Assessment of longitudinal serum neutrophil extracellular trap–inducing activity in anti-neutrophil cytoplasmic antibody–associated vasculitis and glomerulonephritis in a prospective cohort using a novel bio-impedance technique

Abstract

Neutrophil extracellular traps (NET) have been implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Here, we developed a novel, label-free, high-throughput bio-impedance technique to effectively measure serum NET-inducing activity. Using this technique, NET-inducing activity of serum derived from patients with AAV was assessed in a prospective cohort of 62 patients presenting with active AAV with major organ involvement. Thirty-five patients presented with new and 27 patients presented with relapsing AAV, of whom 38 had kidney and/or 31 had lung involvement. NET-inducing activity was assessed at diagnosis of active AAV (time zero), during the first 6 weeks of treatment, and after 6 months of treatment. Forty-seven patients revealed elevated NET-inducing activity at time zero. After initiation of immunosuppressive treatment, NET-inducing activity was reduced at six weeks. A subsequent increase at six months could potentially identify patients with relapsing disease (hazard ratio, 11.45 [interquartile range 1.36-96.74]). NET-inducing activity at time zero correlated with kidney function and proteinuria. Importantly, in kidney tissue, NETs co-localized with lesions typical of ANCA-associated glomerulonephritis and even correlated with systemic serum NET-inducing activity. Thus, our prospective data corroborate the importance of NET formation in AAV and ANCA-associated glomerulonephritis and the potential of longitudinal evaluation, as monitored by our novel bio-impedance assay and detailed histological evaluation. Neutrophil extracellular traps (NET) have been implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Here, we developed a novel, label-free, high-throughput bio-impedance technique to effectively measure serum NET-inducing activity. Using this technique, NET-inducing activity of serum derived from patients with AAV was assessed in a prospective cohort of 62 patients presenting with active AAV with major organ involvement. Thirty-five patients presented with new and 27 patients presented with relapsing AAV, of whom 38 had kidney and/or 31 had lung involvement. NET-inducing activity was assessed at diagnosis of active AAV (time zero), during the first 6 weeks of treatment, and after 6 months of treatment. Forty-seven patients revealed elevated NET-inducing activity at time zero. After initiation of immunosuppressive treatment, NET-inducing activity was reduced at six weeks. A subsequent increase at six months could potentially identify patients with relapsing disease (hazard ratio, 11.45 [interquartile range 1.36-96.74]). NET-inducing activity at time zero correlated with kidney function and proteinuria. Importantly, in kidney tissue, NETs co-localized with lesions typical of ANCA-associated glomerulonephritis and even correlated with systemic serum NET-inducing activity. Thus, our prospective data corroborate the importance of NET formation in AAV and ANCA-associated glomerulonephritis and the potential of longitudinal evaluation, as monitored by our novel bio-impedance assay and detailed histological evaluation. Lay SummaryNeutrophils play an important role in damaging the blood vessels in anti-neutrophil cytoplasmic antibody–mediated vasculitis (AAV) by releasing toxic web-like structures called neutrophil extracellular traps (NETs). We used a new test to easily measure how much NETs are formed using patient blood (serum). In a large group of AAV patients, serum forms many NETs during active disease. The more the NETs formed by serum, the more the NETs are found in kidneys as well. After treatment when the disease is quiet, serum forms substantially less NETs. This new technique can be useful to evaluate treatment effectiveness, monitor patients at risk, and test new therapies. Neutrophils play an important role in damaging the blood vessels in anti-neutrophil cytoplasmic antibody–mediated vasculitis (AAV) by releasing toxic web-like structures called neutrophil extracellular traps (NETs). We used a new test to easily measure how much NETs are formed using patient blood (serum). In a large group of AAV patients, serum forms many NETs during active disease. The more the NETs formed by serum, the more the NETs are found in kidneys as well. After treatment when the disease is quiet, serum forms substantially less NETs. This new technique can be useful to evaluate treatment effectiveness, monitor patients at risk, and test new therapies. Anti-neutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) is a small- to medium-sized vessel vasculitis that is characterized by circulating antibodies against myeloperoxidase (MPO-ANCA) or proteinase-3 (PR3-ANCA).1Jennette J.C. Falk R.J. Bacon P.A. et al.2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides.Arthritis Rheum. 