123. Methionine oxidation compromises protective AAT effects in PR3-ANCA vasculitis
Creators: name: Ebert1, Maximilian
name: Jerke1, Uwe
name: Eulenberg-Gustavus1, Claudia
name: PD Dieter Jenne2
name: Kirchner3, Marieluise
name: Mertins3, Philipp
name: Bieringer4, Markus
name: Saban Elitok5
name: Kai-Uwe Eckardt6
name: Adrian Schreiber1, 6
name: Salama7, Alan
name: Ralph Kettritz1, 6
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> PR3 is a major autoantigen in ANCA-associated vasculitis (AAV) and a neutrophil serine protease (NSP) inhibited by alpha1-antitrypsin (AAT). Increased PR3 and decreased AAT were suggested as disease mechanisfor PR3- but not MPO-AAV. We systematically assessed PR3 and AAT in AAV patients and healthy controls (HC), characterized mechanistic interactions between PR3 and AAT, and identified disease-related modifiers of this interaction. <strong>Methods:</strong> We measured PR3 and AAT in 110 AAV patients and 51 HC and explored correlations with clinical parameters. We produced recombinant wild-type (wt)- and mutant (mu)-AAT to study the interaction of AAT with PR3, including membrane-presented PR3 (mPR3) on the neutrophil surface by FRET assay, flow cytometry, surface plasmon resonance measurements (SPR), and neutrophil superoxide release. Plasma AAT from AAV patients and AAT exposed to ANCA-activated neutrophils were analyzed by quantitative targeted mass spectrometry, namely Parallel Reaction Monitoring (PRM) to detect unmodified and oxidized AAT variants. <strong>Results:</strong> We found that the PR3 and AAT pools were increased in both active PR3- and MPO-AAV. The increased PR3 pool correlated with markers of inflammation (CRP), kidney injury (creatinine, erythrocyturia), and autoimmunity (ANCA ELISA), in PR3- but not MPO-AAV patients. In HC, plasma AAT showed an inverse correlation with neutrophil mPR3 that was not observed in AAV patients. Mechanistically, wt-AAT dose-dependently displaced PR3 from the neutrophil membrane by competing with the PR3-presenting CD177 receptor for PR3 binding thereby reducing neutrophil activation by PR3- but not MPO-ANCA. Neutrophil mPR3 depletion was less efficient when using active PR3-AAV patient plasma compared to remission and HC plasma at the same AAT concentration. We identified AAV-related plasma factors that modified mPR3, AAT, and the PR3:AAT interaction. Compared to HC and remission PR3-AAV, plasma AAT from active PR3-AAV patients showed increased methionine (Met) oxidation at Met residues 351 and 358 in the reactive center loop that are critical for the efficient encounter of mature NSPs. This oxidative modification was recapitulated by AAT exposure to ANCA-activated neutrophils <em>in vitro</em>. Oxidized AAT did not interact with PR3 and did not reduce the proteolytic PR3 activity, mPR3 on neutrophils, or neutrophil activation by PR3-ANCA. Conclusions: PR3 and AAT are increased in active PR3- and MPO-AAV patients. The increased PR3 pool correlates with inflammation, kidney injury, and autoimmunity in PR3- but not MPO-AAV patients. AAT controls mPR3 and thereby PR3- but not MPO-ANCA induced neutrophil activation. We identified AAV-related factors that modify mPR3, AAT, and the PR3:AAT interaction. <strong>Disclosures:</strong> No disclosures with relevance to this study