PONE-D-20-25189

The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation.

PLOS ONE

Dear Dr. Cho,

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.

Both reviewers have issues with the details concerning the numbers of mice used in each experiment and reviewer 2 wants specific information about how mice were handled.  How do the second and third arthritis induction experiments compare to that listed in 1A?

Both reviewers share concerns about the specificity of suppressive effects on B cells, suggesting that the effects may either be broad (covering multiple cell types) or worse, non-specific in nature.  These points will need to be addressed before this manuscript can be considered for publication. 

Reviewer 1's comment that "There is really no experiments in the manuscript documenting that sulforaphane is actually an activator of Nrf2" could be handled either through experimentation or simply through highlighting evidence from the literature, but it should be acknowledged somehow.

I would point out that the flow cytometry data needs attention as well.  It is difficult to determine exactly how the regions outlining specific populations were chosen. I have attached a copy of figure 3 on which annotations have been made to point out specifics.

The first issue is that of gating.  How are the dot plots shown in Figure 3A gated?  The only antibodies listed were those for B220, CD138 and GL-7.  Sometimes it is helpful to include a plot of ex-vivo splenocytes as a comparison to demonstrate that your antibodies have identified the populations that you intended.  

The box used to define B220low/CD138+ plasma cells does not really seem to specify any definable population even in the vehicle group.  It appears to cover B220 negative cells.   If one wants to define B220lo and B220hi populations, then arguably the best division may be the orange line I have indicated on the annotation, which would then make the plasma cells fall into the magenta colored rectangle.  This might change the conclusions.

At any rate, a more complete description of the strategies used to define specific populations should be provided.

--

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David Douglass Brand

Academic Editor

PLOS ONE

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I would personally like to apologize for the length of time it took to get this review completed. Due to current circumstances, finding appropriately qualified reviewers for this work was particularly challenging but those agreeing to do so are indeed highly qualified for this subject area. Please look over their responses carefully as you decide how to proceed with this important work.

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: Yes

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Reviewer #1: The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation

The manuscript investigates the effect of sulforaphane on collagen-induced arthritis. Mice are treated with sulforaphane intraperitoneally and the investigators demonstrate that there is a decrease in severity of arthritis. This is associated with decreases in histologic inflammation, decreases in cytokines IL-6, IL-17, TNF-a, and TRAP. The investigators also demonstrate that human PBMs can have inhibition in production of IL-6, TNF-a, and IL-17 when cultures with sulforaphane. This is an important finding and may have therapeutic implications.

However, there are some problems with the manuscript.

The title is misleading. The effects of sulforaphane appear to be broad and cause inhibition of both T cell cytokines, as well as antibody production and inhibition of differentiation into plasma cells. Therefore, the focus on B cells is misleading.

There is really no experiments in the manuscript documenting that sulforaphane is actually an activator of Nrf2.

There was no clear indication as to how many mice were used in the experiments described in figure 1A. It is important to know the number of mice in order to evaluate the validity of the results.

For the flow experiments, it is also important to know how many mice were evaluated and what are the means of the populations evaluated.

Reviewer #2: This paper shows that sulforophan, known as an inhibitor of Nrf2, suppress collagen induced arthritis. It is claimed it do so based on its effects on B cells. Major points:

1) All data are not shown. Its not acceptable to base a study on an arthritis experiment with n=5. Apparently the experiment has ben run three times so this is a selected experiment. If the same experiment has been done 3 times it should be pooled and calculated together. The pooled data can be shown in the paper and the single experiments int he suppl information.

2) It should be clearly stated that the experiment was done blindly and distributed in the cages randomly, especially as it is well known there is a strong cage effect in DBA/1 mice. It should also be stated that all animal experiments follow the ARRIVE guideline. Of course only if this was the case.