2013; 65: 1-11Crossref PubMed Scopus (4217) Google Scholar Neutrophils play a central role in the development of AAV. Neutrophil extracellular trap (NET) formation, that is, the extrusion of neutrophil chromatin fibers laced with histones and enzymes,2Neubert E. Meyer D. Rocca F. et al.Chromatin swelling drives neutrophil extracellular trap release.Nat Commun. 2018; 9: 3767Crossref PubMed Scopus (114) Google Scholar,3Brinkmann V. Reichard U. Goosmann C. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6434) Google Scholar is thought to be a critical process in AAV and glomerulonephritis (GN) pathogenesis.4Kessenbrock K. Krumbholz M. Schonermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1197) Google Scholar,5Kumar S.V. Kulkarni O.P. Mulay S.R. et al.Neutrophil extracellular trap-related extracellular histones cause vascular necrosis in severe GN.J Am Soc Nephrol. 2015; 26: 2399-2413Crossref PubMed Scopus (133) Google Scholar In recent years, several groups have stated that ANCAs and/or serum components can induce NETs.6Schreiber A. Rousselle A. Becker J.U. et al.Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.Proc Natl Acad Sci U S A. 2017; 114: E9618-E9625Crossref PubMed Scopus (171) Google Scholar, 7Nakazawa 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; 25: 990-997Crossref PubMed Scopus (193) Google Scholar, 8Kraaij T. Kamerling S.W.A. van Dam L.S. et al.Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.Kidney Int. 2018; 94: 139-149Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Accumulating evidence suggests that NET formation is increased during active vasculitis in contrast to patients in remission,8Kraaij T. Kamerling S.W.A. van Dam L.S. et al.Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.Kidney Int. 2018; 94: 139-149Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,9Soderberg 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: 2085-2094Crossref PubMed Scopus (84) Google Scholar implying NET formation can be a valuable biomarker for response to therapy or predicting disease flares. However, longitudinal prospective data are lacking. Previous studies focus on the mechanism of action as opposed to their value in clinical practice. Multiple techniques have been developed to assess NET formation. These techniques have shown drawbacks; for example, quantification of MPO-DNA complexes or free DNA is nonspecific and error-prone and NET visualization with immunofluorescence (IF) markers is time-consuming and can be subjective. Recently, bioimpedance measurement was described to detect and quantify real-time NET formation.10Linnemann C. Venturelli S. Konrad F. et al.Bio-impedance measurement allows displaying the early stages of neutrophil extracellular traps.EXCLI J. 2020; 19: 1481-1495PubMed Google Scholar,11Lv D. Xu Y. Cheng H. et al.A novel cell-based assay for dynamically detecting neutrophil extracellular traps-induced lung epithelial injuries.Exp Cell Res. 2020; 394112101Crossref Scopus (22) Google Scholar The technique uses microelectrode-coated 96-well plates, which can detect changes in electron flow translated to differences in impedance. Cells incubated with stimuli undergo changes, for example, size and surface, like in the process of NET formation. These changes disturb electron flow, which are depicted as real-time experimental readings without the need for IF labeling or image processing techniques.12Hamidi H. Lilja J. Ivaska J. Using xCELLigence RTCA instrument to measure cell adhesion.Bio Protoc. 2017; 7: PubMed Google Scholar NET for example, histones and can endothelial in and components effectively endothelial S.V. Kulkarni O.P. Mulay S.R. et al.Neutrophil extracellular trap-related extracellular histones cause vascular necrosis in severe GN.J Am Soc Nephrol. 2015; 26: 2399-2413Crossref PubMed Scopus (133) Google et of of neutrophil extracellular trap formation in 2017; Full Text Full Text PDF PubMed Google Scholar NET in kidney of AAV patients are and have been to systemic NET K. Krumbholz M. Schonermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1197) Google et of myeloperoxidase in cytoplasmic antibody (ANCA)-associated Int. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Y. et anti-neutrophil cytoplasmic antibody is with the formation of neutrophil extracellular traps in the kidney and vasculitis activity in myeloperoxidase anti-neutrophil cytoplasmic microscopic Google Scholar, H. A. J. et of neutrophils is useful for active and lesions in cytoplasmic PubMed Scopus Google Scholar, S. Y. et al.Neutrophil extracellular trap formation is with in ANCA-associated 2015; PubMed Google Scholar The kidney is, the organ in AAV presenting as kidney to treatment is to and for example, kidney and kidney or A detailed of the of NETs in kidney in with systemic NET formation in AAV patients can the of NETs in In this we assessed NET-inducing activity of serum in a prospective cohort of AAV We bioimpedance is a to assess NET-inducing activity of AAV we the of therapy on NET-inducing activity and the value on by NET-inducing activity at the time of active during treatment, and at the time of we kidney for the of NETs to assess their and to systemic NET formation. in the cohort and presenting with active AAV, to the Chapel Hill Consensus J.C. Falk R.J. Bacon P.A. et al.2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides.Arthritis Rheum. 2013; 65: 1-11Crossref PubMed Scopus (4217) Google Scholar with disease activity major organ or from the van van H. et and of a prospective on Int. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar and data from the and the was at the time of active disease and after therapy at 6 weeks and 6 months and at was on and M. et for the of ANCA-associated PubMed Google Scholar activity was using the C. D. et and of the 2009; PubMed Scopus Google Scholar and clinical was as a of and of data for to controls with kidney from the from with kidney and/or and on kidney The was by the and is in with the of was Neutrophils from of blood from a in by using after which using for at and for Neutrophil was assessed using and neutrophils to the in that is, with serum and Neutrophils in an with at during on the neutrophils with patient serum serum and/or for neutrophils with the or the for necrosis was used to neutrophils to the in AAV IF neutrophil with cell to Neutrophils incubated on with and as described After neutrophils with NETs with and using a DNA was using using a on a to for swelling and cell extracellular and Neutrophils incubated in 96-well as described for with to using the xCELLigence Cell After extracellular DNA was using incubated in in with neutrophils and the as a and and in longitudinal patient in the and to the with NET-inducing activity of a serum was is the value by neutrophils with serum increase in NET-inducing activity at as with was as and with longitudinal and at and kidney to clinical van van H. et and of a prospective on Int. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar kidney for and to assess NET formation. incubated with that is, serum serum in and incubated with antibody and antibody incubated with antibodies and with and with and assessed for the of and the of the of and and the of in and using the using as using the test or as presented as or range using the independent test or U as using the Multiple group was using the test with and for the for kidney and with the test was The correlation was used for correlation A value of was we that bioimpedance measurement was to NET-inducing activity. a of NET formation, a response of The response could be by neutrophils with and by with necrosis used to neutrophils to the in patients in induce a response by or neutrophil response to and serum and the of extracellular DNA was using IF the that extracellular DNA in contrast to serum In neutrophils with complement or induce a response Using we that serum from patients with active disease increased NET formation as with serum swelling and cell with serum with serum serum and serum extracellular DNA to this was more with serum was with AAV serum in contrast to serum and neutrophils with cell and an increased of serum to induce NET formation patients for and The at was patients presented with with or microscopic in and patients, for or in and Thirty-five patients at the time of 27 patients presented with relapsing disease. The at was The major kidney and in 38 and 31 patients, and histological are presented in F. et of ANCA-associated Am Soc Nephrol. PubMed Scopus Google S.R. M. et and of a in ANCA-associated Int. 2018; 94: Full Text Full Text PDF PubMed Scopus Google Scholar during in and patients at and patient and with of kidney for for to et to et anti-neutrophil cytoplasmic anti-neutrophil cytoplasmic anti-neutrophil cytoplasmic antibody are presented as or kidney for for to et F. et of ANCA-associated Am Soc Nephrol. PubMed