PONE-D-21-05124

The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation

PLOS ONE

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Ozkan et al describe a potential caveat of utilising IL-10 reporter mice (VeRT-X) to identify myeloid-derived IL-10 production during an experimental model of LPS-induced lung inflammation.

The manuscript is well written, and the data is presented in a concise manner. The following comments are suggestions which I believe may add clarity to the overall message of the manuscript.

Major comments:

1. Can the authors comment on how the VeRT-X IL-10 reporter line and their results compare to any of the other eight IL-10 reporter lines, described by Bouabe 2012 (https://doi.org/10.1111/j.1365-3083.2012.02695.x)?

2. The original paper describing the VeRT-X mice (Madan et al., 2009) shows IL-10 GFP expression by B cells. Have the authors measured B cell derived IL-10 GFP levels in their model of LPS-induced lung inflammation?

3. It would be helpful to provide representative FACS plots as part of Supplementary Figure 1, to complement the gating strategy.

4. Figure 2 nicely illustrates IL-10 detection by ICCS vs GFP. It would be helpful to show representative FACS plots as part of this figure or as a Supplementary figure.

5. The figure legend for Figure 2 states: “The graph shows combined data from two independent experiments (PBS: n = 6 mice/group. LPS: n=7-9 mice/group)”. Can the authors confirm whether 6-9 mice/group were used in each individual experiment? Or whether the 6-9mice/group refers to the pooled data shown in Figure 2?

Minor comments:

Line 31: please remove ‘s’ from inflammations

Line 49: please change where to were

Line 54: please insert reference 7 after myeloid cells.

Line 54: please check reference 8, since the autofluorescence of myeloid cells was not mentioned in this reference.

Line 90-91: please clarify why data is not shown from WT controls. It might be informative to show this data as a supplementary figure.

Line 103: please remove ‘s’ from inflammations

Line 108: please remove ‘also’

Line 110: please change ‘analysis’ to analysing

Line 114: please remove ‘s’ from inflammations

Line 145: please add ‘d’ to purchase

Line 318: please superscript IL10GFP

Line 321 – 322: please superscript CD45+ CD45R/B220- CD3- Siglec-F+ F4/80+ CD11b- and IL10GFP

Reviewer #2: In this paper, the authors have proposed that IL-10 GFP reporter strain is not an appropriate model to detect IL-10 production by granulocytes. Using C57BL/6 and IL-10 GFP mice and by employing flowcytometry, the authors show that GFP signal seen in granulocytes post LPS challenge is not the actual GFP signal due to IL-10 production. Rather, it is attributable to autofluorescence. I think the authors have used proper controls in the study and results from the study illustrates important technical caveat in using GFP reporter mice for analysing myeloid cells. Overall, the study is good. However, I have few minor suggestions:

1. Line 31: Please change " during inflammations" to during inflammation. Please make this change elsewhere as well.

2. Line 39-40: Please rephrase this sentence as the meaning is not clear.

3. Line 49: Please change "lines where" to lines were.

4. Line 54: Please give reference when you discuss that IL-10 GFP strain was reported to enable identification of IL-10+

myeloid cells.

5. Line 110: Please change"analysis" to analysing.

6. Line 170: Change "where" to were.

7. Line 277-279 and 295-296: Please rephrase these two sentences as they seem incomplete.

8. Line 337-339: I think this sentence " C) Representative histograms from the spleen of PBS-------" is not required as it is a repetition of Supplementary Figure 3 legend.

9. Please check the overall grammar and sentence structure throughout the text.

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Reviewer #1: No

Reviewer #2: No

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The IL-10GFP (VeRT-X) mouse strain is not suitable for the detection of IL-10 production by granulocytes during lung inflammation

PONE-D-21-05124R1

Dear Dr. Wingender,

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.

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Kind regards,

Lynette Beattie, PhD

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

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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: (No Response)

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No