Neutrophil Extracellular Traps In Tissues Research Articles

Neutrophil extracellular traps involved in the pathogenesis of IgA vasculitis: Confirmed in two IgAV rat models.

Neutrophil extracellular traps (NETs) have been found to play a role in the development of autoimmune diseases. In the past two years, studies have demonstrated a significantly increase of NETs in skin tissues during the early stages of IgAV, indicating their involvement in disease activity among children with IgAV. However, the presence of NETs in IgAV animal models has not yet been reported. The objective of this study is to investigate whether NETs are involved in the pathogenesis of IgA vasculitis (IgAV) rats. Twenty-four SD rats were randomly divided into three groups: the ovalbumin group, the gliadin group, and the control group. The IgAV rat models were established administering Indian ink with ovalbumin (ovalbumin group) or gliadin (gliadin group) with Freund's complete adjuvant. The cell-free DNA (cf-DNA) was quantified by using dsDNA quantification kit, while the levels of Immunoglobulins, complement C3 and myeloperoxidase-DNA (MPO-DNA) in serum were tested using enzyme linked immunosorbent assay (ELISA). The IgA, complement C3 and NETs in tissues were detected through multiple immunofluorescences. Both the ovalbumin group and gliadin group showed IgA and C3 deposition in various tissues, including the glomerular mesangial region, skin, and digestive tract, while the control group showed no such deposition. The levels of circulatory cf-DNA and MPO-DNA, which are components of NETs, were significantly elevated in both ovalbumin and gliadin groups compared with the control group. Furthermore, the presence of NETs were found in gastrointestinal and renal tissues of the ovalbumin and gliadin groups, but not in the control group. IgAV model rat can be established through the combination of ovalbumin and gliadin with Indian ink and Freund's complete adjuvant. This study provides the first confirmation that NETs are involved in the pathogenesis of IgAV rat.

Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock

Neutrophil elastase (NE) is a serine protease stored in the azurophilic granules of neutrophils and released into the extracellular milieu during inflammatory response or formation of neutrophil extracellular traps (NETs). Neutrophils release NETs to entrap pathogens by externalizing their cellular contents in a DNA framework decorated with anti-microbials and proteases, including NE. Importantly, excess NETs in tissues are implicated in numerous pathologies, including sepsis, rheumatoid arthritis, vasculitis, and cancer. However, it remains unknown how to effectively prevent NET formation. Here, we show that NE plays a major role during NET formation and that inhibition of NE is a promising approach for decreasing NET-mediated tissue injury. NE promoted NET formation by human neutrophils. Whereas sivelestat, a small molecule inhibitor of NE, inhibited the formation of NETs in vitro , administration of free sivelestat did not have any efficacy in a murine model of lipopolysaccharide-induced endotoxic shock. To improve the efficacy of sivelestat in vivo, we have developed a nanoparticle system for delivering sivelestat. We demonstrate that nanoparticle-mediated delivery of sivelestat effectively inhibited NET formation, decreased the clinical signs of lung injury, reduced NE and other proinflammatory cytokines in serum, and rescued animals against endotoxic shock. Collectively, our data demonstrates that NE signaling can initiate NET formation and that nanoparticle-mediated inhibition of NE improves drug efficacy for preventing NET formation.

Neutrophil Extracellular Traps in Tissue and Periphery in Juvenile Dermatomyositis.

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). 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. 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 differentiation-associated 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). 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.

Immunofluorescent Detection of NET Components in Paraffin-Embedded Tissue.

Neutrophil extracellular traps (NETs) consist of decondensed chromatin fibers studded with granular and cytoplasmic proteins and peptides that are released by stimulated neutrophil granulocytes. If present in abundance (e.g., in large thrombi), NETs are depicted in H&E-stained tissue sections as pale bluish areas. Since no NET-specific antibodies exist, to unambiguously identify even small amounts of NETs in tissue, it is essential to demonstrate colocalization of nuclear and granular/cytoplasmic NET components which in unstimulated neutrophils are clearly separated. This requires good tissue preservation and a very defined immunolocalization, which can be achieved by using 2-3μm thick sections of paraffin-embedded tissue. It provides sufficiently good tissue preservation for subcellular localization of two or more NET components, thereby allow to differentiate stimulated from unstimulated neutrophils and to clearly identify NETs. In this chapter, we will provide protocols for antigen retrieval and immunofluorescent labeling of NET components in paraffin-embedded tissue with commercially available antibodies.

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6-8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.Neutrophils can combat invading microorganisms by phagocytosis,degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.