Saffarzadeh, Mona; Preissner, Klaus T.
Neutrophils, as the first line of defence against microorganisms, attack invading pathogens by release of antimicrobial peptides and production of reactive oxygen species (ROS) as well as by phagocytosis (intracellular pathogen killing) that enables clearance of the invading microorganisms . Moreover, neutrophils apply another extracellular antimicrobial mechanism by expelling their entire chromatin to which granular contents (including antimicrobial peptides and enzymes) are bound and highly concentrated. These structures have been designated as ‘neutrophil extracellular traps’ (NET), which trap microbes and thereby facilitate killing of bacteria, fungi and pathogenic protozoa [2▪▪,3–8].
The beneficial role of NET during infections has been clearly shown, as deficiency in NET production [5,8,9] or destroying the NET scaffold by bacterial DNases, which disintegrate the chromatin backbone of NET [10–12], leads to increased susceptibility towards infections. However, excessive NET formation has also been observed in several pathological situations (Fig. 1) such as preeclampsia , periodontitis , appendicitis [2▪▪], sepsis , asthma , Crohn's disease , cystic fibrosis [18–20], thrombosis [21,22,23▪▪,24–26], atherosclerosis [27▪▪,28▪,29], acute lung injury or acute respiratory distress syndrome [30▪,31], as well as in different auto-inflammatory diseases such as systemic lupus erythematosus (SLE) [32–34,35▪▪,36▪▪], small vessel vasculitis  or psoriasis . As exaggerated NET formation and impairment of NET removal have been correlated with tissue damage and impaired cell functions, regulation of NET formation, dismantling NET structure or inhibiting the activity of NET components is advisable. In this concise review, we will focus on the role of NET in several pathological situations as well as on some of the strategies that have been experimentally approached and that may reduce the unfavourable consequences of NET.
REGULATION OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION
NET formation is dependent on the generation of ROS by NADPH oxidase [8,39,40▪▪,41], although in a few cases NET can also be formed independent of NADPH oxidase, for instance by Staphylococcus aureus or singlet oxygen [42,43]. However, NADPH oxidase inhibition also blocks phagocytosis [44,45], and the lack of these two pivotal antimicrobial mechanisms will result in severe infection [8,9]. Apart from superoxide generation, NET formation requires autophagy (degradation of a cell's own compartments by the lysosomal machinery), and inhibition of either autophagy or NADPH oxidase activity prevents NET formation [40▪▪]. Likewise, inhibition of autophagy not only increases the risk of intracellular infections but also interferes with cellular degradation processes (Fig. 1) [46,47].
Moreover, neutrophil-specific enzymes elastase and myeloperoxidase (MPO) are necessary for regulation of NET formation . Elastase escapes from azurophilic granules and translocates to the nucleus, degrades specific histones and promotes chromatin decondensation. Later, MPO synergizes with elastase in chromatin decondensation. Nevertheless, neutrophil elastase and MPO (as a highly productive ROS generator) not only are involved in NET formation, but they also have important roles in different processes including phagocytosis  as well as degradation of extracellular matrix . Furthermore, the Raf-MEK-ERK pathway is involved in NET formation by activation of NADPH oxidase and upregulation of antiapoptotic proteins [50▪]. As inhibition of the Raf-MEK-ERK pathway may inhibit not only NET formation, inhibition of this pathway would not be a feasible strategy to suppress generation of NET under inflammatory conditions. In addition, histone hypercitrullination catalysed by peptidylarginine deiminase-4 appears to be essential for chromatin decondensation during NET formation [51,52]. However, citrullinated histone also acts as an ‘apoptotic histone code’ to detect damaged cells, and inhibition of citrullination leads to increased tumour size in nonsmall cell lung cancer tissue [53▪▪]. Hence, histone hypercitrullination cannot be recommended to inhibit NET formation in inflammatory diseases, as it might increase the risk of carcinogenesis.
