125. Endothelial cell ferroptosis promotes renal damage in ANCA-induced glomerulonephritis
Creators: name: Rousselle1, Anthony
name: Doerte Lodka1
name: Sonnemann1, Janis
name: Kettritz1, Ralph
name: Schreiber1, Adrian
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases characterized by inflammation of small blood vessels and organ damage, including necrotizing and crescentic glomerulonephritis (NCGN). ANCA are circulating immunoglobulin G (IgG) autoantibodies directed against either myeloperoxidase (MPO) or proteinase 3 (PR3), both antigens being expressed by neutrophil granulocytes and monocytes. ANCA bind to these surface antigens and provoke the activation of both myeloid cell subsets. The persistent inflammation leads to endothelial and renal cell necrosis. In contrast to apoptosis, necrosis has long been thought to be an uncontrolled event. However, this dogma has been challenged over the last decade. Ferroptosis is a new type of programmed cell death characterized by the production of ROS- and iron-dependent lipid peroxidation leading to membrane rupture. The Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) enzyme play a crucial role in the generation of lipid peroxides. Ferroptosis has been described to play a role during acute kidney injury. Our work aimed to investigate the contribution of ferroptosis in endothelial cell death during ANCA-associated NCGN. <strong>Methods:</strong> To analyse the biological significance of ferroptosis in endothelial cells <em>in vivo</em>, we bred ACSL4<sup>lox/lox</sup> mice with transgenic mice expressing the Cre recombinase under the control of the endothelial Tie2 promoter to obtain ACSL4<sup>∆EC</sup> mice. These mice were further crossed with myeloperoxidase<sup>-/-</sup> (MPO<sup>-/-</sup>) mice to generate MPO<sup>-/-</sup> ACSL4<sup>∆EC </sup>chimeric mice. These animals were immunized with murine MPO, irradiated and subsequently transplanted with hematopoietic cells from C/57BL6J (WT) mice. Mice were sacrificed and analysed 6-8 weeks following transplantation. Ferroptosis was investigated <em>in vitro</em> using human umbilical vein endothelial cells and ANCA-stimulated neutrophils. Cell death and lipid peroxidation were detected by flow cytometry. Ferrostatin-1 (Fer-1) and siRNA against ACSL4 were used to inhibit ferroptosis. <strong>Results:</strong> We found increased lipid peroxidation (4-HNE staining) in kidney section of mice with AAV. MPO<sup>-/-</sup> ACSL4<sup>∆EC </sup>chimeric mice were protected from the development of renal damage compared to WT chimeric mice suggesting that ferroptosis in endothelial cells play an important role in the development of ANCA-induced glomerulonephritis. <em>In vitro</em>, we found that ANCA-activated neutrophils induce endothelial cell death and this effect was prevented by inhibition of ferroptosis with Fer-1 and siRNA against ACSL4. In contrast, inhibition of either necroptosis or apoptosis did not have any effects. In addition, ferroptosis inhibition alleviated the accumulation of lipid peroxides and endothelial dysfunction induced by ANCA-activated neutrophils. <strong>Conclusions</strong>: ANCA-activated neutrophils induce ferroptosis in endothelial cells <em>in vitro</em>. Ferroptosis is induced in endothelial cell in vivo and play a major role in the development of vasculitis and ANCA-associated renal damage. Further experiments are warranted to decipher the mechanism(s) by which ferroptosis regulate the development of AAV. Altogether, our data suggest that ferroptosis is an important mediator in the development of AAV and might represent a novel interesting therapeutic target. <strong>Disclosures:</strong> none