Neutrophils are one of the first lines of defense against invading microbes. Neutrophils in circulation are directed by cytokines into infected tissues, where they encounter invading microbes. This encounter leads to the activation of neutrophils and the engulfment of the pathogen into a phagosome. In the phagosome, two events are required for antimicrobial activity. First, the presynthesized subunits of the NADPH oxidase assemble at the phagosomal membrane and transfer electrons to oxygen to form superoxide anions. These dismutate spontaneously or catalytically to dioxygen and hydrogen peroxide. Collectively, superoxide anions, dioxygen, and hydrogen peroxide are called reactive oxygen species (ROS). Second, the granules fuse with the phagosome, discharging antimicrobial peptides and enzymes. In the phagosome, microorganisms are exposed to high concentrations of ROS and antimicrobial peptides. Together, they are responsible for microbial killing. Patients with mutations in the NADPH oxidase suffer from chronic granulomatous disease (CGD). CGD patients are severely immunodeficient, have recurrent infections, often with opportunistic pathogens, and have poor prognosis.
Upon activation, neutrophils also release extracellular traps (NETs), which provide a high local concentration of antimicrobial molecules that kill microbes effectively. Forming the NETs is dependent on the generation of ROS by NADPH oxidase. In an infection, ROS formation may contribute to the following two antimicrobial pathways: intraphagosomal killing in live neutrophils and NET-mediated killing post mortem.
Although naïve cells are round with some membrane folds, neutrophils stimulated with interleukin-8 (IL-8), phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) become flat and form membrane protrusion; activated neutrophils but not naïve cells also make prominent extracellular structures. These fibers, or NETs, are very fragile. High-resolution scanning electron microscopy (SEM) shows that the NETs contain smooth stretches with a diameter of 15 to 17 nm and globular domains of around 25 nm that aggregate into larger threads with diameters of up to 50 nm. Analysis of cross section of NETs by transmission electron microscopy (TEM) reveals they are not surrounded by membranes.
The composition of NETs has been analyzed by immunofluorescence. NETs contain proteins from azurophilic (primary) granules such as neutrophil elastase, cathepsin G and myeloperoxidase. Proteins from secondary and tertiary granules, such as lactoferrin, are also present. In contrast, actin, tubulin and various other cytoplasmatic proteins are excluded from NETs.
DNA is a major structural component of NETs: for example a brief treatment with deoxyribonuclease (DNase) results in a disintegration of NETs. Conversely protease treatment left the DNA of the NETs intact.
NETs associate with both Gram-positive (Staphyloccocus Aureus) and Gram-negative pathogens (Salmonella typhimurium).
Neutrophils make NETs through an active mechanism. NETs disarm pathogens with proteases such as neutrophil elastase. NETs also kill bacteria efficiently, and at least one of the NET components, histones, exerts antimicrobial activity at surprisingly low concentrations. The data presented by Brinkmann et al. indicate that granule proteins and chromatin together form an extracellular structure that amplifies the effectiveness of its antimicrobial substances by ensuring a high local concentration. NETs degrade virulence factors and/or kill bacteria even before the microorganisms are engulfed by neutrophils. In addition to their antimicrobial properties, NETs may serve as a physical barrier that prevents further spread of bacteria. Moreover, sequestering the granule proteins into NETs may keep potentially noxious proteins like proteases from diffusing away and inducing damage in tissue adjacent to the site of inflammation. NETs might also have a deleterious effect on the host, because the exposure of extracellular histone complexes could play a role during the development of autoimmune diseases like lupus erythematosus (whose patients develop antibodies to chromatin and granular components).
"Neutrophil Extracellular Traps Kill Bacteria, 2004": http://www.ncbi.nlm.nih.gov/pubmed/15001782
Figure 1:(A) Reactions initiated by the NADPH oxidase (NOX2) in phagosomes. Upon stimulation, the cytosolic components (light orange) of NOX2 are recruited to the transmembrane subunits (dark orange) on the phagosomal membrane. The active complex catalyzes the oxidation of cytosolic NADPH, produced by the hexose monophosphate (HMP) shunt, resulting in the liberation of cytosolic H+ and the translocation of electrons across the membrane into the lumen. Inside the phagosome, the electrons reduce molecular oxygen to superoxide (O2–), which is subsequently converted to H2O2 and other ROS. The electrogenic nature of NOX2 makes the phagosome lumen negative and facilitates the uptake of H+ (and possibly other ions) through H+-selective voltage-gated channels (blue). Luminal H+ serves as substrate for superoxide dismutation. A fraction of the ROS, which are membrane permeant, escape the phagosome and can oxidize cytoplasmic cysteine-requiring enzymes, such as tyrosine phosphatases (PTP) (green). (B) ROS-induced formation of NETs. Protons liberated during the oxidation of NADPH are extruded from the cell by Na+/H+ exchangers (pink). The net solute content of the cell increases, driving intake of osmotically obliged water and causing cell swelling. At the same time, plasmalemmal NOX2 activity depolarizes the plasma membrane. Depolarization decreases Ca2+ entry through store-operated channels (red), but facilitates H+extrusion through proton channels (blue). ROS also promote generation of NETs. This process begins with the homogenization of nuclear chromatin and the dissolution of nuclear and granular membranes (top). Ultimately, the plasma membrane is ruptured (bottom), releasing fibers of chromatin (gray), decorated with granular content proteins (yellow), into the extracellular space. NETs are able to bind and kill extracellular pathogens (green).
