Aberrant oxidative modifications of neutrophil myeloperoxidase in anti-neutrophil cytoplasmic antibody-associated vasculitis

Abstract

Anti-neutrophil cytoplasmic antibodies directed to myeloperoxidase (MPO-ANCA) are key molecules in the pathogenesis of ANCA-associated vasculitis (AAV), however, the mechanisms of autoantibody production have not been elucidated. We hypothesized that an aberrant PTM occurs in the MPO of MPO-ANCA-positive AAV (MPO-AAV), which induces immune responses to self MPO. To test this, we purified MPO proteins from neutrophils of 8 patients with MPO-AAV and 8 healthy subjects, digested them with trypsin, and comprehensively quantified PTMs of the MPO peptides using the sequential window acquisition of all theoretical fragment ion spectra (SWATH) method of LC-MS. Among the 1034 detected MPO peptides, 38 peptides were increased in the patients with MPO-AAV relative to the healthy subjects, whereas 10 peptides were decreased in the patients (p < 0.05). Interestingly, oxidative modifications were found in 11 of the 38 increased peptides (1.14- to 3.29-fold), but not in the decreased peptides. These included oxidation of Met577, Phe686, Met688 and Met719, dioxidation of Met409, Phe605, Trp679 and Met719, and kynurenylation of Trp255. Conversely, glycosylation was detected in 4 of the 10 decreased peptides (−1.32- to −2.32-fold), but not in the increased peptides. They were O-type glycans at Ser357 and Ser731, and N-type glycans at Asn355 and Asn729. In animal experiments, immunization of mice with in vitro oxidized or unoxidized mouse MPO (mMPO) showed that not only anti-oxidized mMPO antibodies but also anti-unoxidized mMPO antibodies were preferentially produced in the oxidized mMPO-immunized mice relative to the unoxidized mMPO-immunized mice (anti-oxidized mMPO antibodies, 6/8 vs 1/9, p < 0.05; anti-unoxidized mMPO antibodies, 4/8 vs 0/9, p < 0.05). Our results suggest that the increased oxidative modifications of MPO in MPO-AAV may break immune tolerance and trigger the MPO-ANCA production.

Significance

AAV is a systemic and refractory disease that causes life-threatening multi-organ involvement such as necrotizing glomerulonephritis and lung hemorrhage. MPO-ANCA is an autoantibody that plays a key role in the pathogenesis of AAV. Therefore, elucidation of the mechanism of MPO-ANCA production is crucial to overcoming this disease. In this study, we applied a SWATH-MS analysis to the detection of aberrant PTMs, and found increased oxidative modifications of neutrophil MPO in patients with MPO-AAV for the first time. Immunization of in vitro oxidized MPO induced autoantibodies to the intact unoxidized MPO, suggesting that the increased oxidative modifications of MPO may break the immune tolerance in MPO-AAV. This study suggests a novel trigger mechanism for MPO-ANCA production.