Anti-Neutrophil Extracellular Trap Antibodies in Antiphospholipid Antibody-Positive Patients: Results From the Antiphospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking Clinical Database and Repository

Abstract

Objective: This study aimed to elucidate the presence, antigen specificities, and potential clinical associations of anti-neutrophil extracellular trap (anti-NET) antibodies in a multinational cohort of antiphospholipid (aPL) antibody-positive patients who did not have lupus.

Methods: Anti-NET IgG/IgM levels were measured in serum samples from 389 aPL-positive patients; 308 patients met the classification criteria for antiphospholipid syndrome. Multivariate logistic regression with best variable model selection was used to determine clinical associations. For a subset of the patients (n = 214), we profiled autoantibodies using an autoantigen microarray platform.

Results: We found elevated levels of anti-NET IgG and/or IgM in 45% of the aPL-positive patients. High anti-NET antibody levels are associated with more circulating myeloperoxidase (MPO)-DNA complexes, which are a biomarker of NETs. When considering clinical manifestations, positive anti-NET IgG was associated with lesions affecting the white matter of the brain, even after adjusting for demographic variables and aPL profiles. Anti-NET IgM tracked with complement consumption after controlling for aPL profiles; furthermore, patient serum samples containing high levels of anti-NET IgM efficiently deposited complement C3d on NETs. As determined by autoantigen microarray, positive testing for anti-NET IgG was significantly associated with several autoantibodies, including those recognizing citrullinated histones, heparan sulfate proteoglycan, laminin, MPO-DNA complexes, and nucleosomes. Anti-NET IgM positivity was associated with autoantibodies targeting single-stranded DNA, double-stranded DNA, and proliferating cell nuclear antigen.

Conclusion: These data reveal high levels of anti-NET antibodies in 45% of aPL-positive patients, where they potentially activate the complement cascade. While anti-NET IgM may especially recognize DNA in NETs, anti-NET IgG species appear to be more likely to target NET-associated protein antigens.

Trial registration: ClinicalTrials.gov UL1-TR000457.

Figure 1:

Measurement of anti-NET antibodies in 389 aPL-positive patients. A, Schematic illustration of the anti-NET Ab ELISA. B-C, Anti-NET IgG and IgM were measured in the indicated groups; no patients in this cohort had lupus. Patients were grouped based on no criteria manifestations (“aPL positive”), a history of thrombotic manifestations (47 of these 278 patients also had a history of obstetric manifestations), or a history of obstetric manifestations without history of vascular thrombosis. Levels of anti-NET IgG and IgM at 450-nm optical density (OD) were compared to controls by Kruskal–Wallis test corrected for multiple comparisons by Dunn’s method; *p<0.05, **p<0.01, and ****p<0.0001. Horizontal black lines indicate medians and dashed lines indicate 99th percentile cut-offs. D-E, Association between positive anti-NET IgG (E) or IgM (F) and various extra-criteria APS clinical manifestations were assessed by multivariate logistic regression adjusted for demographic variables (age, sex, ethnicity) and aPL profiles.

Figure 2:

Function and potential antigen specificities of anti-NET antibodies. A, Complement C3d decorating NETs. Control neutrophils were stimulated with PMA to generate NETs, which were then incubated with serum from patients with high (top panels) anti-NET IgM or from healthy controls; scale bars=100 microns. B-D, Autoantibody microarray comparing anti-NET IgG-positive and -negative samples (C-D), and anti-NET IgM-positive and -negative samples (E). Volcano plots demonstrate (in red) antigen specificities that were significantly enriched in the anti-NET-positive groups.