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  1. Evaluation Summary:

    This manuscript identifies the DNA sensor AIM2 as a target of auto-antibodies in the human autoimmune disease systemic lupus erythematosus. Importantly, the authors provide evidence that AIM2 protects extracellular DNA from destruction and propose that this property may enhance the autoimmune response to the DNA and associated proteins. The work may therefore provide an important underlying mechanism for a prevalent and important human autoimmune disease.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Autoantibodies that bind nucleic acids and nucleic acid-binding proteins are a hallmark feature of SLE. In this study, Rosen and colleagues identify AIM2, a dsDNA responsive inflammasome forming sensor as a new autoantigen in SLE. Further they show that anti-AIM2 antibodies are associated anti-IFI16 Abs previously linked to SLE and athese ALR Auto antibodies correlate with anti-dsDNA Abs and SLE disease severity. The authors also provide compelling evidence that AIM2 and the ALR family member IFI16 bind NETs and shield NETs from Dnase-mediated nuclease degradation. The finding that AIM2 like IFI16 are autoantigens and that these ALRs might contribute to SLE disease pathogenesis by protecting NETs from destruction thereby enhancing extracellular nucleic acid driven immune stimulation and IFN responses is striking and directly relevant to disease. This is a very interesting study. The identification of AIM2 as an auto antigen and the role of AIM2 in promoting the interferogenic activity of NETs by preventing their clearance has important implications for SLE pathogenesis.

  3. Reviewer #2 (Public Review):

    Antiochos et al. analyzed plasma from 131 Lupus patients, 133 Sjogren's syndrome patients and sera from 49 healthy controls for binding to AIM2, a DNA sensor, IFI16, a related cellular protein, and dsDNA, a characteristic Lupus antigen. These 3 reactivities tended to overlap and their presence tended to correlate with a more severe disease. In addition, the authors considered whether the proteins would be able to associate with NET chromatin from healthy control neutrophils treated with PMA and whether they could protect the DNA from degradation by DNAse 1. Further, the authors identify structures in kidneys from Lupus biopsies that stain with antibodies to AIM2, IFI16, or MPO, a marker for neutrophils. These are visualized relative to DNA, and areas of more decondensed chromatin are considered as NETs. Binding to each is observed and also colocalization is performed using confocal microscopy. The statistics of data analysis are valid and correlations with disease scores are appropriate. Negative controls for microscopy data presumably have been done by the authors (not shown).

    An inherent conundrum is present in the initial data on plasma reactivity determination. The AIM2 protein is prepared in vitro and radiolabeled during in vitro translation. This is then incubated with patient plasma and the counts in the immuno precipitate are used to determine antibody reactivity to AIM2. This would seem to be a sensitive and quantitative way to measure antibody binding. However, the additional observations made by the authors call the straight-forward interpretation into question. The authors show that AIM2 strongly binds to dsDNA and protects it from nuclease digestion. Previous studies from other authors have shown that cell-free DNA is present in plasma and more elevated in severe cases of Lupus. Moreover, immune complexes between autoantibodies and nucleic acid complexes are also found in the plasma of lupus patients. Thus, it is difficult to exclude the possibility that rather than measuring the binding to AIM2, the assay detects other antibodies that bind to a complex to which the AIM2 protein binds as well. This complication does not apply to all samples, as clearly there are plasmas that are anti-dsDNA negative but anti-AIM2 positive. This is also a clear benefit of the SS samples, where binding to DNA is rare. Nevertheless, it is difficult to suggest an alternative route of approach, as the binding of AIM2 to DNA is DNAse resistant. Once a complex of DNA and the recombinant AIM2 forms, it would presumably be able to survive an IP procedure. This represents a major obstacle, at present, to the clear identification of "bona fide" antibodies to AIM2 in the Lupus samples.

  4. Reviewer #3 (Public Review):

    The authors developed an assay to quantify Aim2 autoantibodies and demonstrated that SLE patients with detectable Aim2 autoantibodies, on average, had more severe clinical disease. Previous studies had demonstrated that Ifi16, another cytosolic DNA sensor, was also an SLE autoantigen. There was a correlation between higher AIM2 antibody titers and high SLEDAI scores.

    They found that patients frequently made autoantibodies reactive with Aim2, Ifi16 and DNA and, given the recent interest in neutrophil NETS and SLE, decided to explore a potential structural explanation for this observation. Their immunofluorescent data convincingly documented the ability of both AIM2 and Ifi16 to multimerize into filaments that bind neutrophil NETS, generated in vitro by PMA activation. They also demonstrated co-localization of NET DNA and their DNA sensors in renal sections obtained from patient populations. Their imaging of renal sections from lupus nephritis patients is particularly impressive. Importantly, they further reported that NET DNA bound by AIM2 and Ifi16 was protected from DNase1-mediated degradation. As far as nuclease protection, there is one point that could be addressed further - the images shown in Figure 2D appear to indicate that AIM2 and Ifi16 are not binding comparably across the NET structure. Quantification of sensor binding in additional images would therefore be informative. The authors should also mention other studies in which DNA-binding proteins protect neutrophil NETS from nuclease degradation (eg. Hakkim 2010).

    The authors further documented the presence of AIM2 autoantibodies, but not Ifi16 autoantibodies, in the sera of Sjogren's Syndrome patients. As an explanation for the difference between SLE and SS, they point out that neutrophils are often found in the kidneys of lupus nephritis patients but do not normally infiltrate the salivary glands of SS patients. They raise the possibility of a different scaffold for AIM2. The novelty of the current study could be considerably bolstered if they could detect and identify AIM2 structures by immunofluorescence in tissue from SS patients.

    Overall, this is a highly significant study that establishes the connection between high AIM2 autoantibody titers, resistance of AIM2/Ifi16 bound DNA to nuclease degradation, and the presence of AIM2-bound NETS in renal biopsies of SLE patients. The report could be further strengthened by addressing mechanisms by which autoantigen-bound NETS might promote autoantibody production and immune activation.