Triggers of NET formation. Several microbial and chemical stimuli have been identified. A summary based on [7-10].
1. Introduction
As a response to inflammatory stimuli, polymorphonuclear (PMN, neutrophil) cells are able to expel a mixture of their nuclear and granular elements. These web-like substances are called neutrophil extracellular traps (NETs), structures that are able to entrap invading pathogens. NETs are composed of DNA, histones, granular enzymes and proteins (such as cathepsin G or elastase), and seem to be a universal tool of defense: humans, animals and even plants [1] are capable of extracellular trap formation, indicating that these webs provide an evolutionarily conserved protective mechanism.
Besides their protective function, a role for NETs is emerging in the pathogenesis of many diseases [2,3], and may be of interest regarding the pathogenesis of thrombosis. Stimulation of coagulation by NETs can result in unwanted thrombosis [4] and infection is a common event in the development of deep vein thrombosis [5,6]. Targeting the release of nucleosomes, development of NETs and the availability of circulating histones could be a strategy for prevention or therapeutic intervention in venous thromboembolism, sepsis and other diseases involving cell death and lysis.
This chapter describes the formation and structure of NETs and discusses the possible connections and interrelations between this newly recognized form of innate immunity and components of the haemostatic system.
2. Triggers of NET formation
NETs can be formed in response to all major types of microbes (bacteria, fungi, protozoa, viruses) and their products, as well as inflammatory mediators, ROS, cell-cell interactions, and certain non-infectious or non-physiological stimuli. Table 1. shows a set of examples for various triggers.
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3. Formation of NETs
3.1. NET formation as a form of cell death
NETs are the results of a unique cell death program that is different from apoptosis or necrosis [11]. It is characterized by the loss of intracellular membranes before the plasma membrane integrity is compromised (NETosis). To release NETs, activated neutrophils undergo dramatic morphological changes [12]. Minutes after activation by PMA, they flatten and firmly attach to the substratum, while showing a multitude of granules and a lobulated nucleus [13]. During the next hour, the nucleus loses its lobules, the chromatin decondenses and swells, and the inner and outer nuclear membranes progressively detach from each other. Concomitantly, the granules disintegrate. After one hour, the nuclear envelope seems to disaggregate into vesicles and the contents of nucleoplasm, cytoplasm and granules are able to freely mix. After approximately 4 hours, the cells round up and seem to contract until the cell membrane ruptures and the internal components are ejected to the extracellular space [13,14]. It is important to note, that depending on stimuli and donor, only a certain percentage of the activated neutrophils make NETs [13].
Apoptosis, another form of programmed cell death, is characterized by membrane blebbing, phosphatidylserine exposure on the cell surface, nuclear chromatin condensation and DNA fragmentation
3.2. Alternative ways of extracellular trap formation
Besides the above described, first observed form of NETosis (also called
In contrast with the PMA-induced 3-4 hour-long cell death program, a recently described form,
4. Structure and composition of NETs
NETs released from neutrophils in the extracellular space consist of nuclear DNA and various histones decorated with granular proteins. NETs are fragile, complex structures composed of smooth ‘threads’, approximately 15-25 nm in diameter, which are likely to represent a chain of nucleosomes from unfolded chromatin. High-resolution scanning electron microscopy (SEM) revealed that the NET threads are studded to variable extent with globuli of 30-50 nm [14] that contain the multiple cathelicidin antimicrobial peptides which originate from the neutrophil granules (or lysosomes). Several ‘threads’ can be wound into ‘cables’ that can be up to 100 nm in diameter (Figure 1.).
These cables then form complex three-dimensional structures that, using SEM, can be hard to distinguish from fibrin networks [22]. Analysis of cross sections of NETs by transmission electron microscopy (TEM) revealed that fibers are not surrounded by membranes [23]. When produced in multiwell plates in vitro, NETs float within the medium, rather like a spider’s web does in moving air [24]. The fact that they are ‘sticky’ as a result of their electrostatic charge and that they extend over areas of several microns makes them very effective at trapping [25], and possibly killing microorganisms [24].
