Dear Dr Ferraz-Amaro,

Thank you for submitting your manuscript entitled "Ultrasensitive Quantification of Serum IFN-α, But Not IFN-γ, Reflects Inflammation, Disease Activity, and Autoantibody Status in Systemic Lupus Erythematosus" for consideration by PLOS Medicine.

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Alexandra Tosun, PhD

Senior Editor

PLOS Medicine

Dear Dr Ferraz-Amaro,

Many thanks for submitting your manuscript "Ultrasensitive Quantification of Serum IFN-α, But Not IFN-γ, Reflects Inflammation, Disease Activity, and Autoantibody Status in Systemic Lupus Erythematosus" (PMEDICINE-D-25-02814R1) to PLOS Medicine. The paper has been reviewed by subject experts and a statistician; their comments are included below and can also be accessed here: https://www.editorialmanager.com/pmedicine/l.asp?i=1026346&l=PA4Y7ZOB

As you will see, the reviewers were positive about the manuscript but also offered valuable suggestions to strengthen it further. After discussing the paper with the editorial team, I'm pleased to invite you to revise the paper in response to the reviewers' and editors' comments. We plan to send the revised paper to some or all of the original reviewers, and we cannot provide any guarantees at this stage regarding publication.

In addition to these revisions, you may need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

When you upload your revision, please include a point-by-point response that addresses all of the reviewer and editorial points, indicating the changes made in the manuscript and either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please also be sure to check the general editorial comments at the end of this letter and include these in your point-by-point response. When you resubmit your paper, please include a clean version of the paper as the main article file and a version with changes tracked as a marked-up manuscript. It may also be helpful to check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper.

We ask that you submit your revision by Sep 24 2025. However, if this deadline is not feasible, please contact me by email, and we can discuss a suitable alternative.

Don't hesitate to contact me directly with any questions (atosun@plos.org).

Best regards,

Alexandra

Alexandra Tosun, PhD

Senior Editor

PLOS Medicine

atosun@plos.org

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Comments from the reviewers:

Reviewer #1: Gonzalez-Gay et al. present a very nice data set relating serum interferon-alpha and -gamma levels measured by high sensitivity Simoa assay to a number of demographic and clinical parameters in 313 patients with SLE. While the observations presented are not novel and generally confirm previously published studies, the availability of IFN levels across a large number of patients allows for a multivariate analysis of correlations that emphasize the significance of IFN-a (but not IFN-gamma) as a mediator with potential to assist in characterizing disease activity and possibly organ damage in the context of clinical practice. The effort to identify cutoff IFN values for degree of disease activity or remission are not helpful (could be eliminated from the manuscript) and the ROC curves shown in Supplementary Figure 1 indicate that in the absence of other measurements, quantitation of IFN-a levels will have limited utility as a biomarker in clinical practice. Nonetheless, the data do support IFN-a levels as highly relevant to lupus disease, and they would be useful to incorporate into clinical practice as one of many factors to be considered in patient assessment. As noted by the authors, the Simoa assay is currently rarely available to clinicians and even for investigators it is quite expensive. Perhaps the data presented here could encourage clinical testing facilities to make the Simoa assay available to clinicians.

In addition to the use of the Simoa assay, strengths of the study include the wide spectrum of patients studied, with more than half characterized as in DORIS remission, and the detailed autoantibody data provided. Notable is the very strong positive correlation between autoantibodies specific for RNA-associated particles and the significant negative correlation with certain anti-phospholipid (APL) antibodies. The latter observation has been previously reported, including in a recent publication from Karolinska (Rheumatology, 2025, 64, 1469-1475) describing IFN-a levels in pregnant SLE patients. The distinction between autoantibodies targeting RNA-associated proteins and those among APL specificities in relation to activation of the IFN pathway was noted years ago, and the current manuscript's data suggest an opportunity for further investigation of the role of the antiphospholipid antibodies in regulating IFN expression.

The manuscript might be strengthened by tightening up the Introduction and Discussion. Much of the Introduction simply reviews background related to interferons and is probably unnecessary for most readers. The authors (in the first paragraph) note the role of cytoplasmic nucleic acid sensors in induction of IFN-a but they fail to describe the role of nucleic acid-containing immune complexes in inducing IFN-a through Toll-like receptors, likely the major mechanism. The role of TLRs should be recognized.

