Neutrophil Extracellular Traps in Tissue and Periphery in Juvenile Dermatomyositis

Abstract

Objective. Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). This study was undertaken to investigate the role of neutrophils in juvenile dermatomyositis (JDM).Methods. Electron microscopy was used to identify neutrophils in tissue. NETs were also imaged using fluorescence microscopy and quantified using a myeloperoxidase-DNA enzyme-linked immunosorbent assay (ELISA) in plasma obtained from healthy children (n = 20), disease controls (n = 29), JDM patients (n = 66), and JDM patients with history of calcifications (n = 20). Clinical data included disease activity scores and complement C4 levels. Levels of immune complexes (ICs) and calprotectin were analyzed using ELISA.Results. Using electron microscopy, neutrophils were found to infiltrate affected muscle tissue, engulfing deposited calcium crystals. Uptake of the crystals led to neutrophil activation (P < 0.01) and subsequent phosphatidylinositol 3-kinase-and NADPH oxidase-dependent but peptidylarginine deiminase 4-independent formation of NETs, which contained mitochondrial DNA (P < 0.05), as confirmed in vivo (P < 0.001) and in vitro (P < 0.01). Peripheral NET levels were associated with calcinosis (P = 0.01), ICs (P = 0.008), and interleukin-8 levels (P = 0.004). Children with JDM had impaired NET clearance (P = 0.01), associated with autoantibody profiles including melanoma differentiationassociated protein 5 (P = 0.005), and depressed complement C4 levels (r = −0.72, P = 0.002). Furthermore, children with JDM showed evidence of neutrophil activation, with elevated levels of peroxidase activity (P = 0.02) and calprotectin (P < 0.01), which were associated with disease activity (P = 0.007), and dyslipidemia (odds ratio 4.7, P < 0.05).Conclusion. We found novel mechanisms of both calcium crystal-mediated neutrophil activation and cell death in JDM pathophysiology. Targeting this pathway may reduce the frequency and extent of calcinosis, as well as prevent long-term development of comorbidities, including atherosclerosis.

Neutrophil dysregulation is pathogenic in idiopathic inflammatory myopathies

“…Indeed, previous observations have suggested that impaired NET degradation may also contribute to ILD in IIM (31). Similarly, a recent study reported a link between calcium deposition in tissues, the calcinosis characteristic of JDM, and the development of neutrophil activation and NETs in muscle tissue (15). This may be an additional mechanism that induces NET formation in these individuals.…”

“…A previous study in the Chinese population reported that NETs are associated with ILD in IIM, particularly in subjects who are positive for anti-MDA5 autoantibodies (14), indicating that neutrophils potentially may be involved in the pathogenesis of this group of conditions. Furthermore, a recent association among circulating NETs, neutrophil activation, and calcinosis in juvenile DM (JDM) has been described, implicating calcium deposits characteristic of this disease with the induction of tissue and peripheral NET formation (15).However, whether aberrant neutrophil responses and NET formation play an important role in IIM and associated muscle damage remains to be systematically studied. Here, we investigated whether neutrophil dysregulation is prevalent in IIM and examined its associations with clinical manifestations and immune perturbances as well as its potential role in muscle damage.…”

Serum protein biomarkers for juvenile dermatomyositis: a pilot study

“…That group included TSP-4, Il-23, VCAM-1 and CD48 and fibrinogen. Of note, CD48 binds to the natural killer cell ligand 284; natural killer cells have recently been more rigorously implicated in JDM tissue damage [40], while polymorphonuclear cells have also been recently identified [41].…”

“…A recent study confirmed the involvement of complement in muscle biopsies from JDM patients [43];further investigation is needed to establish the physiological significance of these circulating complement factors in JDM. Not only may complement be bound to circulating immune complexes, present in JDM plasma, [41], but the levels of C4 may be decreased as a consequence of decreased gene copy number [43].…”

Role of Neutrophils in Systemic Vasculitides

“…The IgG ICs induce NET release after binding to FcgRIIA on neutrophils (8,9). The capacity of ICs and inflammatory cytokines to induce NET formation is consistent with excessive NET formation being observed in several autoimmune and inflammatory conditions, including gout, rheumatoid arthritis (RA), psoriasis, systemic lupus erythematosus (SLE), juvenile dermatomyositis (JDM), and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) with levels of NETs often linked to disease activity and severity (10)(11)(12)(13).…”