Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation
Maximilian Ebert, … , Alan D. Salama, Ralph Kettritz
Maximilian Ebert, … , Alan D. Salama, Ralph Kettritz
Published September 20, 2022
Citation Information: J Clin Invest. 2022;132(23):e160089. https://doi.org/10.1172/JCI160089.
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Clinical Research and Public Health Autoimmunity Inflammation

Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation

Abstract

Background Antineutrophil cytoplasmic autoantibody–associated (ANCA-associated) vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCAs directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils, and cause vasculitis. An imbalance between PR3 and its major inhibitor α1-antitrypsin (AAT) was proposed to underlie PR3- but not MPO-AAV. We measured AAT and PR3 in healthy individuals and patients with AAV and studied protective AAT effects pertaining to PR3- and MPO-ANCA.Methods Plasma and blood neutrophils were assessed for PR3 and AAT. WT, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, fluorescence resonance energy transfer assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring.Results We found significantly increased PR3 and AAT pools in patients with both PR3- and MPO-AAV; however, only in PR3-AAV did the PR3 pool correlate with the ANCA titer, inflammatory response, and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active patients with PR3-AAV showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when 2 critical methionines were substituted with valine and leucine.Conclusion Pathogenic differences between PR3- and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-ANCA rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.FUNDING This work was supported by KE 576/10-1 from the Deutsche Forschungsgemeinschaft, SCHR 771/8-1 from the Deutsche Forschungsgemeinschaft, grant 394046635 — SFB 1365 from the Deutsche Forschungsgemeinschaft, and ECRC grants.

Authors

Maximilian Ebert, Uwe Jerke, Claudia Eulenberg-Gustavus, Lovis Kling, Dieter Jenne, Marieluise Kirchner, Philipp Mertins, Markus Bieringer, Saban Elitok, Kai-Uwe Eckardt, Adrian Schreiber, Alan D. Salama, Ralph Kettritz

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Figure 2

PR3 and AAT are both increased in patients with active PR3- and MPO-AAV and normalize with remission.

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PR3 and AAT are both increased in patients with active PR3- and MPO-AAV ...
Plasma from HCs, and patients with active (act) and remission (rem) PR3- and MPO-AAV were analyzed for (A) plasma PR3 (n = 50, 37, 39, 22, 16 from left to right) and (B) plasma AAT (n = 50, 36, 39, 22, 16 from left to right). The normal AAT concentration between 16.2 and 30.2 μmol/L(28) is indicated by the dotted black lines. Red lines indicate the mean. (C) The plasma PR3 to AAT molar ratio was calculated (n = 50, 36, 39, 22, 16 from left to right). (D) Plasma PR3 correlation with plasma CRP in PR3- and MPO-AAV is illustrated by the red regression lines and the gray shadings indicating the 95% confident intervals. (E) Neutrophil PR3 was assessed in HC and patients with active PR3- and MPO-AAV by ELISA (n = 50, 37, 39, 22, 15 from left to right), and (F) by immunoblotting in randomly selected HCs and patients with PR3-AAV. Densitometry measurements of single PR3 bands normalized to a β-actin loading control are indicated. (G) The total PR3 blood pool (n = 50, 37, 39, 22, 15 from left to right) and (H) plasma AAT pool (n = 50, 36, 39, 22, 16 from left to right) was calculated in HCs and patients with AAV. Neutrophils from HCs and patients with AAV were stained with an anti-PR3 mAb and analyzed by flow cytometry. (I) A typical histogram illustrates the bimodal staining pattern with distinct mPR3lo and PR3hi neutrophil subsets. (J) The percentage of mPR3hi neutrophils is depicted (n = 50, 36, 37, 20, 13 from left to right). (K) The mPR3 amount is given as expression index of the MFI (MFI EI, n = 50, 36, 37, 20, 13 from left to right). (L) Spearman correlation between mPR3 amount and plasma AAT is illustrated. HC are depicted as red circles (r = –0.32, P = 0.02), all patients with PR3-AAV as black triangles (r = 0.24, P = 0.04), and all patients with MPO-AAV as gray squares (r=0.23, P = 0.21). Individual results are depicted, and the regression line for each color-coded group is given by the solid lines together with the shaded areas indicating the 95% confident intervals. 1-way ANOVA was performed with Tukey’s posthoc testing. *P < 0.05, ** P < 0.01.