Abnormal conformation and impaired degradation of propylthiouracil‐induced neutrophil extracellular traps: Implications of disordered neutrophil extracellular traps in a rat model of myeloperoxidase antineutrophil cytoplasmic antibody–associated vasculitis

Abstract

Methods. NETs were induced by treating human neutrophils with phorbol myristate acetate (PMA) in vitro. We examined whether the addition of PTU influenced the NET formation induced by PMA and the degradation of NETs by DNase I, which is regarded as a regulator of NETs. Furthermore, we examined whether NETs generated by the combination of PMA and PTU induced MPO ANCA and MPO AAV in vivo in rats.Results. When NETs were induced by PMA with PTU using human neutrophils in vitro, abnormal conformation of NETs was observed. Interestingly, the abnormal NETs were hardly digested by DNase I. Moreover, rats immunized with the abnormal NETs, which had been induced by PMA with PTU using rat neutrophils, produced MPO ANCA and developed pulmonary capillaritis. When rats were given oral PTU with intraperitoneal injection of PMA, pauci-immune glomerulonephritis and pulmonary capillaritis occurred with MPO ANCA production in the serum.Conclusion. Our findings indicate that abnormal conformation and impaired degradation of NETs induced by PTU are involved in the pathogenesis of PTU-induced MPO ANCA production and MPO AAV. These findings suggest that disordered NETs can be critically implicated in the pathogenesis of MPO AAV.

Measurement of NET formation in vitro and in vivo by flow cytometry

“…The most popular method to quantify NETs is the image analysis, which is based on fluorescent staining using DAPI 10, 16, 17. To determine the equivalence in quantification of the flow cytometric detection of neutrophils that formed NETs using SYTOX Green to the image analysis, we compared the two methods.…”

Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission

“…NETs trap and kill microbes, including bacteria, fungi and protozoa (3,(10)(11)(12)(13)(14) but have also been connected to various autoinflammatory or autoimmune diseases, such as preeclampsia, systemic lupus erythematosus (SLE), Felty´s syndrome (FS) and rheumatoid arthritis (RA) (15)(16)(17)(18)(19). NETs have also been suggested to be important in a rodent model of drug-induced vasculitis (20), implying a role of NETs in the pathogenesis of AAV as well. Further, increased levels of NETosis-derived products (or NET remnants) in the circulation have been reported in a small number of patients with active vasculitis (2).…”

NETosis: how vital is it?

“…A recent study demonstrated that propylthiouracil given to NETosing PMNs resulted in disordered NETs, which were resistant to DNase and had the ability to induce vasculitis in rodents. 109 Systemic lupus erythematosus (SLE, lupus) is a complex autoimmune disease, comprising a constellation of clinical manifestations of unknown cause. In addition to important clinical features, serological testing often reveals high levels of antinuclear antibodies and anti-double-stranded DNA antibodies.…”