In general, it would be inappropriate to suppress NET formation completely for the following reasons: First, NET have an important role as an antimicrobial mechanism of neutrophils, and their inhibition would result in the progression of severe infections. Second, an interventional mechanism that merely and specifically inhibits NET formation has not been discovered yet. Blocking the essential processes required for NET formation, including NADPH oxidase, the Raf-MEK-ERK pathway or histone hypercitrullination, may not only inhibit NET formation but would also interfere with other cellular processes (Fig. 1). Therefore, interfering with the structure and/or activity of the generated NET components is deemed to be more feasible in order to dampen the adverse manifestations induced by NET.
INTERFERING WITH NEUTROPHIL EXTRACELLULAR TRAPS STRUCTURE/COMPONENTS
Disintegration of DNA in NET by exogenous DNase has been shown to be protective in several pathological situations associated with exaggerated NET formation. Moreover, more than 20 different proteins have been identified which are tightly bound to DNA or to each other in NET [3,54], and histones comprise the major entity . Neutralization of NET proteins, particularly histones, has been demonstrated to provide protective influence in several in-vivo pathogenicity models with exaggerated NET formation, which will be summarized below.
SLE patients carry auto-antibodies against NET proteins as well as against double-stranded DNA. A subset of SLE patients is hardly able to degrade NET, and they are positive for either DNase-1 inhibitor or anti-NET antibodies that prevent access of DNase-1 to NET. Interestingly, impairment in NET degradation is associated with disease severity in lupus nephritis . Furthermore, in SLE patients, complement components such as C1q can be deposited on NET, resulting in prevention of NET degradation due to a direct inhibition of DNase-1 [36▪▪]. Complement deposition on NET may further facilitate auto-antibody production, as increased levels of antibody against NET epitopes were detectable in patients with impaired ability of NET degradation. Therefore, targeting complement with inhibitors as well as removing NET (as a complement activator) by a DNase, which is resistant to a potential inhibitor, has been suggested to improve the disease outcome in SLE patients [36▪▪].
Several other studies concerned with the involvement of NET in SLE have shown that antimicrobial peptide LL-37 or the early response gene product high-mobility group protein B1 binds to NET and forms nondegradable complexes. These complexes, which could also stimulate production of interferon-α by plasmacytoid dendritic cells (pDCs), were found to be elevated in SLE patients [34,35▪▪]. Moreover, high levels of interferon-α may augment the susceptibility of neutrophils to produce NET [32,35▪▪]. Likewise, using a functional DNase or a protease, which can remove proteins from NET and therefore make NET more susceptible towards NET degradation, may have a beneficial impact on improvement of SLE patients.
Apart from the indirect activation of T cells via NET (i.e., NET-activated pDCs lead to maturation of myeloid dendritic cells that subsequently modulate T-cell function), NET are also able to prime T cells by reducing their activation threshold, and for this purpose direct contact of NET with T cells is required [55▪▪]. As T-cell activation plays a central role in the development of autoimmune diseases such as SLE, vasculitis or psoriasis, DNase treatment of NET can likely disrupt the required NET structure for T-cell contact and activation, and therefore may be recommended as a cotreatment of these diseases.
Recently, it has been shown that auto-antigenic protein (Cramp/LL37)–DNA complexes are also involved in aggravating atherosclerosis lesion formation via activating pDCs, whereas DNase treatment or deficiency in Cramp may have a protective effect [27▪▪]. Pro-atherogenic functions of Cramp require the association with extracellular self-DNA, whereby DNase treatment may interfere with this association. However, further investigation is needed in order to explore to what degree these complexes are degradable by DNase in the context of atherosclerosis.
Transfusion-related acute lung injury
NET are present in the lungs of patients with transfusion-related acute lung injury (TRALI) as well as in their blood, where NET biomarkers (such as circulating DNA or MPO) are detectable. NET are also found in alveoli of mice experiencing antibody-mediated TRALI, whereby platelets were demonstrated to play a causative role in NET formation [30▪,56]. Inhalation of DNase-1 could prevent alveolar NET accumulation and thereby improve arterial oxygen saturation. In these experimental studies, the authors suggested that NET may develop in the lungs during TRALI and contribute to the disease process, and thus could be targeted to avert or treat TRALI. In mice, NET targeting with antihistone antibody as well as inhibition of platelet activation [30▪] can decrease lung oedema and increase survival in experimental TRALI. However, the histone/antihistone antibody complexes can also provide auto-antigenic determinants, which may further activate adaptive immunity. Whether the used antihistone antibodies can also be efficient in human patients needs to be studied.
