The involvement of NETs in ANCA-associated vasculitis

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a serious autoimmune disease that is characterized by vascular necrosis. The pathogenesis of AAV includes ANCA-mediated neutrophil activation, subsequent release of inflammatory cytokines and reactive oxygen species (ROS), and formation of neutrophil extracellular traps (NETs). Excessive NETs could participate not only in ANCA-mediated vascular injury but also in the production of ANCAs per se as autoantigens. Thus, a vicious cycle of NET formation and ANCA production is critical for AAV pathogenesis. Elucidating the molecular signaling pathways in aberrant neutrophil activation and NETs clearance systems will allow specific therapeutics to regulate these pathways. Currently, standard therapy with high doses of glucocorticoids and immunosuppressants has improved outcomes in patients with AAV. However, AAV frequently develops in elderly people, and adverse effects such as severe infections in the standard regimens might contribute to the mortality. Mechanistically, cytokines or complement factors activate and prime neutrophils for ANCA-binding; thus, C5a receptor blocker has garnered attention as potential replacement for glucocorticoids in clinical settings. Recent studies have demonstrated that receptor-interacting protein kinases (RIPK3) and cyclophilin D (CypD), which regulate cell necrosis, may be involved in ANCA-induced NETs formation. Meanwhile, targeting NETs clearance, including the addition of deoxyribonuclease I (DNase I) and macrophage engulfment, may improve vasculitis. In this review, we focus on the pathogenesis of NETs and discuss potential targeted therapies for AAV based on recent experimental evidence

NETosis markers: Quest for specific, objective, and quantitative markers

AbstractMore than 10years have passed since the discovery of neutrophil extracellular traps (NETs) in 2004. NETs are extracellular web-like DNA decorated with antimicrobial proteins, which are released from activated neutrophils. The state of neutrophils with NET formation is called NETosis. It has been realized that NETosis includes suicidal NETosis and vital NETosis. The former state means cell death of neutrophils, whereas the latter state preserves living neutrophilic functions. Although both suicidal and vital NETosis play essential roles in elimination of microorganisms, excessive formation of NETs, especially the ones derived from suicidal NETosis, can harm the hosts. Therefore, the discovery of NETosis markers and development of evaluation methods are important. In this review, we compare the methods for evaluating NETosis, including immunocytological and immunohistological detection of co-localized neutrophil-derived proteins and extracellular DNA, and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometric detection of cell-appendant NET components, with focus on the specificity, objectivity, and quantitativity. Since the gold standard marker of NETosis or method of NET detection has not been established yet, researchers should choose the most appropriate marker or method in each situation based on the knowledge of the respective virtues and faults

Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis

Venous thromboembolic (VIE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils;however, the molecular mechanisms of neutrophil death in VIE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil-platelet aggregate formation induced by tumor necrosis factor-a but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space

Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin

The human body is exposed to a wide range of particles of industrial, environmental or internal origin such as asbestos, alum, silica or crystals of urate, calcium phosphate, calcium oxalate, cystine or cholesterol. Phagocytic clearance of such particles involves neutrophils and macrophages. Here we report that neutrophils encountering such particles of diverse sizes and shapes undergo necrotic cell death, a process associated with the formation of neutrophil extracellular trap (NET)-like extracellular DNA. In human neutrophils receptor-interacting protein kinase (RIPK)-1 inhibition with necrostatin-1s or mixed lineage kinase domain-like (MLKL) inhibition with necrosulfonamide abrogated cell death and associated-neutrophil extracellular DNA release induced by all of the aforementioned particles. Similar results were obtained with Mlkl-deficient mice neutrophils for all particles in vitro. Furthermore, Mlkl-deficient mice lacked tophus formation upon injection of MSU crystals into subcutaneous air pouches. These findings imply that nano-or microparticle-induced neutrophil extracellular DNA release is the consequence of neutrophil necroptosis, a regulated form of cell necrosis defined by RIPK1-RIPK3-MLKL signaling. Interestingly, this finding was consistent across different particle sizes and shapes. The RIPK1-RIPK3-MLKL signaling pathway may represent a potential therapeutic target in nano-or microparticle-related diseases (crystallopathies)

Possible implication of disordered neutrophil extracellular traps in the pathogenesis of MPO-ANCA-associated vasculitis

Neutrophil extracellular traps (NETs) are characterized by the presence of extracellular DNA fibers studded with antimicrobial proteins, including myeloperoxidase (MPO). Although NETs play an important role in the innate immune system, the scattered extracellular enzymes, such as MPO, pose risks to the host. Therefore, NETs are strictly regulated by DNase I in the serum, which prevents them from persisting. Recent studies have demonstrated that dysregulation of NETs could be involved in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. In this review, we interpret the association of disordered NETs with autoimmune diseases, especially propylthiouracil-induced MPO-ANCA-associated vasculitis

Abundant neutrophil extracellular traps in thrombus of patient with microscopic polyangiitis

This is a case study of a patient diagnosed with microscopic polyangiitis (MPA) and complicated with deep vein thrombosis (DVT), who died of respiratory failure despite treatment. Autopsy revealed severe crescentic glomerulonephritis and massive alveolar hemorrhage. The thrombus contained abundant neutrophils. Although it is reported that patients with ANCA-associated vasculitis (AAV) have an increased risk of DVT, it remains elusive why they are prone to thrombosis. A recent study has demonstrated the presence of neutrophil extracellular traps (NETs), a newly recognized mode of neutrophil cell-death, in glomerular crescents of MPA patients. Interestingly, NETs were identified in the thrombus as well as in the glomerular crescents in the present case. When compared to other thrombi unrelated to MPA, the amount of NETs was significantly greater in the MPA patient. On the other hand, NETs are critically involved in thrombogenesis because histones within NETs can bind platelets and blood coagulants. Although this is important in regard to containment of microbes within NETs, excessive NETs could cause thrombosis. The collective findings suggest the possibility that thrombosis could be critically associated with MPA via NETs, and that NETs could be a therapeutic target in MPA patients