Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA

Neutrophil extracellular traps (NETs) are auto-antigenic strands of extracellular DNA covered with myeloperoxidase (MPO) and proteinase3 (PR3) that can be a source for the formation of anti-neutrophil cytoplasmic autoantibodies (ANCAs). The presence of NETs was recently demonstrated in renal tissue of patients with ANCA-associated vasculitis (AAV). NET formation was enhanced in AAV, suggesting that MPO-ANCA could trigger NET formation, supporting a vicious circle placing NETs in the center of AAV pathogenesis. Here we investigated NET formation in 99 patients with AAV by a novel highly sensitive and automated assay. There was a significant excess of ex vivo NET formation in both MPO-ANCA– and PR3-ANCA–positive patients with AAV compared to healthy individuals. Excessive NET formation did not correlate with serum ANCA levels. Likewise, immunoglobulin G depletion had no effect on excessive NET formation in patients with AAV, indicating an ANCA-independent process. Next, we explored the relation of excessive NET formation to clinical disease in ten patients with AAV and showed that excessive NET formation was predominantly found during active disease, more so than during remission. Excessive NET formation was found in patients with AAV hospitalized for disease relapse but not during severe infection. Thus, excessive NET formation in AAV is independent of ANCA, and an excess of ex vivo NET formation was related to active clinical disease in patients with AAV and a marker of autoimmunity rather than infection. Neutrophil extracellular traps (NETs) are auto-antigenic strands of extracellular DNA covered with myeloperoxidase (MPO) and proteinase3 (PR3) that can be a source for the formation of anti-neutrophil cytoplasmic autoantibodies (ANCAs). The presence of NETs was recently demonstrated in renal tissue of patients with ANCA-associated vasculitis (AAV). NET formation was enhanced in AAV, suggesting that MPO-ANCA could trigger NET formation, supporting a vicious circle placing NETs in the center of AAV pathogenesis. Here we investigated NET formation in 99 patients with AAV by a novel highly sensitive and automated assay. There was a significant excess of ex vivo NET formation in both MPO-ANCA– and PR3-ANCA–positive patients with AAV compared to healthy individuals. Excessive NET formation did not correlate with serum ANCA levels. Likewise, immunoglobulin G depletion had no effect on excessive NET formation in patients with AAV, indicating an ANCA-independent process. Next, we explored the relation of excessive NET formation to clinical disease in ten patients with AAV and showed that excessive NET formation was predominantly found during active disease, more so than during remission. Excessive NET formation was found in patients with AAV hospitalized for disease relapse but not during severe infection. Thus, excessive NET formation in AAV is independent of ANCA, and an excess of ex vivo NET formation was related to active clinical disease in patients with AAV and a marker of autoimmunity rather than infection. Activated neutrophils have been shown to release neutrophil extracellular traps (NETs), a novel antimicrobial mechanism coexisting with the classical and well-known enzyme degranulation and reactive oxygen species (ROS) production. NETs are net-like structures extruded by neutrophils and consist of decondensed DNA with nuclear and granular proteins, such as histones, proteinase 3 (PR3), and myeloperoxidase (MPO)1Brinkmann V. Reichard U. Goosmann C. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Google Scholar. They can be released upon stimulation with pathogens,1Brinkmann V. Reichard U. Goosmann C. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Google Scholar, 2Pilsczek F.H. Salina D. Poon K.K.H. et al.A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.J Immunol. 2010; 185: 7413-7425Google Scholar, 3Urban C.F. Ermert D. Schmid M. et al.Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.PLoS Pathog. 2009; 5e1000639Google Scholar, 4McCormick A. Heesemann L. Wagener J. et al.NETs formed by human neutrophils inhibit growth of the pathogenic mold Aspergillus fumigatus.Microbes Infect. 2010; 12: 928-936Google Scholar immune complexes,5Behnen M. Leschczyk C. Möller S. et al.Immobilized immune complexes induce neutrophil extracellular trap release by human neutrophil granulocytes via FcγRIIIB and Mac-1.J Immunol. 2014; 193: 1954-1965Google Scholar, 6Chen K. Nishi H. Travers R. et al.Endocytosis of soluble immune complexes leads to their clearance by FcgammaRIIIB but induces neutrophil extracellular traps via FcgammaRIIA in vivo.Blood. 