DNA is a major structural component, because several intercalating dyes stain NETs strongly, and deoxyribonuclease (DNAse) treatment results in the disintegration of NETs, whereas protease treatment has no such effect [23]. Accounting for approximately 70%, the most abundant component of NETs are histones [26]. All core histones as well as linker histones can be found in NETs (H1, H2A, H2B, H3, H4), although in an enzymatically processed form (see later). The aforementioned globuli contain proteins and enzymes from the primary (azurophilic) granules (e.g. neutrophil elastase, cathepsin G, myeloperoxidase, bactericidal permeability increasing protein BPI), secondary (specific) granules (e.g. lactoferrin), and tertiary granules (e.g. gelatinase or MMP-9, peptidoglycan recognition proteins PGRPs [27]) of neutrophils [28]. Cytoplasmic components, like calprotectin, a heterodimer of cytosolic S100A8 and S100A9, are rarely found in NETs [26].
These proteins exert various antimicrobial actions [29]: MPO is responsible for microbicidal HOCl generation; serine proteases (neutrophil elastase NE, cathepsin G, proteinase 3, tryptase, neutrophil serine protease 4 NSP4 [30]) are able to inactivate bacteria by cleaving their virulence factors [23]; cathelicidin LL37, BPI, defensins, and histones can disintegrate pathogen cell membranes challenging their viability [31,32]; calprotectin [26,33], calgranulin and lactoferrin chelate ions that are vital for microbial growth, altogether making NETs an effective tool virtually against all types of microbes.
NETs produced from mitochondrial DNA release have a slightly different structure [21]. NE and MPO co-localize with mitochondrial DNA, but certain nuclear (lamin B, nuclear matrix protein-45, poly-ADP-ribose polymerase, histones) and other (cytoplasmic caspase-3, beta-actin, mitochondrial cytochrome c, membrane markers CD15 and 16) elements are absent, which suggests a different type of host-NET interaction in the case of mitochondrion-derived NETs.
5. Intracellular events leading to NET formation
A unifying theory describing the subsequent steps of NET formation is still missing, but many mechanisms have been identified to contribute to NET expulsion.
5.1. Signaling events
The signaling mechanisms leading to the formation of NETs are poorly understood, and it is very likely that different triggers are able to induce NETosis through different pathways (Figure 2. [34]).
The protein kinase C (
PK-C activation (e.g. by PMA) is upstream of the
The monomeric G-protein (rho small GTPase)
The role of
Certain triggers of NETosis act through a PKC/ROS-independent pathway, possibly mediated by
Cytoskeletal elements may also play a role in transmitting signals from the cell surface to the nucleus, e.g. inhibition of the cell surface receptor integrin
5.2. NADPH oxidase and ROS formation
Most pathways converge to activate
5.3. Chromatin decondensation
One option to weaken the interaction between DNA and highly positively charged histones is the enzymatic processing. At this moment, two enzymes seem to be of greatest importance: PAD4 (peptydilarginine deiminase 4) and NE (neutrophil elastase).
PAD4, a 74 kDa protein that exists as a head-to-tail dimer [58,59] is the only member of the peptydilarginine deiminase family containing a nuclear localization signal that ensures its trafficking to the nucleus [58,60,61] (although not the only one to be found inside, e.g. PAD2 is also reported to be localized intranuclearly [62]). The activation of PAD4 is calcium-dependent: binding of calcium to the C-terminal catalytic domain induces conformational changes that lead to the adequate positioning of critical active site residues [58]. The calcium-dependency of the enzyme also serves as a possible connection between ROS generation (possibly leading to calcium release from the endoplasmic reticulum) and PAD4 activation. In addition, ROS are possible direct regulators of PAD4 [63]. Cytoskeletal activity and autophagy may also be involved in PAD4 activation, since both processes have been shown to be required for chromatin decondensation during NET generation.
The main nuclear substrates of PAD4 are arginyl residues of PRMT1 (protein arginine methyltransferase 1) [61], PAD4 itself (autocitrullination downregulating the activity of the enzyme [64,65]), and, most importantly regarding the process of NETosis, histones (H2A, H3Arg-8 and-17 or H4Arg3) [66]. Hypercitrullination of arginil residues in histones [67] weakens their interactions with DNA resulting in the dissociation of heterochromatin protein 1-β [57], and the extensive chromatin decondensation that leads to nuclear delobulation and swelling of the nuclear content [66,68].