The Discussion is also excessively long and does not provide any major insights into the biologic significance of the findings. Shortening the manuscript to just present the data with a brief acknowledgement of other relevant publications would be helpful.

Reviewer #2: I only reviewed the methods and statistics of this paper. There are several major concerns:

1. Confounders selected based on p-values are not appropriate for 2 reasons: p-values are not designed for variable selection and doing so could inflate type 1 errors; this practice does not account for the causality between variables - confounders by definition should be a variable causing exposure and outcome. If the author intend for a prediction (which has not been clear) they should consider either using AIC for variable selection or LASSO.

2. Relatedly, if that analysis aims for causal inference, it should be cautioned that cross-sectional design would mean reverse causation is possible.

3. Log-transformation was done to account for non-normally distributed variables but have the authors examine whether the transformed variables are 'sufficiently normal'?

4. It is clear how the 'optimal cut-off' is determined - is it by maximising the sum of sensitivity ana specificity?

5. The AUC and sensitivity shown are really low - I'm not sure whether this is clinically relevant / useful.

6. (minor) would be good to show Table 1 by the outcome.

Reviewer #3: I carefully read the article by Gonzalez-Gay et al. entitled "Ultra-sensitive quantification of serum IFN-α, but not IFN-γ, reflects inflammation, disease activity, and autoantibody status in systemic lupus erythematosus." This study demonstrates that serum IFN-α, measured with an ultra-sensitive immunoassay, is a valuable biomarker of SLE activity. It confirms findings from other groups in the field, using a large cohort of patients with this disease. The data also show that IFN-γ is a less reliable marker of SLE activity compared to IFN-α. As studies using ultra-sensitive immunoassays to quantify IFN-α in SLE remain scarce, this work is an important contribution to the scientific community. However, the clinical utility of ultra-sensitive assays appears less critical for IFN-γ than for IFN-α, given the higher circulating serum concentrations of the former. The manuscript is well-structured, concise, and easy to follow. However, several aspects require revision and, hopefully, improvement from the authors.

Major points

* For clinicians, perhaps the most striking—and disappointing—result is that serum IFN-α levels, although correlated with disease activity, do not reliably discriminate between clinically active and inactive patients, regardless of the activity scores used. This finding contrasts with previous reports (Mathian, Arthritis Rheumatol 2019, PMID: 30507062; Wahadat MJ, Rheumatology 2023, PMID: 36515466). It is surprising that the present study identifies discriminant thresholds as high as >10-15 pg/mL, whereas earlier studies reported much lower cut-offs (266 fg/mL in Mathian et al., 225.9 fg/mL in Wahadat et al.). Such high IFN-α levels are usually observed in clinically overtly active SLE and are rarely found in patients with minimal disease manifestations. These discrepancies likely reflect differences in assay technologies, pre-analytical processes, and possibly patient characteristics. Of note, the Gonzalez-Gay cohort included relatively few clinically active patients, with poorly detailed distribution of activity levels and no description of clinical involvement types. The authors acknowledge these limitations, including the inability to test their platform on healthy controls.

Furthermore, the use of SLEDAI and SLEDAS in this context raises concerns, as these scores include serological markers (complement and anti-dsDNA), which are not clinically meaningful indicators of clinical activity. For instance, the authors report 45% inactive versus 55% active patients. Yet, they also report 68% patients in remission. This implies that, at most, only 32% had clinical evidence of activity—likely fewer, since many of those not in remission were classified so merely due to prednisone >5 mg/day, despite clinical inactivity. It is probable that the 23% discrepancy (55%-32%=23%) represents patients with only serological abnormalities (complement consumption and/or elevated anti-dsDNA), which are of little clinical relevance.