2012; 120: 4421-4431Google Scholar and chemical compounds and are able to trap and kill pathogens.1Brinkmann V. Reichard U. Goosmann C. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Google Scholar, 7Khandpur R. Carmona-Rivera C. Vivekanandan-Giri A. et al.NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.Sci Transl Med. 2013; 5: 178ra40Google Scholar, 8Kessenbrock K. Krumbholz M. Schönermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Google Scholar Although NETs clearly act as an important first-line immune defense mechanism, they express potential autoantigens and have therefore been implicated in the pathogenesis of ANCA-associated vasculitis (AAV). Anti-neutrophil cytoplasmic antibodies (ANCAs) against MPO and PR3 have been shown to induce NET release,8Kessenbrock K. Krumbholz M. Schönermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Google Scholar, 9Nakazawa D. Shida H. Tomaru U. et al.Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.J Am Soc Nephrol. 2014; : 1-8Google Scholar and NETs are present in kidney biopsies of AAV patients.8Kessenbrock K. Krumbholz M. Schönermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Google Scholar, 10O’Sullivan K.M. Lo C.Y. Summers S.A. et al.Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.Kidney Int. 2015; 88: 1030-1046Google Scholar Furthermore, in MPO-ANCA–positive AAV a reduced degradation of NETs has been observed, suggesting that ANCAs arise from a possible break of tolerance against NET-derived antigens such as MPO and PR3.9Nakazawa D. Shida H. Tomaru U. et al.Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.J Am Soc Nephrol. 2014; : 1-8Google Scholar The latter was further corroborated by showing that myeloid dendritic cells (mDCs) loaded with extracellular DNA derived from NETotic neutrophils led to MPO-ANCA and PR3-ANCA production with concomitant vasculitis-like renal lesions in lpr/lpr mice.11Sangaletti S. Tripodo C. Chiodoni C. et al.Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity.Blood. 2012; 120: 3007-3018Google Scholar In addition, the pathogenicity of NETs was demonstrated by NET-mediated damage to the vascular endothelium and its surrounding tissues.12Saffarzadeh M. Juenemann C. Queisser M.A. et al.Neutrophil extracellular traps directly induce epithelial and endothelial cell death: A predominant role of histones.PLoS One. 2012; 7Google Scholar, 13Villanueva E. Yalavarthi S. Berthier C.C. et al.Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.J Immunol. 2011; 187: 538-552Google Scholar, 14Nakazawa D. Kumar S.V. Marschner J. et al.Histones and neutrophil extracellular traps enhance tubular necrosis and remote organ injury in ischemic AKI.J Am Soc Nephrol. 2017; 28: 1753-1768Google Scholar Lastly, levimasole has recently been shown to induce NETs resulting in drug-induced systemic vasculitis strongly resembling AAV.15Lood C. Hughes G.C. Neutrophil extracellular traps as a potential source of autoantigen in cocaine-associated autoimmunity.Rheumatology (Oxford). 2016; 56: 638-643Google Scholar Taken together, these studies provide evidence for a central role for NETs in the pathophysiology of AAV. Supported by the accumulating evidence that NETs are important to the initiation and perpetuation of vasculitis, the present study applied a newly developed, highly sensitive, and automated assay16Kraaij T. Tengström F.C. Kamerling S.W.A. et al.A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.Autoimmun Rev. 2016; 15: 577-584Google Scholar to quantify ex vivo NET formation in PR3- and MPO-ANCA–positive AAV patients to study the interaction between ANCAs, NET formation, and clinical disease in AAV. Upon stimulation with serum from AAV patients, the formation of NETs was visualized by the expression of NET-specific citrullinated histon3 (citH3) and neutrophil elastase (NE) on Hoechst-positive extracellular DNA from healthy neutrophils (Figure 1a). Additionally, inhibition of reduced NAD phosphate oxidase with diphenyleneiodonium (DPI) and peptidyl arginine deiminase (PAD) with Cl-amidine confirmed that AAV-induced extracellular DNA were part of NETs: co-incubation with DPI demonstrated a significant reduction of AAV-associated NET induction (mean reduction ± SEM: 61% ± 10%, P < 0.01; Figure 1b) as well as co-incubation with Cl-amidine, which led to a significant reduction of AAV-derived NET formation (mean reduction ± SEM: 40% ± 11%, P < 0.05; Figure 1c). Ex vivo NET formation was determined in a large cohort of AAV patients positive for anti-PR3 or anti-MPO. To measure NET formation by serum from AAV patients, we used a novel, highly sensitive, and automated method.16Kraaij