Background/Purpose: Neutrophil extracellular traps (NETs) play an important role in the pathogenesis of ANCA-associated vasculitis (AAV). Sera of MPO-ANCA or PR3-ANCA positive patients can induce NETs In vitro. The present study aimed to investigate whether NET induction could serve as a biomarker in MPO- and PR3-positive AAV patients.

Methods: Healthy neutrophils were stimulated with 10% serum from 62 GPA patients, 37 MPA patients and 18 healthy subjects. NETs were stained with Sytox and imaged by automated 3D confocal laser scanning microscopy and quantified with digital image analysis. NET-inducing capacity was defined as the fold increase of quantified NETs relative to healthy control. To investigate NET induction by ANCA autoantibodies, IgG was isolated from sera using protein G agarose beads after which depletion of IgG in the flow through was confirmed with ELISA.

Results: Both GPA and MPA samples showed significantly higher NET-inducing capacity (fold change mean±SEM for GPA 40±7.4, p<0.0001 and for MPA 153.2±44.9, p<0.01). MPA sera had a significantly higher NET-inducing capacity than GPA (p<0.0001). In 14 AAV patients who had seroconverted, we observed that the NET-inducing capacity returned to levels of healthy controls (1.40±0.42, p=0.37). However, we found no correlation of NET-inducing capacity with titers of MPO and PR3 antibodies (r=0.17, p=0.35 for MPO and r=-0.04, p=0.79 for PR3). To further determine whether NET release was mediated by ANCA autoantibodies, we isolated MPO-ANCA IgG and PR3-ANCA IgG from 5 different sera. These purified IgG antibodies did not show NET-inducing capacity (1.40±0.33, p=0.45). In contrast, corresponding IgG-depleted sera had similar NET-inducing capacity as whole sera (12.91±4.5 for IgG-depleted sera and 18.88±7.26 for whole sera, p=0.51).

Conclusion: These data show that NET release in AAV patients is independent of MPO-ANCA or PR3-ANCA. Therefore, these data indicate that NET release might be a novel biomarker of AAV that is independent of ANCA autoantibodies. Our data supports the hypothesis that NETs are a source of autoantigens for the production of ANCA.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/net-inducing-capacity-is-a-biomarker-in-anca-associated-vasculitis-independent-of-anca-antibodies/