To clarify these issues and assist readers in interpreting the data, I suggest the following:

o Provide detailed graphical presentations of individual IFN-α values and medians across subgroups (by SLEDAI activity level: none/mild/moderate/high; by SLE-DAS activity levels; remission/mild/moderate/severe; remission vs non-remission; LLDAS vs non-LLDAS). This would allow readers to assess dispersion and subgroup sizes, as well as to visualize how many patients fall above or below ROC-derived thresholds.

o For ROC analyses, repeat calculations excluding purely serological activity by using the clinical SLEDAI (i.e., omitting complement and anti-dsDNA components). Many clinically inactive patients are misclassified as active under standard SLEDAI ≥4, while some with overt manifestations fall below this cut-off. Similar considerations may apply to SLE-DAS if a "clinical" version exists. Additionally, for comparison, I strongly encourage ROC analyses of C3, C4, and anti-dsDNA against clinical SLEDAI, DORIS, and LLDAS.

o Expand the discussion by comparing the proportion of active patients in this study with other published cohorts.

* The discussion misrepresents the work of Mathian et al. (Mathian, Arthritis Rheumatol 2019, PMID: 30507062). The threshold of 266 fg/mL was derived using ROC curves with clinical activity (clinical SLEDAI >0) as the gold standard, not SLEDAI ≥4. This point requires correction.

* Damage indices such as SDI integrate cumulative disease and treatment consequences over many years (mean disease duration ~19 years in this cohort). It is biologically implausible that a short-term biomarker such as serum IFN-α, which can fluctuate within minutes, reflects cumulative damage. Without longitudinal IFN-α data, such analyses should be avoided.

* Additional analyses of modified LLDAS (≤5 mg/day prednisone instead of 7.5 mg) would be informative, given its status as a current treatment target (Fanouriakis A, Ann Rheum Dis 2024, PMID: 37827694).

* In Table 2, cytokine levels should be reported for each subgroup, including measures of dispersion, to fully inform the reader.

* Table 2 would be more clinically interpretable if results were expressed as odds ratios rather than beta coefficients.

Minor points

* Pre-analytical conditions must be detailed (sample type, storage, and handling), as IFN-α is labile and requires strict preservation protocols.

* In Table 1, several percentages appear inconsistent. My recalculations yield: dyslipidemia 56% (vs 58%), metabolic syndrome 44% (vs 45%), SLEDAS remission 76% (vs 78%), anti-ENA 70% (vs 71%), anticardiolipin IgG 15% (vs 16%). These discrepancies should be corrected.

* The claim that this is the first multivariable analysis of IFN-α clinical associations is inaccurate; such analyses have been reported (Mathian, Arthritis Rheumatol 2019, PMID: 30507062).

* The study does not reference Chasset et al. (Ann Rheum Dis 2022, PMID), one of the earliest to compare ultra-sensitive immunoassays for IFN-α, IFN-γ, and type I IFN scores in relation to SLE activity. This work should be discussed.

Any attachments provided with reviews can be seen via the following link: https://www.editorialmanager.com/pmedicine/l.asp?i=1026346&l=PA4Y7ZOB

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Attachments

Attachment

Submitted filename: Gonzalez-Gay.docx

Dear Dr. Ferraz-Amaro,

Thank you very much for re-submitting your manuscript "Ultrasensitive Quantification of Serum IFN-α, But Not IFN-γ, Reflects Inflammation, Disease Activity, and Autoantibody Status in Systemic Lupus Erythematosus" (PMEDICINE-D-25-02814R2) for review by PLOS Medicine.

Thank you for your detailed response to the reviewers' and editors’ comments. I have discussed the paper with my colleagues, and it has also been seen again by all three original reviewers. The changes made to the paper were mostly satisfactory to the reviewers. As such, we intend to accept the paper for publication, pending your attention to the reviewers' and editors' comments below in a further revision. When submitting your revised paper, please once again include a detailed point-by-point response to the editorial comments. The remaining issues that need to be addressed are listed at the end of this email.

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper.

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We look forward to receiving the revised manuscript.

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Senior Editor 

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Comments from Reviewers:

Reviewer #1: The authors have made a good effort to respond to all reviewer comments. While the study has limitations - including the high number of patients in remission (although that is also a strength as it demonstrates that IFN-a is present in inactive as well as active patients) and the absence of information on the relationship of IFN-a to clinical features of SLE (such as organ involvement), it provides a nice dataset for consideration by other investigators. It also supports the utility of the Simoa platform for detection of IFN-a. The manuscript strongly makes the case for an association of autoantibodies targeting RNA-associated proteins with serum IFN-a and a negative association of anti-phospholipid antibodies with IFN-a, with the latter an association (negative) that cries out for more investigation.