T. Tengström F.C. Kamerling S.W.A. et al.A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.Autoimmun Rev. 2016; 15: 577-584Google Scholar Experimental studies supporting the assay’s approach to use healthy neutrophils as the basis to quantify excessive NET formation are summarized in Supplementary Figure S1A–C. As such, Figure 2a shows excessive NET formation in PR3-ANCA–positive patients (median fold induction ± interquartile range: 13.5 ± 52) as well as MPO-ANCA–positive patients (38.8 ± 160.1) compared with healthy controls. The difference between MPO-ANCA– and PR3-positive ANCA patients was statistically significant (P < 0.01). Unexpectedly, neither PR3-ANCA titers (R = –0.17, P = 0.19; Figure 2b) nor MPO-ANCA titers (R = 0.22, P = 0.21; Figure 2c) correlated with the amount of NET formation. NET formation was not found to correlate with serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, or IL-8 (data not shown) or classical markers of inflammation (i.e., C-reactive protein and erythrocyte sedimentation rate ; Supplementary Figure S2). To investigate whether ANCA IgG was responsible for the excessive NET formation by AAV serum, we next depleted IgG, and later also IgA, from serum of 10 PR3-ANCA–positive patients, 5 MPO-ANCA–positive patients, and 3 healthy controls. Figure 3a summarizes the serum characteristics demonstrating IgG depletion, including depletion of ANCA IgG. Also, after elution of IgG from the depleting beads, IgG including PR3-ANCA and MPO-ANCA were retrieved. Figure 3b illustrates for 1 representative patient that despite IgG depletion, excessive NET formation remained present and IgG alone was not able to induce NET formation. Quantifying all patients together, for PR3-ANCA–positive patients we observed no significant difference in NET formation after IgG depletion (mean ± SEM fold induction: 8.1 ± 1) compared with whole serum (9.3 ± 2.4; P = 0.60). For MPO-ANCA–positive patients we also observed no significant difference in NET formation after IgG depletion (18.3 ± 5.4) compared with whole serum (25.9 ± 6; P = 0.06). Moreover, isolated IgG from PR3-ANCA–positive patients was unable to induce NET formation (fold induction ± SEM: 1.5 ± 0.4 for 250 μg/ml and 1.3 ± 0.6 for 25 μg/ml), and neither was isolated IgG from MPO-ANCA–positive patients (1.4 ± 0.7 for 250 μg/ml and 1.1 ± 0.3 for 25 μg/ml) (Figure 3c and d). To exclude that IgA-derived ANCA was responsible for the observed NET formation in IgG-depleted sera, IgA was subsequently depleted. IgA depletion was confirmed with enzyme-linked immunosorbent assay (ELISA). No significant difference was found after both IgA and IgG depletion (mean fold induction ± SEM: 17 ± 5.5) compared with corresponding serum samples (20.2 ± 10.1; P = 0.74; Figure 3e and f). Next, we investigated whether upregulation of PR3 and MPO expression on neutrophils could influence NET formation by ANCA IgG. To do so, neutrophils were primed with TNF-α, after which PR3 and MPO upregulation was confirmed by flow cytometry (Figure 4a). Incubation of unprimed and TNF-α-primed neutrophils with isolated IgG from AAV patients did not show a significant difference: using 25 ug/ml IgG, the mean fold NET induction ± SEM in unprimed cells was 1.5 ± 0.5 versus 1.5 ± 0.3 (P = 0.69) in primed cells. Also, when increasing IgG concentration to 250 ug/ml, the mean fold NET induction ± SEM in primed cells (2.4 ± 0.4) was not significantly different from that in unprimed cells (2.8 ± 1.2, P = 0.67). As a positive control, NET formation was observed with whole serum from AAV patients before and after IgG depletion in unprimed as well as primed cells (Figure 4b). Because neutrophils of AAV patients are activated through C5a receptor triggering,17Xing G.Q. Chen M. Liu G. et al.Complement activation is involved in renal damage in human antineutrophil cytoplasmic autoantibody associated pauci-immune vasculitis.J Clin Immunol. 2009; 29: 282-291Google Scholar, 18Wang H. Wang C. Zhao M.H. et al.Neutrophil extracellular traps can activate alternative complement pathways.Clin Exp Immunol. 