In concert with PAD4,
5.4. Reorganization of membrane structures-the role of autophagy in NETosis
While the decondensated nuclear content expands, the space between the two membranes of the delobulated nuclear envelope starts growing, this which eventually leads to formation of vesicles and disintegration of nuclear membranes. During the final stage, nuclear and granular integrity is completely lost, which allows mixing of the chromatin and the granular components, and a rupture in the plasma membrane causes the release of extracellular chromatin traps.
However, vesicle formation is also seen in neutrophils isolated from CGD patients, which are unable to produce NETs [73]. This observation suggests that vesicles do not necessarily originate from the nuclear envelope, but ER membranes are likely to be assembled as a source of autophagic vesicles [34], in addition to possible
6. NETs and haemostasis
NETs are a newly recognized scaffold of venous [74] and arterial [75,76] (Figure 3.) thrombi (besides fibrin and von Willebrand Factor [vWF]) that allows cell localization (neutrophils, red blood cells), platelet activation and aggregation, and promotion of both (extrinsic and intrinsic) pathways of coagulation. Thus, NETs are a focus of cross-talk between immunity, inflammation and haemostasis. Here we discuss the interaction among the various players of the haemostatic system and NET components.
6.1. NETs and the vessel wall
The classic view of the intact endothelial surface emphasizes its anticoagulant role. While endothelial damage is a common initiator of arterial thrombosis, in the case of deep vein thrombosis (DVT), activation of endothelium and Weibel-Palade Body (WPB) release play a crucial role. NETs induce endothelial cell damage and death [17,77-79], an effect that is likely to be assigned to NET-associated proteases, defensins and, most importantly, histones [78,80]. Binding of histones to membrane phospholipids results in pore formation and influx of ions [81-83], this may lead to elevated endothelial calcium levels, vWF release from WPB [84], activation of endothelium, or even endothelial cell death. Endothelial ROS formed under these circumstances may, in turn, trigger NET formation by neutrophils [77]. Perfusion of iliac artery cross sections with NE results in increased thrombogenicity of the arterial wall [85], although it is not clear if NET-bound NE is able to reproduce this effect at the site of vascular damage.
NETs also contribute to the progression of atherosclerotic plaque formation in the subendothelial layer of arteries: neutrophils infiltrate arteries during early stages of atherosclerosis [86], and NETs can be detected in murine and human atherosclerotic lesions [87].
6.2. NETs and platelets
NET fibres bind platelets directly and/or indirectly, and support their aggregation [88]. When perfused with blood, NETs bind platelets serving as an alternative scaffold for platelet adhesion and activation [89].
The first step of platelet binding involves either electrostatic interactions between NET histones and platelet surface phospholipids [81]/carbohydrates [90], or histone binding to Toll-like receptors 2 and 4 [91]. Platelets also bind double and single stranded DNA in vitro [92,93]. Adhesion molecules may also play a role in thrombocyte-NET interactions, such as vWF (binding histones through its A1 domain) [94], fibronectin or fibrinogen [89,84]. The interaction of histones with platelets results in calcium influx either by pore formation [95] or by opening of existing channels [96], a process, which triggers activation of αIIbβ3 [97]. This chain of events raises the possibility of a sequential histone-induced activation of platelets (first binding to platelet surface, then, following activation, binding to adhesion molecules [88]), which could explain the unsaturable nature of histones binding to platelets [88]. When infused into mice, histones co-localize with platelets and induce thrombocytopenia and thrombosis [83,84,88], possibly partially through potentiation of thrombin-dependent platelet-activation [98].
Serine proteases may also play a role in platelet activation: NETs contain enzymatically active neutrophil elastase NE and cathepsin G [23], and these proteases potentiate platelet aggregation through proteolitically activating platelet receptors [99,100]. Some of these elements, however, play an ambiguous role in the modulation of platelet functions: e.g. NE is also an effective enzyme for the cleavage of vWF under high shear stress [101], helping the detachment of platelets from thrombogenic surfaces.