Upon activation (by IL-8, lipopolysaccharide (LPS), bacteria, fungi or activated platelets) neutrophils start a programme that leads to their death and the formation of NETs.
This activation pathway can involve different receptors, such as the Toll-like receptors (TLRs), as well as cytokine and Fc receptors.
Microorganisms induce more NETs than are induced following stimulation with single TLR activators, and beads covered with antibodies, but not antibodies in solution, also induce NETs.
Stimulation of the above-mentioned receptors activates protein kinase C (PKC), which initiates a signal transduction cascade inducing the assembly and activation of the NADPH oxidase complex. The kinetics of kinase activation and the consequences of kinase inhibition and silencing have revealed a critical role for a PKCδ-PAK-class I PI3K/Akt1 cascade in the regulation of p40phox activation upon bacterial challenge.
There are four lines of evidence that indicate that ROS are required for NET formation. First, H2O2, which is the product of the dismutation of O2-, is a potent inducer of NETs at physiological concentrations. It is important to note that H2O2, as opposed to O2-, is membrane permeable and has a longer half-life than O2-, suggesting that exogenous H2O2 might target the same molecules as the endogenously produced H2O2. The second line of evidence for the requirement of ROS in NET formation is pharmacological. The oxidase inhibitor diphenylene iodonium (DPI), at the minimal concentration needed to block the respiratory burst, also blocks the formation of NETs. The third line of evidence is that exogenous catalase, which degrades H2O2 to molecular oxygen and water, blocks NET formation, whereas an inhibitor of catalase, 3-amino-1,2,4-triazole, increases NET formation. Last, and most importantly, neutrophils that are isolated from CGD patients fail to produce NETs when stimulated with either bacteria or phorbol myristate acetate (PMA), that is, when stimulated upstream of NADPH oxidase. Interestingly, these neutrophils do produce NETs when they are stimulated downstream of NADPH oxidase with H2O2.
The requirement for ROS in NET formation indicates that ROS also act as second messengers in this pathway, promoting the downstream signalling events that culminate in NET formation.
Beneficial suicide: why neutrophils die to make NETs, 2007
Unconventional Roles of the NADPH Oxidase: Signaling, Ion Homeostasis, and Cell Death, 2007
"Novel cell death program leads to neutrophil extracellular traps, 2007": http://www.ncbi.nlm.nih.gov/pubmed/17210947
NET formation is an active process that involves the rearrangement of the nuclear and granular architecture. Under NET activation conditions, in vitro, neutrophils flatten and become motile and phagocytic, and the respiratory burst peaks within the first hour of activation. One hour after activation, the nucleus starts to lose the lobules and the chromatin segregates into transcriptionally active (euchromatin) and inactive (heterochromatin) regions. The result is a nearly globular structure filled with amorphous chromatin. The two membranes of the nuclear envelope gradually separate. One hundred and twenty minutes after stimulation, the continuity of the nuclear envelope is lost and it disintegrates into a chain of individual vesicles. Thus, the karyoplasm and cytoplasm are no longer separated and the chromatin comes into direct contact with the cytoplasmic components. Concurrently, the granules gradually dissolve. As a result, after 180 minutes of activation, NET components can freely mix in the cell and 15–60 minutes after that, NETs are released. Using markers for cell death and NET formation in video microscopy experiments, it has been demonstrated that NET components are released at the moment the cell membrane ruptures and the cell dies; depending on the experimental conditions, this occurs 45–240 minutes after activation. This form of cell death, which in a recent review was termed 'NETosis', is clearly different from both apoptosis and necrosis. Although the nuclei of necrotic cells lose the segregation between eu- and heterochromatin inside an intact nuclear envelope, apoptotic nuclei exhibit strongly condensed chromatin and are separated into several membrane-bound apoptotic bodies. By contrast, activated neutrophils gradually dissolve their nuclear membranes and the granules, thus allowing mixing of NET components in the cytoplasm. In addition to the morphological characteristics described above, the cell death that leads to NET formation is unique, because it does not require caspases and it is not accompanied by DNA fragmentation, but it is not the result of a direct disruption of the cell membrane, similarly to what happens when cells are attacked by bacterial toxins or complement.
At this point, the molecular mechanisms connecting ROS production and the morphological changes described above are not understood. So far, the collapse of the nuclear envelope has only been described in relation to mitosis and meiosis. Whether the formation of NETs involves a molecular machinery similar to that used during cell division is unknown. The lack of cell lines with true neutrophil characteristics and the lack of genetic accessibility to these terminally differentiated cells complicate the analysis of the process.