2015; 181: 518-527Google Scholar we investigated whether complement activation had a role in excessive NET formation in AAV. Therefore, we pre-incubated healthy neutrophils with a C5aR antagonist, after which NET formation was induced with serum from ANCA-positive AAV patients. Figure 5a shows that excessive NET formation was independent of C5aR inhibition (mean ± SEM of 11 ± 3.8 without and 12.1 ± 3.6 with C5aR inhibition). Next, we pre-incubated serum from ANCA-positive AAV patients with eculizumab, a C5 inhibitor, after which NETs were induced in healthy neutrophils. Neutralization of C5 by 100 ug/ml eculizumab was functionally confirmed (Supplementary Figure S3); however, the abrogation of activated complement with eculizumab did not result in a significant change in NET formation (mean ± SEM of 11 ± 3.8 without and 13.5 ± 4.5 with 10 μg/ml eculizumab and 13.4 ± 4 with 100 μg/ml eculizumab) (Figure 5b). From these data it can be concluded that activated complement pathways were not involved in NET formation in AAV. Because NET formation appeared to be independent of the presence of circulating ANCA, a well-established biomarker in AAV, we explored whether excessive NET formation related to clinical disease in AAV. We found excessive NET formation in paired samples from 10 AAV patients during active disease (mean fold induction ± SEM was 9.8 ± 2.5), which was significantly less when disease was in remission (2.2 ± 0.3, P < 0.01), suggesting excessive NET formation associated with clinical disease activity (Figure 6a). Moreover, at time of hospitalization of patients with an established AAV diagnosis, we observed a significant excess in NET formation (mean fold induction ± SEM of 10 ± 3.2) in patients who were admitted with relapsing AAV disease compared with patients who were admitted with a severe infection (2.9 ± 0.5; P < 0.05) (Figure 6b). Details of the clinical characteristics and infectious events are summarized in Supplementary Table S1. Taken together, we observed that excessive NET formation was associated with clinical disease activity in AAV patients and possibly distinguished autoimmunity from infection. This study demonstrated that excessive NET formation in a large cohort of MPO-ANCA– and PR3-ANCA–positive AAV patients was independent of the presence of PR3- and MPO-ANCA. Additionally, we observed that excessive NET formation was predominantly seen in AAV patients during active disease rather than in remission or during infection. Thus, the present study conceptualized measuring excessive NET formation in AAV patients, which could be instrumental to increase our understanding of NETs in relation to clinical disease. Although several studies have previously described NET formation in MPO-ANCA–positive patients,8Kessenbrock K. Krumbholz M. Schönermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Google Scholar, 9Nakazawa D. Shida H. Tomaru U. et al.Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.J Am Soc Nephrol. 2014; : 1-8Google Scholar the present study is the first to describe excessive NET formation in a large number of PR3-ANCA–positive patients. In contrast to the pioneering studies on NETs in AAV, we demonstrate by using a nonbiased automated quantification method of NETs that ANCAs do not contribute to the induction of NETs in AAV. Thus far, it was a matter of debate whether ANCAs mediated NET formation in AAV because Söderberg et al.19Söderberg D. Kurz T. Motamedi A. et al.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.Rheumatology (Oxford). 2015; 54: 2085-2094Google Scholar demonstrated that circulating NET remnants were higher in AAV patients with an inverse correlation to serum ANCA levels in AAV patients in remission and a negative correlation in active MPO-ANCA–positive patients. Additionally, ANCA-negative patients showed higher levels of circulating NET remnants compared with ANCA-positive patients, supporting the notion that mechanisms other than ANCA might be responsible for excessive NET formation in AAV. Until now, methods for NET quantification (e.g., using fluorescence microscopy8Kessenbrock K. Krumbholz M. Schönermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Google Scholar or ELISA measurements of NET components8Kessenbrock K. Krumbholz M. Schönermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Google Scholar, 19Söderberg D. Kurz T. Motamedi A. et al.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.Rheumatology (Oxford). 2015; 54: 2085-2094Google Scholar, 20Wang H. Sha L.L. Ma T.T. et al.Circulating level of neutrophil extracellular traps is not a useful biomarker for assessing disease activity in antineutrophil cytoplasmic antibody-associated vasculitis.PLoS One. 2016; 11: 1-10Google Scholar, 21Surmiak M. Hubalewska-Mazgaj M. Wawrzycka-Adamczyk K. et al.Neutrophil-related and serum biomarkers in granulomatosis with polyangiitis support extracellular traps mechanism of the disease.Clin Exp Rheumatol. 