NETs also seem to bind certain interleukins that may enhance platelet activation and aggregation: the presence of IL17A and-F was shown in NET regions of acute myocardial infarction thrombus specimens [102].
Platelet-NET interaction seems to be bidirectional in many ways. Serotonin released from platelets promotes the recruitment of neutrophils [103]. Activated platelets generate ROS, such as superoxide [104], and secrete human β-defensin 1 [105], both of which can trigger formation of NETs [13,106]. Platelets pre-stimulated with LPS or collagen also induce NETosis in neutrophils [17,108], contributing to the formation of a vicious cycle of NET formation and platelet activation [74].
Interaction between platelets and NETs might also be involved in pathological situations like transfusion-related acute lung injury (TRALI) [108,109], thrombotic microangiopathies [110], or heparin-induced thrombocytopenia (HIT). During HIT, possible binding of NETs to PF4 forming an antigenic complex may offer an explanation for disease progression even after immediate removal of heparin [111].
6.3. NETs and red blood cells
Red blood cells are no longer considered as passively entrapped elements of thrombi, but cells that may promote thrombosis by exposing phosphatydilserine and altering blood viscosity [112]; furthermore, their presence modulates structural parameters of the forming fibrin meshwork through integrin-mediated fibrin(ogen)-red blood cell interactions [113].
Similarly to platelets, RBCs avidly bind to NETs after perfusion of whole blood [89], possibly through direct and indirect mechanisms. RBCs can bind DNA, since it was eluted from the surface of isolated RBCs from cancer patients [114]. Activated neutrophils or platelets (e.g. in NETs) can also recruit RBCs at very low venous shear in vitro [115]. NETs are predominantly found in the red, RBC-rich part of experimental mice DVT thrombus, suggesting that NETs could be important for RBC recruitment to venous thrombi [84].
6.4. NETs and the coagulation system
NETs offer a variety of activators for both the extrinsic and the intrinsic (contact-) pathways of the coagulation cascade [116,107] stimulating fibrin formation and deposition in vitro [89,107,116] (Figure 4.).
NE and cathepsin G, two serine proteases that are in the NETs, degrade inhibitors of coagulation [12]. NE is known to cleave tissue factor pathway inhibitor (TFPI) of the
NETs also bind factor XII and stimulate fibrin formation via the
Besides its crucial role in NET-driven thrombosis [125], PAD4 has also been shown to citrullinate antithrombin (ATIII) in vitro [126], which weakens its thrombin-inhibiting efficiency and this may be an additional factor contributing to increased thrombin generation associated with NETs. Histones also bind to fibrinogen and prothrombin [127], and can aggregate vWF [94], the significance of which is not clear.
NET components also interfere with the
6.5. NETs, thrombolysis, NET lysis
Whilst there are extensive studies on the interaction between NET components and coagulation, little is known about their effects on fibrinolysis.
Histones and DNA, representing the main mass of NETs, seem to have
Nevertheless, certain NET components may
In vitro and in vivo observations indicate that fibrin, vWF and chromatin form a co-localized network within the thrombus that is similar to extracellular matrix [84,82,116], and it is likely that each of these components should be cleaved by their own appropriate enzyme (plasmin, ADAMTS-13, and DNAses), therefore it is important to assess current knowledge on the possible ways of NET degradation in blood plasma.
NETs can be degraded by
As a possible counter-regulatory mechanism, NETs seem to protect themselves from bacterial and perhaps human DNases by limiting the availability of divalent cations (see calprotectin) and consequently the activity of these enzymes [155].
7. Conclusion
NETs are ‘double-edged swords’ of innate immunity. While they seem to be protective against a wide range of pathogens, their contribution to various diseases, and their clear prothrombotic role in the circulation may have dangerous consequences to the host. In terms of thrombosis, they seem to serve as a fundamental scaffold that supports thrombus integrity by providing a surface for activation of procoagulant proteins and platelets, in both venous and arterial thrombi. Further investigation is indispensable to determine their exact role in the process of thrombi dissolution, and to test whether breakdown of NETs (e.g. by DNases) increases the therapeutic efficiency of the current thrombolysis protocols.
Acknowledgments
This work was supported by the Hungarian Scientific Research Fund [OTKA 83023].
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