3) The treatment has a profound effect on the inflammatory response. It is likely to ha ve very broad effects and it will be difficult to say exactly what is the specific effect. Basically all readouts a re secondary effects to something that this high dose of sulforophan is dong, whatever that is. Thus, it is not possible to claim that the effect on arthritis is due to effects on B cells as there is no evidence for this. The treatment is given 3 weeks after priming which means that the B cells have been primed and a full antibody response been developed. What will happen is that if these mice, due to this unknown "toxic" effect of the treatment does not develop arthritis it will secondarily, dur to less exposure of inflamed cartilage as well as a less powerful immune system give lower antibody titres. It can be predicted whatever is given to a mice leading to such a suppression of arthritis development.

In conclusion. If the arthritis data holds its a valuable report. But the authors need to make it very clear that they cannot say anything about the specific effects about sulforophan action as all evidenced data are secondary to the arthritis effect per se. Regarding human cells it seems to have profound inhibitory effect on cytokine production and I am afraid that these cells are not very happy.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Figure 3 (Comments).pdf

PONE-D-20-25189R1

The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of B cell differentiation and inflammation cytokine.

PLOS ONE

Dear Dr. Cho,

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.

==============================

• Address the subject of the under-powered nature of the CIA study (both reviewers) either through the pooling of data or through new CIA experiments.  Your response did not properly address these previously.

• Provide the missing flow cytometric data in Figure three showing the nature of the splenocytes as taken from each group (sulforaphane treated and untreated) of CIA mice. 

==============================

Please submit your revised manuscript by Jan 22 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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We look forward to receiving your revised manuscript.

Kind regards,

David Douglass Brand

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Both reviewers showed concerns regarding the numbers of mice used in the CIA experiment. The second reviewer suggested pooling data and supplying individual experiments as supplementary data. These concerns are shared by this editor in that under no circumstances will five mice per group properly power a CIA experiment. A minimum of twice that number is required, with even larger numbers necessary in experiments where the detection of subtle differences are required. However, your response to the reviewers did not address their concerns. I am afraid that providing more information than that or new data using larger numbers per group will be required before these answers can be met.

There is an additional problem in that it appears that some data are inadvertently missing from this publication. Figure 3 suggests that there are flow data available from cells analyzed ex vivo from sulforaphane treated mice. This is very confusing because the section is entitled "Effect of sulforaphane on B-cell differentiation *in vitro* and Ig production". The second and third sentences say "The results showed that the population of CD138+B220low plasma cells was significantly decreased by sulforaphane treatment in CIA mice, whereas the population of GL7+B220+ germinal-center B cells did not differ between the two groups (Fig. 3a). Then, in vitro experiments were performed to determine the effects of sulforaphane on B cell differentiation. " This suggests that the first set of data was a flow cytometric analysis of splenocytes from each group of mice demonstrating a reduction in CD138+B220low plasma cells but no reduction in GL7+B220+ germinal-center B cells. Figure 3a, which is referred to twice in this section appears to only show the in vitro data. The labeling demonstrates the concentrations (5 and 10 µM sulforaphane) and clearly shows in vitro data due to the LPS stimulated absence of T cells (very few double negative cells) under any condition. What happened to the flow data demonstrating a reduction in CD138+B220low plasma cells but no reduction in GL7+B220+ germinal-center B cells taken from CIA mouse spleens?

Other points are

The title that you suggested including the words "and inflammation cytokine" is not proper English usage. Perhaps "The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines"

Strike the word "etiological" from the last sentence of the abstract or provide more information about what your were trying to say.

I am aware that this decision is a very difficult one to resolve, but since this editor agrees with the concerns of the two reviewers regarding properly powering the study, it cannot be ignored as you have done in your response.

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The anti-arthritis effect of sulforaphane, an activator of Nrf2, is associated with inhibition of both B cell differentiation and the production of inflammatory cytokines.

PONE-D-20-25189R2

Dear Dr. Cho,

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.

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Kind regards,

David Douglass Brand

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

So long as the two additional severity scatter plots/Arthritis score graphs which were provided in your second response (Revision 2) are provided to the readers in the supplemental data section, the manuscript is acceptable.

However,

The wording is still confusing on page 11 would suggest removing lines 2 through 5 entirely.

Instead, start the section with line 6 but remove the word "Then"

Reviewers' comments:

Attachments

Attachment

Submitted filename: Screen Shot 2021-01-11 at 4.07.43 PM.png