therefore, it is surprising that the complex SP-D-H4 is pelleting at 1,200 x g. Is a visible pellet obtained in that centrifugation? How much are the volumes for each reagent and final volume that allow to differentiate and pipet supernatant and pellet?

I would recommend the authors to perform the binding experiments in presence of EDTA and maltose (Figure 5E, which in the text is pointed as 5D -see last figure mentioned in that paragraph in page 14) by the ELISA method as a more robust technique to confirm that the binding is not calcium dependent.

The authors show that the binding site of SP-D to H4 is not in the collagen or N-terminal domain because binding is observed with the NCRD mutant. At the same time, binding is reported to be non-calcium dependent and non-CRD mediated. However, looking at the ELISA results (Figure 4) and comparing the binding of NCRD and *NCRD (the mutant with increased affinity to mannan) to H4, it is intriguing that the *NCRD mutant seems to bind more or with higher affinity to H4 than the NCRD peptide, taking into account that the binding is not dependent of the lectin activity of the protein. How do the authors explain it? Could it be related to the charge variation also induced by the amino-acids that are substituted in the mutant?

5. A significant inhibitory effect of CRP/H4 in neutrophil uptake of IAV is observed at 40 µg/mL of H4 in combination with PCR, but inhibition is not observed at 20 µg/mL of H4 at the same CRP concentration. Do the authors have a proposed explanation? Why at a lower concentration of H4, there isn’t an inhibitory effect of CPR in neutrophil uptake of IAV? (In addition, review the figure legend of figure 6 C-D, since the concentration of H4 indicated does not match the figure and does not indicate 2 different testing concentrations, besides PCR concentration is 8 µg/mL when in the following experiments is 80 µg/mL).

6. In table 1 and 2, why higher concentrations (ng/mL) were tested for the mutant NCRD* than for SP-D. Is it the effect observed with the mutant NCR* due to the higher concentrations of protein there in comparison to SP-D (full length)?

7. The reason behind the selection of the different concentrations of CRP, NCRD* and SP-D in the experiments in combination with H4 (results section B and C) should be, at a minimum, indicated or commented in the discussion. How the authors explain the different effects observed between SP-D and NCRD* in intracellular free calcium and MPO release?

Statements as “data not shown” should be avoided and the data/figure provided in supplemental material.

8. Are any of the peptides (CRP, H4, SP-D, NCRD and NCRD*) in an EDTA-containing buffer? In case they are, is the EDTA concentration being compensated with additional calcium before being added to the cells to exclude any EDTA-related effect in the experiments (for example in the MPO release, figure 10)?

9. Recently, it has been shown that SP-D reduces LPS-induced NETosis in vivo and the detrimental effect of NETs in pulmonary surfactant (in mice lacking SP-D) (Arroyo, R., et al., 2020, Comm Biology), which should be referred in the discussion of SP-D ability to reduce NET mediated injury if authors want to discuss that idea.

**********

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: Yes: Taruna Madan

Reviewer #2: Yes: Raquel Arroyo

[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.

Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: down-modulation by H4 binding to C-reactive protein and Surfactant protein D.

PONE-D-20-38304R1

Dear Dr. Hartshorn,

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,

Nades Palaniyar, MSc., PhD.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: The study and the revisions have been done, well.