2016; 34: S98-S104Google Scholar) incorporated some limitations, such as the inability to dissect in vivo NET formation from NET degradation and the inability to correct for extracellular DNA derived from other death pathways (such as necrosis), and depended upon the relatively low-frequency expression of specific markers such as MPO or citH3. The method used in this study overcame these issues by using automated laser scanning microscopy, enabling us to detect and quantify large series of imaged neutrophils, optimizing the reliability and reproducibility of the amount of NET released by neutrophils.16Kraaij T. Tengström F.C. Kamerling S.W.A. et al.A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.Autoimmun Rev. 2016; 15: 577-584Google Scholar A potential limitation of our assay was the use of healthy neutrophils instead of (autologous) neutrophils from AAV patients. Besides the obvious challenge to perform an NET quantification with freshly obtained AAV patients, we addressed this issue of intrinsic differences between healthy neutrophils compared with AAV neutrophils in 2 ways. First, we observed that spontaneous NET release in AAV-derived neutrophils was not significantly different from healthy neutrophils (Supplementary Figure S1A). Second, with respect to quantifying ex vivo NET formation, we observed no significant differences in the excessive NET formation induced by serum from AAV patients (Supplementary Figure S1B). These studies supported the approach used for the reported NET assay to use healthy neutrophils as the basis to quantify serum factors that induce excessive NET formation. Thus, our study provided unique, novel insights in the dynamics of excessive NET formation in AAV, notably that it was independent of ANCAs. Because our study could exclude ANCA-IgA or complement activation as a potential factor to induce NET formation in AAV, future research will be directed at identifying the exact molecular triggers and pathways underpinning this process. Because we observed higher NET formation during active disease than during remission, it is tempting to speculate that excessive NET formation is induced by a combination of pro-inflammatory cytokines and/or damage-associated molecular patterns. In recent years, additional molecules have been described to be enhanced in AAV, including calprotectin,22Ehrchen J.M. Sunderkötter C. Foell D. et al.The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer.J Leukoc Biol. 2009; 86: 557-566Google Scholar GM-CSF, and others.23Monach P.A. Warner R.L. Tomasson G. et al.Serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ANCA-associated vasculitis.Ann Rheum Dis. 2013; 72: 1342-1350Google Scholar For future research, it will be critical to investigate these factors, either alone or in combination, in relation to their effect on NET formation. ANCAs play an important role in the pathogenesis of ANCA-associated vasculitis.24Jennette J.C. Falk R.J. Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease.Nat Rev Rheumatol. 2014; 10: 463-473Google Scholar In vitro, ANCAs can activate neutrophils via Fcγ receptors and binding of ANCA to the ANCA target antigens, leading to the release of reactive oxygen species (ROS) and granule enzymes,25Falk R.J. Terrell R.S. Charles L. et al.Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro.Proc Natl Acad Sci U S A. 1990; 87: 4115-4119Google Scholar mediating damage to the endothelium and inducing more neutrophil activation via complement activation. In addition, AAV patients have higher availability of ANCA target antigens on the resting neutrophil membrane.26Schreiber A. Luft F.C. Kettritz R. Membrane proteinase 3 expression and ANCA-induced neutrophil activation.Kidney Int. 2004; 65: 2172-2183Google Scholar, 27van Rossum A.P. Limburg P.C. Kallenberg C.G.M. Membrane proteinase 3 expression on resting neutrophils as a pathogenic factor in PR3-ANCA-associated vasculitis.Clin Exp Rheumatol. 2003; 21: S64-S68Google Scholar Also, neutrophil adhesion molecules are upregulated on ANCA-stimulated neutrophils and migration, and translocation of neutrophils is enhanced.28Radford D.J. Savage C.O.S. Nash G.B. Treatment of rolling neutrophils with antineutrophil cytoplasmic antibodies causes conversion to firm integrin-mediated adhesion.Arthritis Rheum. 2000; 43: 1337-1345Google Scholar In vivo, anti-MPO splenocytes cause pauci-immune crescentic glomerulonephritis in mice.29Xiao H. Heeringa P. Hu P. et al.Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Google Scholar From a clinical perspective, support for pathogenicity of ANCAs has come from the positive predictive value of ANCA serology,30Christiaan Hagen E. Daha M.R. Hermans J. et al.Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis for the EC/BCR Project for ANCA Assay Standardization.Kidney Int. 1998; 53: 743-753Google Scholar its association with disease activity,31Tomasson G. Grayson P.C. Mahr A.D. et al.Value of ANCA measurements during remission to predict a relapse of ANCA-associated vasculitis-a meta-analysis.Rheumatology (Oxford). 