Scopus Google to et S.R. M. et and of a in ANCA-associated Int. 2018; 94: Full Text Full Text PDF PubMed Scopus Google Scholar in a new AAV, anti-neutrophil cytoplasmic anti-neutrophil cytoplasmic anti-neutrophil cytoplasmic antibody are presented as or could effectively detect NET-inducing activity in serum of AAV In with IF AAV serum increased NET formation by bioimpedance as with serum and and NET-inducing activity was by neutrophils with was that is, serum from AAV patients for NET formation. serum more NET-inducing activity with serum and patients presenting with microscopic more NET-inducing activity patients with with NET-inducing activity a correlation with the kidney and a correlation with NET-inducing activity organ or relapsing patient and with or and serum at and in 62 and patients, NET-inducing activity was to assess the of treatment on NET-inducing activity of NET-inducing activity at and as with In patients with and longitudinal the of patients 6 patients and more as with and a more kidney and/or In patients with NET-inducing activity with patients with NET-inducing activity serum and to be and data are depicted in and differences in treatment and their on NET-inducing activity of serum patients developed a 6 patients developed kidney and patients patients after a of months and with patients and patients are presented in at and with without could be be anti-neutrophil cytoplasmic anti-neutrophil cytoplasmic time of active after therapy at 6 are presented as or could be be in a new and and at 6 patient 6 to to to kidney to anti-neutrophil cytoplasmic are presented as or patient 6 in a new AAV, anti-neutrophil cytoplasmic anti-neutrophil cytoplasmic time of active after therapy at 6 are presented as or AAV, anti-neutrophil cytoplasmic are presented as or patients, serum at time patients a NET increase at with of patients developed a during in contrast to of the patients without an increase (hazard 11.45 patients with a NET increase after and and with patients with the time of patients presented with and/or patients with and without a NET an NET increase and a and are presented in NETs have been shown to be in K. Krumbholz M. Schonermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1197) Google Scholar and we found a correlation with kidney and and NET-inducing activity of serum, we assessed NET in AAV patients with AAV without for and that is, with and in from active patients of in at in contrast to kidney from AAV patients without of and of NET formation in with active characterized by in and formation The of NETs was in patients less NETs in the of patients and in and cell to was in active with AAV without and in the and In AAV patients AAV without serum for bioimpedance The of and NETs correlated with serum NET-inducing which was for neutrophil extracellular traps and as on anti-neutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) glomerulonephritis AAV without and NETs as assessed bioimpedance The of and that NETs correlated with systemic NET-inducing activity as assessed with bioimpedance which was for without image we that bioimpedance technique using xCELLigence is to detect NET-inducing activity of AAV of NET formation using bioimpedance was C. Venturelli S. Konrad F. et al.Bio-impedance measurement allows displaying the early stages of neutrophil extracellular traps.EXCLI J. 2020; 19: 1481-1495PubMed Google Scholar,11Lv D. Xu Y. Cheng H. et al.A novel cell-based assay for dynamically detecting neutrophil extracellular traps-induced lung epithelial injuries.Exp Cell Res. 2020; 394112101Crossref Scopus (22) Google Scholar and is by our using of NET formation and IF NET formation. We used the bioimpedance technique to measure NET-inducing activity in a prospective AAV with active disease had serum NET-inducing and longitudinal that NET-inducing activity reduced after immunosuppressive treatment. A NET increase 6 months of diagnosis increased the of relapsing disease during NET-inducing activity correlated with and with as as with the of and NETs. In NETs with The bioimpedance several used techniques to detect as electron and V. Reichard U. Goosmann C. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6434) Google K. Krumbholz M. Schonermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1197) Google U. Goosmann C. et cell to neutrophil extracellular Cell PubMed Scopus Google Scholar, H. et of neutrophil extracellular traps is a useful biomarker for disease activity in cytoplasmic Scholar, V. Goosmann C. et quantification of in NET Google Scholar, T. Kamerling et al.A novel for high-throughput and quantification of neutrophil extracellular traps NET with 15: PubMed Scopus Google Scholar The bioimpedance is label-free, and less which can by of E. Meyer D. Rocca F. et al.Chromatin swelling drives neutrophil extracellular trap release.Nat Commun. 