Another disease that is associated with exaggerated DNA accumulation and NET formation is cystic fibrosis [18–20]. DNase inhalation as a therapeutic intervention is one of the successful treatment strategies for many but not all cystic fibrosis patients, as it improves lung function and reduces infectious exacerbations . Dissociation and dilation of accumulated extracellular DNA and NET in the airways of patients via DNase is probably responsible for the improvement of lung function. Interestingly, the combination of poly(aspartic acid) with DNase has been reported to be more effective to disrupt DNA/F-actin bundles present in the biofilm (produced by bacteria in cystic fibrosis patients) in comparison to DNase alone [58,59]. This effect is probably due to the fact that polyvalent anions will expose more cleavage sites in the extracellular DNA to DNase. Moreover, whether antihistone approaches can also improve lung function in cystic fibrosis needs to be investigated.
Involvement of NET in different experimental in-vivo models of thrombosis has been clearly demonstrated by several groups [21,22,23▪▪,24,25]. NET not only provide a ‘foreign’ surface that allows binding and activation of the contact-phase system of the intrinsic coagulation pathway [22,60] but also the extrinsic coagulation pathway is triggered by NET. Here, NET-associated serine proteases elastase and cathepsin-G may enhance coagulation indirectly by proteolytic degradation of tissue factor pathway inhibitor , the major trigger protein in the onset of blood clotting. As therapeutic approaches, interference with histone components of NET (e.g., by an antihistone antibody), disintegration of NET by DNase-1 or using the anticoagulant heparin can prevent thrombus formation [21,22]. As DNase-1 preferentially degrades protein-free DNA , a combination of DNase-1 with plasmin, which degrades histones, was found to be more efficient in removing the blood clot than DNase-1 alone [26,61]. Despite its harmful nature in thrombus generation in large vessels, NET production (mediated by platelet–neutrophil aggregate formation) in, for example, microvessels of the liver may trap circulating bacteria and thereby prevent their dissemination into tissue , thus assisting the bacterial killing properties of NET.
Apart from plasma DNase-1, monocytes and macrophages may also play an important role in DNA degradation by providing their lysosomal DNase-2 [26,62]. However, the presence of DNA–protein (histones and nonhistones) complexes in NET may generate an inefficient environment for these deoxyribonucleases. Moreover, hypoxic conditions or ischaemic stroke models are associated with increased levels of circulating nucleosomes and DNA , which may originate from dying neurons or from NET. Also, in such models, antihistone antibodies as well as DNase-1 had protective effects on cerebral ischaemia/reperfusion injury, even after onset of reperfusion. It was suggested that DNase-1 may attenuate blood clotting by cleaving long DNA polymers into smaller fragments to become less procoagulant.
Platelets also play an important role in the orchestrated action of NET in thrombosis and inflammation, as activated platelets are among the strongest and fastest inducers of NET formation [15,21,30▪]. Recently, von Bruhl et al. [23▪▪] have demonstrated that NET are also involved in the propagation of deep vein thrombosis (DVT) via binding to coagulation factor XII and providing a scaffold for its activation. The important role of activated platelets in this concerted action is emphasized by the fact that in their presence, the capacity of neutrophils to trigger factor XII activation (and subsequently intrinsic blood coagulation) was significantly increased. The role of NET in DVT propagation was confirmed, as neutropenia, genetic ablation of factor XII or disintegration of NET by DNase could protect against DVT progression [23▪▪].
