SAT0010 Excessive formation of neutrophil extracellular traps have a different role in the pathogenesis of anca-associated vasculitis and systemic lupus erythematosus
Authors: L.S. van Dam, T. Kraaij, S.W. Kamerling, H.U. Scherer, T.J. Rabelink, C. van Kooten, Y.O. Teng
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Renal involvement in ANCA-associated vasculitis (AAV) and systemic lupus erythematosus (SLE) manifests as autoimmune-mediated glomerulonephritis (AIGN). In AAV, crescentic lesions and a pauci-immune immunofluorescence is typically seen while in SLE endo- and extracapillary proliferative lesions and a full-house immunofluorescence is seen. Although these are clinically divergent autoimmune diseases, neutrophil extracellular traps (NETs) are postulated to be involved in their pathogenesis. NETs are immunogenic, extracellular DNA structures harbouring relevant ANCA- and nuclear auto-antigens. However, it is still unclear how and if NETs can act as a common pathway for both AAV and SLE. <h3>Objectives</h3> To increase our understanding of the potential pathogenic role of NETs in AAV and SLE, the aim of this study was to compare the characteristics of <i>ex vivo</i> AAV- and SLE-induced NET formation. <h3>Methods</h3> <i>Ex vivo</i> NET formation was quantified by our highly-sensitive NET quantification assay using 3D-confocal microscopy<sup>1</sup> for 82 AAV, 56 SLE patients and 10 healthy controls (HC). Live cell imaging was used to study the morphology and kinetics. Qualitative characteristics of NETs were investigated by immunofluorescence microscopy that detected co-localisation of NET-markers, including citrullinated histon-3 (CitH3) and high mobility group box-1 (HMGB1). Also, the presence of IgG, IgM or IgA autoantibodies on AAV- and SLE-induced NETs was studied. Autoantibodies as trigger of NET formation were investigated by depleting serum from IgG and NET inhibition assays were performed using peptidylarginine deiminase-4 (PAD4) and NADPH inhibitors. <h3>Results</h3> Quantifying <i>ex vivo</i> NET formation demonstrated excessive NET formation for both AAV and SLE as compared to HC. AAV-induced NET formation (median [Q1 – Q3]: 20.7 [9.6–74.1]) was significantly higher compared to SLE-induced NET formation (5.6 [2.3–14.3]; p&lt;0.0001). Secondly, live cell imaging revealed lytic NET formation in AAV peaking after 2–4 hours while in SLE non-lytic NET formation with neutrophil clustering occurred within minutes. Thirdly, the presence of CitH3 was significantly higher on AAV-induced NETs, whereas SLE-induced NETs contained significantly more HMGB1. AAV-NETs were triggered independent of IgG, in contrast to IgG dependence of SLE-NETs. Intriguingly, immunofluorescence staining of immunoglobulins revealed a pauci-immune expression on AAV-NETs compared to a full-house expression of IgG, IgM and IgA on SLE-NETs. Both PAD4 and NADPH were involved in AAV- but not in SLE-induced NET formation. To further corroborate on these differences, we found that SLE-NETs were enriched for oxidised mitochondrial DNA as demonstrated by TOMM20 and MitoSOX. <h3>Conclusions</h3> This study demonstrates that excessive NET formation in AAV is intrinsically different to NET formation in SLE. AAV-NETs are characterised by a suicidal lytic PAD4- and NADPH-dependent expulsion of citrullinated NETs, whereas SLE-NETs are characterised by rapidly-induced clusters with HMGB1, enriched for mitochondrial DNA and enhanced immune complex formation altogether supporting a pro-inflammatory role of NETs in the pathophysiology of SLE, including immune-complex mediated, full-house lupus nephritis. <h3>Reference</h3> [1] Kraaij T, et al. Autoimmunity reviews2016. <h3>Disclosure of Interest</h3> None declared