2018; 9: 3767Crossref PubMed Scopus (114) Google M. et is a in neutrophil extracellular trap formation by anti-neutrophil cytoplasmic PubMed Scopus Google Scholar of and/or V. U. et al.Neutrophil extracellular how to and Google Scholar We found increased NET-inducing activity by patient serum with with at an However, evidence on the of to induce NET A. Rousselle A. Becker J.U. et al.Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.Proc Natl Acad Sci U S A. 2017; 114: E9618-E9625Crossref PubMed Scopus (171) Google Scholar, 7Nakazawa 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; 25: 990-997Crossref PubMed Scopus (193) Google Scholar, 8Kraaij T. Kamerling S.W.A. van Dam L.S. et al.Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.Kidney Int. 2018; 94: 139-149Abstract Full Text Full Text PDF PubMed Scopus (64) Google M. M. Y. et anti-neutrophil cytoplasmic antibody is with the formation of neutrophil extracellular traps in the kidney and vasculitis activity in myeloperoxidase anti-neutrophil cytoplasmic microscopic Google Scholar We used bioimpedance to in patient serum for NET-inducing activity. to neutrophils during necrosis complement and to induce or increase NET-inducing activity However, using AAV serum we found that the for NET-inducing activity are to their NET-inducing activity the could be by using a with a of In to the described to measure the vascular by vascular to patient et of the vascular and blood to measure vascular PubMed Scopus Google Scholar we that the circulating active in active AAV is a of serum components that can be by neutrophils and a studies could bioimpedance to which serum components are for NET formation in AAV. longitudinal that quantification of NET-inducing activity can be to monitor disease response to treatment, and of relapsing disease. In patients in serum NET-inducing could be was shown that serum components for are to induce NET formation in T. Kamerling S.W.A. van Dam L.S. et al.Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.Kidney Int. 2018; 94: 139-149Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar We to that patients have serum components with NET-inducing activity by AAV treatment without vasculitis disease activity. NET-inducing activity be to identify patients with disease activity at of kidney are by studies NET remnants elevated at the time of active AAV in contrast to K. Krumbholz M. Schonermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1197) Google T. Kamerling S.W.A. van Dam L.S. et al.Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.Kidney Int. 2018; 94: 139-149Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,9Soderberg 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: 2085-2094Crossref PubMed Scopus (84) Google Scholar can NET formation by A. et are less blood to the of Scopus Google A. M. et neutrophil extracellular traps for the disease Scopus Google Scholar of R.J. et cytoplasmic induce neutrophils to and in Natl Acad Sci U S A. PubMed Scopus Google Scholar and E. M. et neutrophils on their in and in PubMed Google Scholar U. Goosmann C. et cell to neutrophil extracellular Cell PubMed Scopus Google A. H. et of on of Full Text PDF PubMed Scopus Google Scholar and neutrophil and by A potential role for as a of from PubMed Scopus Google Scholar However, novel AAV for example, P.A. et for the treatment of ANCA-associated Med. PubMed Scopus Google Scholar are to In data from treatment H. C. et of in cytoplasmic vasculitis by and neutrophil extracellular 2015; PubMed Google Scholar evidence T. Kamerling S.W.A. van Dam L.S. et al.Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.Kidney Int. 2018; 94: 139-149Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar studies on for example, Y. D. Shida H. et neutrophil extracellular trap formation and 7: PubMed Scopus Google Scholar, H. Y. S. et and clinical of in of neutrophil extracellular traps by of on the pathogenesis of microscopic 2018; PubMed Scopus Google Scholar, K. H. K. et mediates neutrophil extracellular traps formation in ANCA-associated PubMed Scopus Google Scholar are that NET formation is critical in AAV prospective studies are to and are in NET formation. the importance of NET formation in NET formation with active lesions and can be to systemic NET-inducing activity of AAV Previous studies NET components in K. Krumbholz M. Schonermarck U. et al.Netting neutrophils in autoimmune small-vessel vasculitis.Nat Med. 2009; 15: 623-625Crossref PubMed Scopus (1197) Google et of myeloperoxidase in cytoplasmic antibody (ANCA)-associated Int. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Y. et anti-neutrophil cytoplasmic antibody is with the formation of neutrophil extracellular traps in the kidney and vasculitis activity in myeloperoxidase anti-neutrophil cytoplasmic microscopic Google Scholar, H. A. J. et of neutrophils is useful for active and lesions in cytoplasmic PubMed Scopus Google Scholar, S. Y. et al.Neutrophil extracellular trap formation is with in ANCA-associated 2015; PubMed Google Scholar to systemic NET-inducing activity. NET are elevated in the circulation of active AAV M. M. K. et and serum in with extracellular traps mechanism of the Google Scholar In studies that NET for example, and neutrophil endothelial and to development in and S.V. Kulkarni O.P. Mulay S.R. et al.Neutrophil extracellular trap-related extracellular histones cause vascular necrosis in severe GN.J Am Soc Nephrol. 2015; 26: 2399-2413Crossref PubMed Scopus (133) Google A. Rousselle A. Becker J.U. et al.Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.Proc Natl Acad Sci U S A. 2017; 114: E9618-E9625Crossref PubMed Scopus (171) Google Scholar or endothelial and development S.V. Kulkarni O.P. Mulay S.R. et al.Neutrophil extracellular trap-related extracellular histones cause vascular necrosis in severe GN.J Am Soc Nephrol. 2015; 26: 2399-2413Crossref PubMed Scopus (133) Google A. Rousselle A. Becker J.U. et al.Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.Proc Natl Acad Sci U S A. 2017; 114: E9618-E9625Crossref PubMed Scopus (171) Google Scholar NETs a and for increased S. C. C. et al.Neutrophil extracellular traps of cytoplasmic neutrophil to and PubMed Scopus Google Scholar complement A. Rousselle A. Becker J.U. et al.Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis.Proc Natl Acad Sci U S A. 2017; 114: E9618-E9625Crossref PubMed Scopus (171) Google Scholar and with for example, D. Shida H. Y. et of in with neutrophils that undergo PubMed Scopus Google Scholar In a NETs an K. T. et action of neutrophil extracellular traps in to after PubMed Scopus Google Scholar We that with active lesions in active S. et and disease activity in ANCA-associated Am Soc Nephrol. 2020; 15: PubMed Scopus Google Scholar a NET formation is thought to play a central and role in the development and of The of our is the of a prospective cohort with serum and kidney A of our is the of neutrophils AAV neutrophils are and to of the of large prospective NET studies AAV neutrophils to the differences in patient In AAV serum NET-inducing which can effectively be by that treatment effectively serum NET-inducing which is during and is a potentially useful technique to monitor patients during The of NETs in with active lesions and with systemic NET-inducing activity. the The data this can be to the We A. and A. of The for their we S. of of and The and M. of The of the and the for data and/or for the of this was presented at the International and on in and the and and/or and the and the revised and the of the with of patients and serum during clinical initiation of immunosuppressive neutrophils incubated for with in the or of to assess their potential to a response using A reduced of was used to assess or of the necrosis complement and assessed in and a response by or the response of neutrophils to Neutrophil extracellular trap (NET) formation could be by neutrophils with neutrophils with with anti-neutrophil cytoplasmic antibody and serum NET formation as by the of cell and and cell on bioimpedance assay for and immunofluorescence of and extracellular as in of therapy on neutrophil extracellular trap activity. differences treatment in the to NET-inducing activity of anti-neutrophil cytoplasmic vasculitis patients and and and and as time treatment. with and immunofluorescence of a image of a of an anti-neutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV) patient with glomerulonephritis patient without and for myeloperoxidase and DNA with in AAV with in which was in AAV patients without and for Anti-neutrophil cytoplasmic vasculitis (AAV) serum with to or by serum to for to AAV incubated with neutrophils for to assess neutrophil extracellular trap cell by bioimpedance using bioimpedance After AAV serum a in NET-inducing of serum to for NET-inducing that NET-inducing of AAV serum is on of and AAV serum for after and patients in the and of of relapsing of for neutrophil extracellular trap of and

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