PONE-D-22-27387

Increased levels of immature and activated low density granulocytes and altered degradation of neutrophil extracellular traps in granulomatosis with polyangiitis

PLOS ONE

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PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

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

Reviewer #1: No

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Reviewer #1: Yes

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Reviewer #1: 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: This is a small inception cohort study examining NET production in GPA (n=12) and SLE (n=21) receiving a wide array of medications. The healthy donor control group was 95% female and much younger than the GPA cohort, making it difficult to separate out gender and age effects. There was significant variability in virtually all readouts, indicating that the study is underpowered to assess most parameters studied. The study confirms previous findings of LDG expansion and impaired NET degradation in GPA.

• The cohorts should be separated into those with active and remission disease (the BVAS range included 0 so remission patients were apparently included). It is likely that disease activity (and, more importantly, the high dose corticosteroids associated with this active disease, will have a big effect on NETosis).

• Note that, although table 1 describes the full cohort, studies examining NETs had fewer participants; these cohorts should be described in a supplementary table.

• Fig S1A: it’s not clear what the y-axis represents. Neutrophils in the PBMC layer are LDGs, but the figure refers to NDGs.

• I suggest that, for all figures, the individuals receiving high dose corticosteroids (say >20mg/day) are identified using, for example, open circles. Also, in addition to correlating NET degrading capacity with BVAS, figure 5 should also correlate against steroid dose in both SLE and GPA patients.

• Are flow cytometry data available for whole blood samples? This is important to clarify the effect of cell isolation on markers such as CD16 and CD63. Indeed, it is not clear why the results pertaining to LDGs are emphasised, given that NETOsis, the focus of the paper, was not studied in this cell population.

• The anti-PR3 antibody levels, rather than c-ANCA levels, should be reported. I presume that none were anti-MPO positive.

• Fig 4: There does appear to be a decline in NET degrading capacity with increasing disease activity, although BVAS is a poor means of quantifying the degree of vasculitic injury. Is the same observed with SLEDAI in the lupus participants? Again, it is important to consider the confounding effect of concurrent corticosteroid therapy, which may in fact be the main reason for this decline in NET degradation capacity.

• The authors comment on some unexpected null findings in the discussion, for example, no increased circulating NETs in GPA. The most likely reason for this is the fact that the study is underpowered, which should be alluded to in the discussion.

• The methods section contains descriptions of quantification of DCs, B cells and other cell types that have no counterpart in the results section. Were these experiments performed? If not, it is not necessary to describe the respective methods.

• The strongest result is the reduction in NET degradation capacity of GPA serum. Were DNAse-1 levels measured in serum? This would be an obvious next step in determining the mechanism of this observation.

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Reviewer #1: Yes: Mark Little

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Increased levels of immature and activated low density granulocytes and altered degradation of neutrophil extracellular traps in granulomatosis with polyangiitis

PONE-D-22-27387R1

Dear Dr. Tobias,

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,

Prof. Pierre Bobé

Academic Editor

PLOS ONE