2269: Anti-NET Antibodies in Antiphospholipid Antibody-positive Patients: Results from the Antiphospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking (APS ACTION) Clinical Database and Repository (“Registry”)

Yu (Ray) Zuo¹, Sherwin Navaz¹, Alex Tsodikov¹, Katarina Kmetova², Claire Hoy¹, Srilakshmi Yalavarthi¹, Danieli Castro Oliveira de Andrade³, Maria G. Tektonidou⁴, Savino Sciascia⁵, Vittorio Pengo⁶, Guillermo Ruiz-Irastorza⁷, H Michael Belmont⁸, maria gerosa⁹, Paul fortin¹⁰, Guilherme de Jesús¹¹, D. Ware Branch¹², Laura Andreoli¹³, Esther Rodriguez Almaraz¹⁴, Michelle Petri¹⁵, Ricard Cervera¹⁶, Rohan Willis¹⁷, Doruk Erkan¹⁸ and Jason S Knight¹⁹, ¹University of Michigan, Ann Arbor, MI, ²Division of Rheumatology, University of Michigan, Ann Arbor, MI, ³Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil, ⁴National and Kapodistrian University of Athens, Athens, Greece, ⁵University of Turin, Torino, Italy, ⁶Padova University Hospital, Padova, Italy, ⁷Autoimmune Diseases Research Unit, Biocruces Bizkaia Health Research Institute, Hospital Universitario Cruces, UPV/EHU, Barakaldo, Spain, ⁸NYU School of Medicine, New York, NY, ⁹University of Milan, Milano, Italy, ¹⁰CHU de Québec, Pavillon CHUL, Québec, Canada, ¹¹Universidade do Estado do Rio de Janeiro, Rio De Janeiro, Brazil, ¹²University of Utah, Salt Lake City, UT, ¹³Rheumatology and Clinical Immunology Unit, ASST Spedali Civili and University of Brescia, Brescia, Italy, ¹⁴Hospital Universitario 12 de Octubre, Madrid, Spain, ¹⁵Johns Hopkins University School of Medicine, Division of Rheumatology, Baltimore, MD, ¹⁶Hospital Clínic de Barcelona, Barcelona, Spain, ¹⁷University of Texas Medical Branch, Galveston, TX, ¹⁸Hospital for Special Surgery, New York, NY, ¹⁹University of Michigan, Division of Rheumatology, Ann Arbor, MI

Background/Purpose: The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils plays a role in the thromboinflammatory phenotype of APS. Previous work has demonstrated that some patients with APS have autoantibodies targeting NETs, which impair NET clearance and potentially activate complement. Here, we aimed to elucidate the presence, clinical associations, and antigen specificities of anti-NET antibodies in a large, ethnically diverse cohort of antiphospholipid antibody (aPL)-positive patients who did not have lupus.

Methods: Levels of anti-NET IgG/IgM were determined in sera of 308 patients with primary APS and 81 patients who were aPL positive but without "criteria" APS manifestations or other systemic autoimmune disease diagnosis. A positive cut-off for anti-NET level was established at the 99^th percentile optical density of healthy-control samples. Multivariate logistic regression with best variable model selection was used to determine clinical associations. For a subset of patients (n=214), we profiled autoantibodies with a microarray platform that included 120 potential autoantigens (66 associated with NETs based on literature mining).

Results: We found elevated levels of anti-NET IgG or IgM in 45% of aPL-positive patients, whether they met criteria for primary APS or not (Figure 1A-B). There was a strong relationship between anti-NET IgG and anti-NET IgM (r=0.52, p< 0.0001). High anti-NET antibody levels correlated with a circulating marker of NETs, myeloperoxidase (MPO)-DNA complexes (IgG r=0.12, p=0.02; IgM r=0.16, p=0.001). Anti-NET activity also demonstrated a positive correlation with levels of traditional aPL (anti-beta-2 glycoprotein I IgG: r=0.21, p< 0.0001) and anticardiolipin IgG: r=0.19, p=0.0001). When considering clinical manifestations among aPL-positive patients, positive anti-NET IgG was strongly associated with brain white matter changes after adjusting for demographic variables and criteria aPL profiles (OR=11, 95% CI 1.9 to 62), but not with other aPL-related manifestations. By immunofluorescence microscopy, we determined that high anti-NET sera more efficiently deposited complement C3d on NETs. In pursuit of the antigen specificity of anti-NET antibodies, 214 samples were analyzed by autoantibody microarray. Anti-NET IgG positivity significantly associated with the following antigens: citrullinated-histone H1 and H4, MPO-DNA complexes, nucleosomes, centromere protein A (a histone H3 variant), heparan sulfate proteoglycan, laminin, and collagen VI. Meanwhile, anti-NET IgM positivity associated with the following: single-stranded DNA, double-stranded DNA, and proliferating cell nuclear antigen.

Conclusion: In summary, these data reveal high levels of anti-NET antibodies in 45% of aPL-positive patients wherein they potentially activate the complement cascade and contribute to white matter changes. While anti-NET IgM may especially target DNA in NETs, anti-NET IgG appear more likely to target protein antigens.
Figure 1. A-B, Anti-NET IgG and IgM were measured in the indicated groups; no patients in this cohort had lupus. As compared with healthy controls, high levels of anti-NET IgG and IgM were found in aPL-positive patients with no criteria manifestations (“aPL positive”), thrombotic manifestations, or obstetric manifestations. Levels of anti-NET IgG and IgM at 450-nm optical density (OD) were compared to Control 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.
Disclosures: Y. Zuo, None; S. Navaz, None; A. Tsodikov, None; K. Kmetova, None; C. Hoy, None; S. Yalavarthi, None; D. Castro Oliveira de Andrade, None; M. Tektonidou, None; S. Sciascia, None; V. Pengo, None; G. Ruiz-Irastorza, None; H. Belmont, None; m. gerosa, None; P. fortin, None; G. de Jesús, None; D. Branch, None; L. Andreoli, GlaxoSmithKlein(GSK), Janssen, Novartis, UCB, Werfen; E. Rodriguez Almaraz, None; M. Petri, Exagen, AstraZeneca, Alexion, Amgen, AnaptysBio, Argenx, Aurinia, Biogen, Caribou Biosciences, CVS Health, EMD Serono, Eli Lilly, Emergent Biosolutions, GlaxoSmithKline (GSK), IQVIA, Janssen, Kira Pharmaceuticals, MedShr, Sanofi, SinoMab, Thermofisher, BPR Scientific Advisory Committee; R. Cervera, None; R. Willis, None; D. Erkan, None; J. Knight, Jazz Pharmaceuticals, Bristol Myers Squibb.