2012; 51: 100-109Google Scholar and the beneficial effects of B-cell–targeted therapies and plasma exchange.32Jayne D.R.W. Gaskin G. Rasmussen N. et al.Randomized trial of plasma exchange or high-dosage methylprednisolone as adjunctive therapy for severe renal vasculitis.J Am Soc Nephrol. 2007; 18: 2180-2188Google Scholar, 33Stone J.H. Merkel P.A. Spiera R. et al.Rituximab versus cyclophosphamide for ANCA-associated vasculitis.N Engl J Med. 2010; 363: 221-232Google Scholar, 34Guillevin L. Pagnoux C. Karras A. et al.Rituximab versus azathioprine for maintenance in ANCA-associated vasculitis.N Engl J Med. 2014; 371: 1771-1780Google Scholar Although we show that ANCA has only a minor role in NET formation in AAV, our current data do not discredit the vast literature on ANCA pathogenicity. There is an unmet need for biomarkers for disease monitoring in AAV. ANCAs are present in 90%35Kallenberg C.G.M. Key advances in the clinical approach to ANCA-associated vasculitis.Nat Rev Rheumatol. 2014; 10: 484-493Google Scholar of patients with active disease and are commonly used as a biomarker for diagnosis but not for disease activity. Research on the value of ANCA titers to predict relapse is controversial and evolving.31Tomasson G. Grayson P.C. Mahr A.D. et al.Value of ANCA measurements during remission to predict a relapse of ANCA-associated vasculitis-a meta-analysis.Rheumatology (Oxford). 2012; 51: 100-109Google Scholar, 36Yates M. Watts R.A. Bajema I.M. et al.EULAR/ERA-EDTA recommendations for the management of ANCA-associated vasculitis.Ann Rheum Dis. 2016; 75: 1583-1594Google Scholar In a cohort of 201 patients,37Kemna M.J. Damoiseaux J. Austen J. et al.ANCA as a predictor of relapse: useful in patients with renal involvement but not in patients with nonrenal disease.J Am Soc Nephrol. 2015; 26: 537-542Google Scholar an ANCA rise was significantly associated with a relapse in patients with renal involvement, but less so in patients without renal involvement. Therefore, several studies have attempted to investigate whether NETs can be used as a biomarker in AAV. Nakazawa et al.9Nakazawa D. Shida H. Tomaru U. et al.Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.J Am Soc Nephrol. 2014; : 1-8Google Scholar showed a correlation between Cit H3-positive NETs and Birmingham Vasculitis Activity Score (BVAS) in MPO-ANCA–positive patients only. Wang et al.20Wang H. Sha L.L. Ma T.T. et al.Circulating level of neutrophil extracellular traps is not a useful biomarker for assessing disease activity in antineutrophil cytoplasmic antibody-associated vasculitis.PLoS One. 2016; 11: 1-10Google Scholar found no association between ELISA-measured cell-free DNA or MPO-DNA complexes and disease activity in AAV patients, likely because the quantification measured the net result of NET formation and NET degradation in vivo. The present study explored a novel and more sensitive assay for NET formation, a potential autoantigen for the production of ANCA, in relation to clinical disease in AAV. Based on our study, it remains speculative whether quantifying excessive NET formation can truly serve as a biomarker in AAV because our study was limited by retrospective design lacking contemporary BVAS, and patient numbers with longitudinal sampling were small. However, despite these important limitations, this study presents for the first time intriguing associations between excessive NET formation and disease activity, as well as the ability to distinguish between disease relapse and severe infection. Both illustrate the need to better establish the potential of measuring excessive NET formation for diagnostic as well as clinical purposes in larger, prospective studies. Lastly, the serendipitous finding that excessive NET formation was significantly higher in MPO-ANCA– compared with PR3-ANCA–positive patients is of interest. Intriguingly, we know from genome-wide associated studies that there is a distinct genetic association between GPA and MPA.38Lyons P.A. Rayner T.F. Trivedi S. et al.Genetically distinct subsets within ANCA-associated vasculitis.N Engl J Med. 2012; 367: 214-223Google Scholar Also, a neutrophil gene signature has been shown in AAV that strongly correlated with disease activity.39Grayson P.C. Carmona-Rivera C. Xu L. et al.Neutrophil-related gene expression and low-density granulocytes associated with disease activity and response to treatment in antineutrophil cytoplasmic antibody-associated vasculitis.Arthritis Rheumatol. 2015; 67: 1922-1932Google Scholar This gene signature overlapped