Abstract: TH-OR064

Ex Vivo Induced Neutrophil Extracellular Traps Are Intrinsically Different in ANCA-Associated Vasculitis- and Systemic Lupus Erythematosus

Session Information

Category: Glomerular

  • 1001 Glomerular: Basic/Experimental Immunology and Inflammation

Authors

  • van Dam, Laura Sophie, LUMC, Leiden, Netherlands
  • Kraaij, Tineke, LUMC, Leiden, Netherlands
  • Kamerling, Sylvia, LUMC, Leiden, Netherlands
  • Scherer, Hans Ulrich, LUMC, Leiden, Netherlands
  • Pusey, Charles D., Imperial College London, London, United Kingdom
  • Rabelink, Ton J., LUMC, Leiden, Netherlands
  • van Kooten, Cees, LUMC, Leiden, Netherlands
  • Teng, Yoe Kie Onno, LUMC, Leiden, Netherlands
Background

Neutrophil extracellular traps (NETs) are immunogenic, extracellular DNA structures that harness important autoantigens to be recognized by the adaptive immune system. NETs are thought to play a pivotal role in the pathogenesis of AAV and SLE. However it is still unclear how and if NETs act as a common pathway in the pathophysiology of these clinically divergent autoimmune diseases. The aim of the present study is to characterize AAV- and SLE-induced NETs.

Methods

Healthy neutrophils were stimulated with 10% serum of AAV (n=101) and SLE (n=59) patients to induce NETs. Ex vivo NET induction by serum and IgG-depleted serum was measured by a novel, highly-sensitive NET quantification assay using 3D-confocal microscopy. Qualitative characteristics of NETs were studied by immunofluorescence to detect NET-related auto-antigens. Additionally, the morphology and kinetics of AAV- and SLE-induced NETs were visualized by live cell imaging and electron microscopy.

Results

Ex vivo NET induction by AAV sera was 20.74 [9.56 – 74.14], (median [Q1 - Q3]) fold higher than sera of healthy controls (n=10) and also significantly higher than NET induction by SLE sera 5.02 [1.88 – 14.33]). Depletion of IgG from serum did not reduce NET induction in AAV, whereas it was significantly decreased in SLE. Colocalisation of NET-related auto-antigens was different: citrullinated histon-3 (CitH3) was predominantly found on AAV-induced NETs, whereas high mobility group box protein-1 (HMGB1) was exclusively found on SLE-induced NETs. Live cell imaging demonstrated that the kinetics of SLE-induced NETs peaked at 30 minutes, while AAV-induced NETs peaked at 4 hours. Intriguingly, SLE sera induced immediate clustering of neutrophils surrounding NETs whereas AAV sera induced NETs composed of long, thin DNA-fibres through lytic expulsion.

Conclusion

We demonstrate distinct features of ex vivo AAV- and SLE-induced NETs, indicating that NET formation in AAV and SLE is based on different mechanisms. Future studies should be directed at unravelling how different NETs are involved in causing SLE- or AAV-associated glomerulonephritis.