NADPH oxidase NOX2 (the form of nicotinamide adenine dinucleotide phosphate oxidase expressed in phagocytes) is an enzyme complex that generates reactive oxygen species (ROS). It consists of a membrane-bound b-type cytochrome, made up of integral gp91phox (glycosilated subunit; phox stands for phagocyte oxidase) and p22phox (nonglycosylated subunit) subunits, to which the cytosolic factors p67phox, p47phox, and Rac are recruited after activation. NOX2 contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O2), resulting in the generation of superoxide ion (O2−).
When neutrophils bind and internalize bacteria or other particulate prey, NOX2 assembles on the membrane of the phagocytic vacuole, delivering ROS into its lumen.
Generating ROS in order to eliminate pathogens has traditionally been considered the primary role of NOX2. However, in addition to its antimicrobial function, the oxidase plays a number of less well-known roles in neutrophil physiology, such as regulation of NET formation.
Fuchs et al. in 2007 demonstrated the role of NADPH oxidase in the formation of NETs. Stimulation with live bacteria or PMA triggers the assembly and activation of NADPH oxidase and the production of ROS. Therefore, they tested whether this enzyme was required to make NETs or not. The NADPH oxidase inhibitor diphenylene iodonium (DPI) prevented NET formation, ROS production, and cell death upon activation with S. aureus or PMA. To test whether stimulation of neutrophils downstream of NADPH oxidase produces NETs, they generated hydrogen peroxide exogenously using glucose oxidase (GO) and showed that stimulation with hydrogen peroxide, which is membrane permeable, induces NETs. To determine whether ROS were regulating the process of NET formation they tested the role of catalase in this process. Catalase converts hydrogen peroxide into water and dioxygen. Accordingly, the presence of exogenous catalases reduced NET formation in response to PMA activation.
They tested neutrophils isolated from five patients that had mutations in the NADPH oxidase. They confirmed that neutrophils from each of these patients are unable to generate ROS upon PMA activation. Interestingly, neutrophils isolated from CGD patients activated with S. Aureus or PMA did not show NETs and lacked the morphological changes characteristic of NET formation, such as breakdown of the nuclear envelope and the mixing of NET components within the cytoplasm. However, neutrophils from these patients, when stimulated with GO, formed NETs that were similar to those made by neutrophils from healthy donors.
Later, in 2011, Metzler et al. described the role of granule enzyme myeloperoxidase (MPO) in neutrophil extracellular trap formation. Patients completely deficient in MPO fail to form NETs upon exposure to PMA. The observation that singlet oxygen is essential for NET formation further substantiates the involvement of MPO and MPO-derived hypoclorous acid (HOCl) in NET formation. In addition to NADPH oxidase, the mitochondrial electron transport chain is another source of intracellular ROS.
Under experimental conditions by Palmer et al., inhibition of MPO by 4-ABAH (4-Aminobenzoic acid hydrazide) inhibits the formation of NETs. Therefore they supplemented the product of MPO, HOCl, directly to neutrophils and it was observed to elicit NET release. Then neutrophils obtained from patients with CGD were exposed to HOCl to ascertain whether NET release could be evoked, despite the absence of a functional NADPH oxidase system. Neutrophils from CGD patients did not release NETs when stimulated with PMA, but were able to release NETs upon exposure to exogenous HOCl.
Taurine is found abundantly within the cytoplasm of neutrophils (at ~50 mM ) and is known to neutralize HOCl by forming taurine chloramine and essentially removing H2O2 and HOCl to promote cell survival. Indeed, taurine chloramine activates Nrf2 (transcription factor that induces the expression of various genes including those that encode for several antioxidant enzymes) and a battery of downstream cytoprotective anti-oxidant enzymes (including haem-oxygenase-1; glutathione-transferase; peroxiredoxin; thioredoxin), thus promoting cell survival. Therefore the role of taurine was examined by its addition prior to stimulation of NET release using both PMA (to stimulate endogenous HOCl generation) and also following direct addition of HOCl (0,75 mM). Taurine treatment reduced NET release significantly in response to PMA at 100 mM and in response to HOCl at only 10 mM. This difference is likely to be due to both taurine and HOCl being added exogenously, and therefore the HOCl was likely to have been neutralized prior to entering the cell, unlike PMA which stimulates HOCl generation by direct intracellular activation of PKC.
These data confirm previous studies performed prior to the advent of NET biology, that taurine is capable of rescuing neutrophils from programmed cell death. There were also functional studies that showed a significant decrease in neutrophil chemiluminescence responses and a decrease in phagocytosis of S. Epidermidis in taurine-deficient cats compared to cats fed the same diet supplemented with taurine.
The data reported by Palmer demonstrate a pivotal role for HOCl in NET release by peripheral blood neutrophils, identifying for the first time the trigger-point downstream of H2O2. Further studies of this pathway may provide opportunities for therapeutic developments in patients with CGD or in sepsis where NET production may enhance the resolution of infection or, conversely, may contribute to autoimmune and/or autoinflammatory disease mechanisms.
"Hypochlorous acid regulates neutrophil extracellular trap release in humans, 2012": http://www.ncbi.nlm.nih.gov/pubmed/22236002
