Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

Neutrophil extracellular traps generation and degradation in patients with granulomatosis with polyangiitis and systemic lupus erythematosus.

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Recent discoveries try to connect NETs formation with autoimmune diseases, like systemic lupus erythematosus (SLE) or granulomatosis with polyangiitis (GPA) and place them among one of the factors responsible for disease pathogenesis. The aim of the study was to assess the NETotic capabilities of neutrophils obtained from freshly diagnosed autoimmune patients versus healthy controls. Further investigation involved assessing NETs production among treated patients. In the latter step, NETs degradation potency of collected sera from non-treated patients was checked. Lastly, the polymorphisms of the DNASE I gene among tested subjects were checked. NETs formation was measured in a neutrophil culture by fluorometry, while degradation assessment was performed with patients' sera and extracellular source of DNA. Additionally, Sanger sequencing was used to check potential SNP mutations between patients. About 121 subjects were enrolled into this study, 54 of them with a diagnosed autoimmune disorder. Neutrophils stimulated with NETosis inducers were able to release NETs in all cases. We have found that disease affected patients produce NETs more rapidly and in larger quantities than control groups, with up to 82.5% more released. Most importantly, we showed a difference between the diseases themselves. NETs release was 68.5% higher in GPA samples when compared to SLE ones while stimulated with Calcium Ionophore. Serum nucleases were less effective at degrading NETs in both autoimmune diseases, with a reduction in degradation of 20.9% observed for GPA and 18.2% for SLE when compared with the controls. Potential therapies targeting neutrophils and NETs should be specifically tailored to the type of the disease. Since there are significant differences between NETs release and disease type, a standard neutrophil targeted therapy could prevent over-generation of traps in some cases, while in others it would deplete the cells, leaving the immune system unresponsive to primary infections.

Potent NETosis inducers do not show synergistic effects in vitro.

INTRODUCTION: NETosis is a process whereby neutrophils release chromatin into the surrounding extracellular matrix to form neutrophil extracellular traps (NETs). Under physiological conditions NETosis can be initiated by a variety of stimuli, including immune complexes, complement activation products, and a milieu of proinflammatory cytokines. Because overproduction of NETs is often related to the promotion or aggravation of autoimmune responses, we decided to assess how simultaneous activation of NETosis by different stimuli affects NET production. MATERIAL AND METHODS: NET formation was initiated by using combinations of three different NETosis inducers: phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (fMLP) and calcium ionophore (CaI). We measured fluorometry in real time, while microscopic visualisation served as an additional control for NET release. In total, 30 subjects free from infections or chronic diseases were enrolled in this study. RESULTS: We were able to demonstrate that in all cases NETosis induced by a combination of two stimuli resulted in diminished NETs production when compared to PMA and CaI single stimulations (p ≤ 0.001). The only cases in which double stimulation showed similar results to single stimulation were when we compared fMLP + CaI stimulation with fMLP single stimulation. Furthermore, when neutrophils were exposed to all three stimuli NETosis was almost entirely inhibited, compared to any single stimulation (p ≤ 0.001). CONCLUSIONS: Our results show that simultaneous stimulation of neutrophils by different NETosis-inducing agents results in diminished formation of NETs compared to a single stimulation. This indicates that cells may possess an internal regulatory mechanism that prevents overgeneration of NETs among healthy people.

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.

A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 µM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.

Biochemistry of Oxidative Stress.

Generation of reactive oxygen species is a physiological process that take place in every aerobic organism. Oxidative stress is defined as a disturbance in the balance between the production of free radicals and antioxidants in favor of the oxidants. The imbalance between those two fractions may potentially lead to cell damage at molecular level. Since oxidants are formed at a different rate as a normal product of aerobic metabolism, complex biochemical mechanisms are required to regulate the entire process.

Neutrophil extracellular traps (Nets) impact upon autoimmune disorders.

Friend or foe? This is often asked question when it comes to neutrophil extracellular traps studies. There is no simple answer to that. At the time of their discovery they were considered to be protectors of our well-being. Excellent pathogen fighting skills were described as purely beneficial. But it was not long before those guardians of immunity reveal their dark side. What seemed to be profitable could also take its toll. They are perfectly constructed, made from nucleic deoxyribonucleic acid ornamented with cytoplasmic and granular proteins, to fight invaders. But this unique build is prone to become considered by our body as a threat. Since there is a thin line which when crossed turns a savior into enemy, it was postulated that Nets can play a significant role in autoimmune disorders pathogenesis and disease exacerbation. Recent years have brought a new insight into autoimmune disorders trying to connect the old knowledge and suspicions with modern discoveries.

Reactive Oxygen Species, Granulocytes, and NETosis.

When pathogens invade the body, neutrophils create the first line of defense. Basic weaponry consists of phagocytosis and degranulation, but these cells have yet another ace in the sleeve, a unique strategy in which invading microorganisms are being destroyed. These cellular warriors are able to release nuclear chromatin and form extracellular structure, known as neutrophil extracellular traps (NET). NET formation is connected with the presence of free radicals. Research has shown that inhibition of free radical formation leads to suppression of NET release. The exact mechanisms controlling cooperation of free radicals with NET still remain unclear. New investigations in this field may contribute to discovery of NET etiology and put a new light on related disorders.

Neutrophil extracellular traps in physiology and pathology.

Neutrophil extracellular traps (NETs) are developed by nature to protect the body from furious invaders. On the other hand NET s can play an important role in human pathology. Recent studies have shown that neutrophils are able to perform beneficial suicide to create an unique microbicidal net composed from cellular content attached to chromatic frame. It is a powerful tool that primary serve as protector from severe infections, but this weapon is also a double ended sword of the immunity. If overproduced NET s provoke certain autoimmune diseases, coagulation disorders and even cancer metastases. Moreover, due to the competition between host and pathogens, the microorganism have developed a width repertoire of sophisticated evading mechanisms, like creation of polysaccharide capsule or changes in cell wall charge. Therefore it is important to increase the knowledge about paths underlying NET s formation and degradation processes if we want to efficiently fight with bacterial infections and certain diseases.

Life of neutrophil: from stem cell to neutrophil extracellular trap.

Neutrophils are one of the main types of effector cells in the innate immune system. Neutrophils play a major role in fighting diseases and are recruited almost immediately to sites of infection. The neutrophils have a variety of defensive mechanisms and their high affinity to chemotactic agents makes them ideal in the defense against pathogens. New functions of neutrophils have been discovered over the years. The latest role of neutrophils is neutrophil traps, which are a new component of innate anti-microbial immunity. Before neutrophils can effectively kill microorganisms they undergo a series of complex developmental processes.

Neutrophil extracellular traps in bacterial infections: strategies for escaping from killing.

Neutrophils are among the first responders to virulent factors. They kill microbes by phagocytosis, oxidative burst, and as neutrophil extracellular traps (NETs). NETs production leads to unique cell death depending on, inter alia, reactive oxygen species (ROS). Recently a number of studies highlight the mechanism of bacterial escape from extracellular traps; the process that may influence the outcome of bacterial infections.

Neutrophil extracellular trap in human diseases.

NETosis is a unique death pathway that differs from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. During this process, neutrophil extracellular traps (NETs) are created. NETs are extracellular structures composed of chromatin and variety of proteins from cells granules that bind and kill microorganisms. Recently, novel functions of NET have been proposed. It seems that neutrophil traps play an essential role during autoimmunity. They can induce and exacerbate diseases based on immune system malfunction.