Moreover, extracellular histones (independent of NET) may also induce thrombin generation and rapid thrombocytopenia. Neutralization of histones using heparin, activated protein C (APC) or antihistone antibodies bears an appreciable therapeutic impact [64–67]. On the basis of their interaction with platelets or red blood cells as well as polyphosphate, fibrinogen and other proteins of the coagulation system, histones may serve as procoagulant and clot-promoting cofactors [21,22,23▪▪,66,68–70].
Other pathological situations
Apart from the aforementioned models, antihistone antibodies have protective effects in pathogenetic models of sepsis  or liver injury [64,71]. Extracellular histones have cytotoxic activity [54,68], and it seems that Toll-like receptors 2, 4 and 9 are involved in histone-mediated cytotoxicity [64,71].
Although in the aforesaid models, the targeting of histones could have protective effects and the presence of NET was also correlated with disease severity, this may not rule out the contribution of histones and/or nucleosomes from events other than NET formation, such as apoptosis or perhaps necrosis [72,73]. In fact, in a purified system, APC-mediated degradation of isolated histones could dramatically abolish their cytotoxicity, although APC was ineffective to reduce NET-mediated cytotoxicity, suggesting that histones in NET are protected against APC . In addition, high APC doses, which were used to suppress the histone cytotoxicity in a mouse model of sepsis, cannot be used in human patients due to the high risk of bleeding . As a novel approach, polysialic acid was shown to dramatically reduce both isolated histone and NET-mediated cytotoxicity . Whether the reduction of cytotoxicity by polysialic acid is merely due to its anionic interaction with histones needs to be further investigated, as there are many other positively charged proteins in NET to which polysialic acid may bind and interfere with or neutralize their functions.
In studies dealing with NET, mostly histones have been targeted to reduce the cytotoxic or thrombotic properties of NET. Due to the presence of other proteins in NET, extensive studies need to be carried out to decipher different cytotoxic components of NET. Furthermore, DNA–protein or protein–protein complexes as well as posttranslational modifications of proteins in NET may serve to protect these components from degradation by DNase or proteases. All of these aspects should be considered in studies dealing with NET to design the most efficient inhibitors, antibodies or proteases in order to dampen the inauspicious consequences of exaggerated NET formation.
Controlling the unfavourable consequences of NET needs careful consideration due to the complexity of NET proteins, which are entangled within the DNA fibres. As inhibition of NET formation is associated with adverse effects such as increasing the risk of infection, it may only be recommended in situations such as auto-inflammatory diseases or severe sepsis in which the detrimental consequence of NET formation outweighs its beneficial function. In addition, intervention with compounds that would target only single proteins in NET may not be sufficiently effective, as NET proteins may be trapped within protein complexes or are posttranslationally modified. Administration of DNase in combination with compounds that will increase its activity by providing more exposed cleavage sites on DNA, together with antihistone approaches, may lead to improved outcome for patients (Table 1).
We apologize for not being able to include all the specific references contained in the cited reviews for space constraints. This work was supported in part by the Bundesministerium für Bildung und Forschung (BMBF, Berlin, Germany) (Clinical Research Group ‘Pneumonia’, 01-KI-1004), and the Deutsche Forschungsgemeinschaft (Bonn, Germany) (Excellence Cluster Cardio-Pulmonary System, ECCPS) as well as by the German Federal State of Hesse (LOEWE Research Network ‘Insect Biotechnology’).
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 66–67).
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A remarkable study showing that NET are also involved in the propagation of DVT via binding to coagulation factor XII. The role of NET in DVT propagation was confirmed, as neutropenia, genetic ablation of factor XII or disintegration of NET by DNase could protect against DVT progression. The important role of activated platelets was emphasized, because in their presence the capacity of neutrophils to trigger factor XII activation was significantly increased.
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A noteworthy study which demonstrates that (Cramp/LL37)–DNA complexes are involved in aggravating atherosclerotic lesion formation via activating pDCs. Also, pro-atherogenic functions of Cramp require the association with extracellular self-DNA.
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An interesting study showing that CRAMP associated with atherosclerotic lesions is neutrophil-derived.
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