126. Mitochondrial Reactive Oxygen Species Inhibition Attenuates Neutrophil Extracellular Trap Formation And Inflammation In MPO-ANCA Vasculitis
Creators: name: O'Sullivan1, Kim Maree
name: CaoLe1, Anne
name: Cheong1, Daniel K.Y.
name: Poh-Yi Gan1
name: Stephen Holdsworth1, 2
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> A key feature of myeloperoxidase anti-neutrophil cytoplasmic antibody associated (ANCA) vasculitis (MPO-AAV) is the observation of neutrophil extracellular traps (NETs) in the glomerular lesions of kidney biopsies. NETs are a unique form of cell death that release highly injurious proinflammatory mediators that are destructive to nearby tissue. The DNA back bone of the NET acts as a reservoir which biologically enhances the activity of myeloperoxidase the autoantigen in MPO-AAV. NETs contain highly proinflammatory mtDNA, mitochondrial reactive oxygen species (mtROS) and histones which contribute to inflammation. Oxidative stress is a key inducer of NET formation mediated by (ROS). These studies aim to investigate the therapeutic potential of a mtROS inhibitor SKQ1 to inhibit NET formation and reduce inflammation in an experimental model of MPO-AAV, without impairing host defence. <strong>Methods</strong>: C57Bl/6 mice were used to isolate peritoneal thioglycolate elicited neutrophils. Neutrophils were stimulated to NET <em>in vitro </em>with either ANCA, LPS, PMA or Ionomycin (to stimulate all pathways to NET formation) and compared to SKQ1 treated neutrophils. Confocal imaging was used to analyse NET formation and contents (MPO, citrullinated histones, Mitotracker, MitoSox, and Sytox green). <em>In vitro</em> Zymosan phagocytosis assays were used to investigate if SKQ1 interfered with neutrophil phagocytosis. Experimental anti-MPO GN was induced in C57Bl/6 mice by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin (anti GBM Ig). Therapeutic intervention with (SKQ1 daily <em>n</em>=8 or vehicle <em>n</em>=8) was administered after the establishment of MPO autoimmunity until termination of the experiment. <strong>Results:</strong> <em>In vitro</em> NET formation was significantly attenuated with SKQ1 regardless of NET stimuli (all, P&gt;0.05, compared to control). Confocal microscopy demonstrated that NETs contained large quantities of mtDNA, and mtROS colocalised with MPO. <em>In vitro</em> nanomolar concentrations of SKQ1 inhibited NET formation without interfering with neutrophil phagocytosis via zymosan phagocytosis assays. SKQ1 <em>in vivo</em> in an experimental model of MPO-AAV was able to significantly reduce inflammatory cells recruited to the glomerulus with a reduction in neutrophils, CD4 T cells, CD8 T cells and macrophage influx (all, P &lt;0.05 compared to vehicle control). SKQ1 significantly reduced glomerular segmental necrosis, autoimmunity to MPO [measured by delayed type hypersensitivity reaction (DTH)] and MPO specific T cells from the draining lymph nodes (all, P&gt;0.05). SKQ1 significantly attenuated the number of glomerular NETs and extracellular deposition of MPO when compared to the control group. <strong>Conclusions:</strong> SKQ1 therapeutically targets NET formation without impairing neutrophil bactericidal killing capacity <em>in vitro. In vivo </em>administration of SKQ1 prevented NET formation and attenuated inflammation in an experimental model of MPO-AAV. This data provides strong pre-clinical evidence that SKQ1 could have potential for clinical translation. <strong>Disclosures:</strong> No disclosures.