P0340CYPD DEFICIENCY AMELIORATES AUTOIMMUNE VASCULITIS IN MOUSE MODEL VIA THE INHIBITION OF PROGRAMMED NECROSIS
Authors: Kudo Takashi , Nakazawa Daigo , Watanabe Kanako , Shiratori Satoka , Saori Nishio , Tomaru Utano , Ishizu Akihiro , Atsumi Tatsuya 
Published: June 2020

Abstract

Background and Aims
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a necrotizing vasculitis that affects systemic organs. Although the standard immunosuppressive therapy in AAV could control disease activity, these existing treatments have no effect to regulate the necrosis, which could cause the persistent organ damage. ANCA binds to neutrophils, leading to neutrophil extracellular (NETs) formation. The formed NETs induce vascular injury and become auto-antigens against ANCA, resulting in an auto-amplification loop of necrosis and inflammation. NETs work as innate immunity and simultaneously undergo programmed cell necrosis. However, the signaling pathway of ANCA-mediated NETs remains elusive. Cyclophilin D (CypD) regulates program necrosis via the opening of mitochondrial permeability transition pores (mPTP) with the consequence of mitochondrial ROS release. The Inhibition or genetic deletion of CypD has been reportedly to protect organ damage in brain and kidney ischemia-reperfusion injury mice. Herein, we hypothesized that CypD would be involved in ANCA-mediated NETs pathway and the pathogenesis of AAV.

Method
Animal experiment;
Autoimmune vasculitis was induced in 10 week-old CypD(+/+) or CypD deficient (-/-) mice by the intravenous injection with anti-mouse MPO serum. At 24 hours after injection, we evaluated the renal and lung involvements. The severity of glomerulonephritis was assessed by the levels of BUN, haematuria, histological findings and TUNEL positivity. The lung injury was evaluated by histopathological alveolar haemorrhage scores and TUNEL positivity. The mPTP related necrosis in tissues was identified by immunostaining against cytchrome C. The NETs-forming neutrophils in peripheral bloods were detected as Sytox Green (SG)-positive neutrophils by flow cytometry (FCM).
In vitro experiment;
We investigated whether CypD inhibitor and genetic ablation suppresses ANCA-mediated NETs. Healthy human neutrophils were incubated with ANCA-IgG derived from AAV patients with pre-treatment of CypD inhibitor (cyclosporine A) or PBS for 4 hours. Next, WT or CypD (-/-) mice neutrophils were cultured with anti mouse MPO antibody for 4 hours. The NETs induction was assessed as Sytox green positive neutrophil using FCM and live imaging.

Results
The amount of haematuria, histological severity including glomerulonephritis and pulmonary haemorrhage in CypD(-/-) mice significantly decreased compared to those in CypD(+/+) mice. Furthermore, TUNEL and cytochrome C positivity of tissues in CypD(-/-) mice was lower than that of CypD(+/+) mice, respectively. The level of BUN in AAV-induced CypD (-/-) mice was significantly lower than that of AAV-induced CypD(+/+) mice (92.4 ± 41.8 vs 36.8 ± 21.5 mg/ dL, p &lt;0.05). FCM analysis showed that circulating NETs and mitochondrial ROS in CypD(-/-) mice were lower than those in CypD(+/+) mice (NETs; 4.7 ± 2.4 vs 7.5 ± 2.6 %, p &lt;0.05, ROS; 1.9 ± 0.38 vs 4.7 ± 0.78%, p &lt;0.05).
In vitro experiments, CypD inhibitor suppressed ANCA-induced NETs formation (Control; 0.45 ± 0.14 vs CypD inhibitor; 0.16 ± 0.04%, p &lt;0.05). In addition, CypD(-/-) mice neutrophils-treated with ANCA showed lower NETs induction rate compared to CypD(+/+) neutrophils (CypD+/+: 4.27 ± 2.09 vs CypD-/-: 0.63 ± 0.35%, p &lt;0.05).

Conclusion
Our data indicate that the inhibition of CypD might ameliorate autoimmune vasculitis via the suppression of programmed NETs formation.