Iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils.

Neutrophil extracellular trap (NET) formation is a significant innate immune defense mechanism against microbial infection that complements other neutrophil functions including phagocytosis and degranulation of antimicrobial peptides. NETs are decondensed chromatin structures in which antimicrobial components (histones, antimicrobial peptides and proteases) are deployed and mediate immobilization of microbes. Here we describe an effect of iron chelation on the phenotype of NET formation. Iron-chelating agent desferrioxamine (DFO) showed a modest but significant induction of NETs by freshly isolated human neutrophils as visualized and quantified by immunocytochemistry against histone-DNA complexes. Further analyses revealed that NET induction by iron chelation required NADPH-dependent production of reactive oxygen species (ROS) as well as protease and peptidyl-arginine-deiminase 4 (PAD4) activities, three key mechanistic pathways previously linked to NET formation. Our results demonstrate that iron chelation by DFO contributes to the formation of NETs and suggest a target for pharmacological manipulation of NET activity.

Völlger L ，Akong-Moore K ，Cox L ，Goldmann O ，Wang Y ，Schäfer ST ，Naim HY ，Nizet V ，von Köckritz-Blickwede M ... -  《-》

Neutrophil extracellular traps - the dark side of neutrophils.

Sørensen OE ，Borregaard N 《-》

Neutrophil Extracellular Traps Activate Proinflammatory Functions of Human Neutrophils.

Neutrophil extracellular traps (NETs) consist of decondensed nuclear chromatin that is associated with proteins and are released by neutrophils during an inflammatory response. Released NETs are able to capture pathogens, prevent their dissemination and potentially kill them via antimicrobial peptides and proteins that are associated with the decondensed chromatin. In addition to their antimicrobial functions, NETs have also been shown to exert immunomodulatory effects by activation and differentiation of macrophages, dendritic cells and T cells. However, the effect of NETs on neutrophil functions is poorly understood. Here we report the first comprehensive study regarding the effects of NETs on human primary neutrophils in vitro. NETs were isolated from cultures of PMA-exposed neutrophils. Exposure of neutrophils to isolated NETs resulted in the activation of several neutrophil functions in a concentration-dependent manner. NETs induced exocytosis of granules, the production of reactive oxygen species (ROS) by the NADPH oxidase NOX2, NOX2-dependent NET formation, increased the phagocytosis and killing of microbial pathogens. Furthermore, NETs induced the secretion of the proinflammatory chemokine IL-8 and the B-cell-activating cytokine BAFF. We could show that the NET-induced activation of neutrophils occurs by pathways that involve the phosphorylation of Akt, ERK1/2 and p38. Taken together our results provide further insights into the proinflammatory role of NETs by activating neutrophil effector function and further supports the view that NETs can amplify inflammatory events. On the one hand the amplified functions enhance the antimicrobial defense. On the other hand, NET-amplified neutrophil functions can be involved in the pathophysiology of NET-associated diseases. In addition, NETs can connect the innate and adaptive immune system by inducing the secretion of the B-cell-activating cytokine BAFF.

Dömer D ，Walther T ，Möller S ，Behnen M ，Laskay T ... -  《Frontiers in Immunology》

Iron-chelating agent, deferasirox, inhibits neutrophil activation and extracellular trap formation.

Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

Kono M ，Saigo K ，Yamamoto S ，Shirai K ，Iwamoto S ，Uematsu T ，Takahashi T ，Imoto S ，Hashimoto M ，Minami Y ，Wada A ，Takenokuchi M ，Kawano S ... -  《-》

Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?

The discovery of neutrophil extracellular traps in 2004 opened a fascinating new chapter in immune-mediated microbial killing. Brinkman et al. demonstrated that neutrophils, when catastrophically stimulated, undergo a novel form of programmed cell death (neutrophil extracellular trap formation) whereby they decondense their entire nuclear chromatin/DNA and release the resulting structure into the cytoplasm to mix with granule-derived antimicrobial peptides before extruding these web-like structures into the extracellular environment. The process requires the activation of the granule enzyme peptidyl arginine deiminase-4, the formation of reactive oxygen species (in particular hypochlorous acid), the neutrophil microtubular system and the actin cytoskeleton. Recent work by Yousefi et al. demonstrated that exposure to different agents for shorter stimulation periods resulted in neutrophil extracellular trap release from viable granulocytes, and that such neutrophil extracellular traps comprised mitochondrial DNA rather than nuclear DNA and were also capable of microbial entrapment and destruction. Deficiency in NADPH-oxidase production (as found in patients with chronic granulomatous disease) results in an inability to produce neutrophil extracellular traps and, along with their failure to produce antimicrobial reactive oxygen species, these patients suffer from severe, and sometimes life-threatening, infections. However, conversely the release of nuclear chromatin into tissues is also potentially autoimmunogenic and is now associated with the generation of anti-citrullinated protein antibodies in seropositive rheumatoid arthritis. Other neutrophil-derived nuclear and cytoplasmic contents are also pathogenic, either through direct effects on tissues or via autoimmune processes (e.g. autoimmune vasculitis). In this review, we discuss the plant origins of a highly conserved innate immune method of microbial killing, the history and biology of neutrophil extracellular traps and their role in defence and in human diseases. We attempt to resolve areas of controversy and propose roles for excess neutrophil extracellular trap release from hyperactive/reactive neutrophils and for the unique peptidyl arginine deiminase enzyme of Porphyromonas gingivalis in the pathogenesis of periodontitis, and subsequently a role for periodontitis/the peptidyl arginine deiminase enzyme of P. gingivalis in the causal pathway of autoimmune diseases such as rheumatoid arthritis. We propose that neutrophil extracellular trap and peptidyl arginine deiminase release may propagate tissue-destructive mechanisms rather than provide protection in susceptible individuals and that release of host-derived DNase may play an important role in the digestion and removal of neutrophil extracellular traps within tissues.

Cooper PR ，Palmer LJ ，Chapple IL 《-》