Reviewer #2: Thanks for addressing my comments and provide clarification to the methods and interpretation. One thing that I do not agree on is the use of 'data-driven' in selecting confounders. Variables that influence the exposure-outcome associations could be confounders (which should be adjusted) as well as mediators and colliders (should not be adjusted). This method does not tell us anyone on these. The authors should at least acknowledge that the method they used to induce overadjustment and collider bias.

Schisterman EF, Cole SR, Platt RW. Overadjustment bias and unnecessary adjustment in epidemiologic studies. Epidemiology. 2009 Jul 1;20(4):488-95.

Holmberg MJ, Andersen LW. Collider bias. Jama. 2022 Apr 5;327(13):1282-3.

Reviewer #3: I have carefully read the authors' responses to the questions raised during the first submission, as well as re-reviewed the revised manuscript in its entirety. The authors have satisfactorily addressed the concerns raised and modified the manuscript accordingly.

I believe that two very minor adjustments would further improve the quality of the paper:

* In the new Figure 2: please add the sample size for each subgroup (n = X). In the legend, indicate explicitly that this is a Box-and-Whisker plot, thus showing medians, quartiles, and extremes. It would also be helpful to include on the figure the thresholds derived from the ROC curve AUC values.

* Page 13, line 360: unless I am mistaken, the authors state that "the cutoff values for remission according to DORIS and LLDAS were 10,800 and …". However, in the corresponding table the value of 11,400 is reported. Could the authors clarify this apparent discrepancy?

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* l.62, “demographic characteristics and traditional cardiovascular risk factors” – please specify.

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* Table 1/2: Please change ‘Female, n (%)’ to ‘Sex, female, n (%)’.

* l.268ff, “While most SLE patients had no activity (45%), mild or moderate-high activity was present in respectively 46% and 9% of the patients as indicated by the SLEDAI score.” – It seems that most patients had mild or moderate-high activity.

* l.272, “The SDI was 0 (IQR 0-1) and an SDI score of 1 or higher was found in 46% of patients.” – We assume this refers to "SLICC-DI" in Table 1. We recommend using the same abbreviation consistently throughout.

* Figure 1: Please note that in the graph the p-value is 0.000. Also, would it be useful to align the y axis for the two distribution graphs to facilitate comparison?

* l.292ff: Please clarify that you are discussing the results of the multivariable adjustment.

* l.293ff: Please ensure to present the results with sufficient detail, e.g. SLEDAI-2K category ‘mild’ was not significantly associated with elevated IFN-α levels.

* S1 Table: For SLEDAS, it says ‘2.08’ while in the main text you write ‘2.8’ (l.316). Please check.

* l.321, “while levels above 29,000 femtograms/ml discriminated high disease activity” – Should the reader be able to find this number in Table S1? Please carefully check the numbers reported in the paragraph (vs. the table).

* l.322, “The cutoff values for remission according to DORIS and LLDAS were 10,800…” – according to S1 Table the cutoff value for DORIS is 11,400.

* Figure 1: Please include the n-numbers for each graph. Are the underlying numbers available in the Zenodo files?

* Figure 2: Please define all elements of box plots in the figure caption - center line, box limits and whiskers. Please include the n-number. Please specify ‘no activity’ (‘no disease activity’). Given a broad medical audience, we recommend that you provide a more detailed definition of "DORIS remission" and "LLDAS remission."

* Please provide the unadjusted comparisons as well as the adjusted comparisons in all relevant Tables (or as supplementary analysis).

* Please confirm that you have specified the variables controlled for in all relevant Tables.

DISCUSSION

* When revising the discussion, please aim to streamline it and keep in mind that the journal is intended for a broad medical audience.

* l.361, “In this regard, our study represents the largest investigation to date…” – please add, ‘to our knowledge’.

* l.459, “In conclusion, elevated serum levels of IFN-α identify patients with SLE exhibiting higher

460 disease activity, alongside increased ANA expression.” – please revise to focus on the associational aspect of the study.

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Dear Dr Ferraz-Amaro, 

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