Neutrophil extracellular trap formation is associated with autophagy-related signalling in ANCA-associated vasculitis

Abstract

SummaryIncreasing evidence indicates that aberrant neutrophil extracellular trap (NET) formation could contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Recent research has provided evidence that a novel type of ANCA autoantibody, anti-lysosomal membrane protein-2 (LAMP-2) antibody, may have a pathogenic role in AAV. We have shown previously that anti-LAMP-2 antibody-stimulated NET formation contains autoantigens and antimicrobial peptides. The current study sought to determine whether LAMP-2, as a novel antigen of ANCA, was present on NETs in AAV patients, the influence of the anti-LAMP-2 antibody on the neutrophil apoptosis rate and the role of autophagy in anti-LAMP-2 antibodyinduced NET formation. NET formation was assessed using immunofluorescence microscopy, scanning electron microscopy or live cell imaging. The neutrophil apoptosis rate was analysed using fluorescence activated cell sorting (FACS). Autophagy was detected using LC3B accumulation and transmission electron microscopy. The results showed that enhanced NET formation, which contains LAMP-2, was observed in kidney biopsies and neutrophils from AAV patients. The apoptosis rate decreased significantly in human neutrophils stimulated with anti-LAMP-2 antibody, and this effect was attenuated by the inhibitors of autophagy 3-methyladenine (3MA) and 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002). The anti-LAMP-2 antibody-stimulated NET formation was unaffected by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) and necrostatin-1 (Nec-1), which are inhibitors of apoptosis and necrosis, respectively, but was inhibited by 3MA and LY294002. Moreover, the proportion of LC3BI that was converted to LC3BII increased significantly (P 5 0Á0057), and massive vacuolizations that exhibited characteristics typical of autophagy were detected in neutrophils stimulated with anti-LAMP-2 antibody. Our results provide further evidence that autophagy is involved in ANCAinduced NET formation in human neutrophils.

The role of neutrophils and NETosis in autoimmune and renal diseases

“…Furthermore, immunostaining of renal biopsy specimens from patients with AAV revealed the presence of NETs and NET-associated molecules in areas of inflammation, around areas of fibrinoid necrosis in kidneys with necrotizing glomerulonephritis, and along interlobular arterial walls 96,100 . These observations suggest that NET formation is involved in vascular damage and immune system activation in AAV 96,97,99,100 . Vascular inflammation could be initiated and perpetuated by ANCA-induced activation of primed neutrophils and monocytes.…”

“…Neutrophils from patients with AAV display enhanced NET formation in vitro 96,97 . In addition, levels of NET remnants, such as MPO–DNA complexes 96 , and neutrophil granular proteins, such as calprotectin (a dimer of S100-A8 and S100-A9) 98 , were increased in sera from patients with AAV.…”

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders

“…Stimulation of human neutrophils with vasculitis‐associated antibodies led to massive vacuolization, increased LC3BI degradation and could be reduced with the inhibitors of autophagy 3‐methyladenine (3MA) and LY294002 74. Similarly, LC3B containing vacuoles were observed preceding NETosis in LPS or septic plasma‐induced NETosis in human neutrophils which was also effectively inhibited by 3MA and bafilomycin A1 75.…”

NETopathies? Unraveling the Dark Side of Old Diseases through Neutrophils

“…Additionally, it was shown that neutrophils release NETs when activated with ANCA (107). Further studies confirmed the deposition of NETs in affected tissues from patients with AAV (14, 61, 108–110), whereas increased levels of circulating NET remnants were observed in patients with AAV (14, 22). Additionally, a recent study correlated AAV disease activity with the presence of NET-prone LDGs in peripheral blood (110).…”