Polymorphonuclear (neutrophil) granulocytes (PMNs) are an essential part of the innate immune responses and key instigators and effectors of the underlying pathological mechanisms (endothelial damage, interstitial histolysis, cytokine production, phagocytosis) leading to post-injury inflammation and secondary tissue injury. In 2004, the formation of neutrophil extracellular traps (NETs) was identified as an additional defence mechanism of PMN against microbes. The understanding of complex regulation of neutrophil functions and NET formation is essential for differentiating between healthy and pathological inflammatory response, which frequently determines if patient recovers uneventfully or develops catastrophic complications. Recent discoveries have revealed the potential role of NETs in the pathogenesis of a wide range of non-infectious diseases, including post-injury sterile inflammation. In such conditions, both spontaneous NET formation and impaired NETosis are documented. In this chapter, we review the evidence for the role of NETs in post-injury inflammation, the key molecular and cellular participants in pathological NET formation, the clinical relevance of NETs in post-injury complications and the therapeutic potential of NET inhibition/clearance.
- neutrophil granulocyte
- post-injury inflammation
- neutrophil extracellular traps
- multiple organ failure
Despite recent improvements in the care of the injured, severe trauma remains a major burden on our society, resulting in the annual death of more than five million people worldwide (World Health Organisation. Injuries and violence: the facts. 2014.
2. Post‐injury inflammation
2.1. Complications of post‐injury inflammation
Major trauma patients universally develop systemic inflammatory response syndrome (SIRS) criteria within 72 h of injury. SIRS is defined by the following criteria:
Temperature greater than 38°C or less than 36°C.
Heart rate greater than 90 beats/min.
Respiratory rate greater than 20/min.
White blood cell count (WBC) greater than 12.0 × 109 L−1, or less than 4.0 × 109 L–1 .
The degree of the dysfunctional post‐injury inflammation is further complicated by the invasive nature of surgical procedures. Moreover, those who survive the initial severe tissue injury and traumatic shock are at an increased risk of acute respiratory distress syndrome (ARDS), multiple organ failure (MOF), nosocomial infections and sepsis. These complications lead to excessive resource utilisation and increased risk of death [7–9]. The systemic inflammatory response to major trauma can lead to the development of early MOF, which progresses to a state of immune paralysis and is viewed as a major factor underlying the increased susceptibility of trauma patients to hospital‐acquired infections [10, 11]. The possible involvement of NETs in post‐injury inflammation has been evaluated in several recent studies. Margraf and co‐workers published in 2008 that NET quantities in plasma may predict MOF and sepsis on the ICU in patients after multiple trauma , and more recently, cell free‐DNA neutrophil extracellular traps (cf‐DNA/NETs) were used in the prediction of mortality in a population of 32 patients with severe burn injury . These associations warrant further research into the precise role and impact of NETosis in the post‐injury inflammatory response. While many aspects of the post‐injury inflammatory response have been characterised over the past decades, our understanding of how NETosis fits into the picture is still rudimentary.
2.2. Mechanisms of post‐injury inflammation
In the bigger picture of the post‐injury inflammatory response, NETosis is considered a later phenomenon than the classical neutrophils functions [14–16]. Before the induction of NETosis, inflammatory reactions triggered by mechanical injury or disturbances of homeostasis are mainly propagated by intravascular events, summarised in Figure 1. The acute phase is characterised by dramatic changes in the diameter of the capillaries and the activation of innate immune cell responses. It is followed by a delayed, subacute reaction, most prominently characterised by oxido‐reductive burst, hypoxic metabolic pathways, the infiltration of leukocytes and phagocytic cells and early cytokine production, while in the late proliferative phase, reperfusion injury, further production of late inflammatory agents, tissue remodelling and fibrosis occur.
Injury leads to the release of damage‐associated molecular patterns (DAMPs) with high immunomodulatory potential (extracellular DNA, mitochondrial remnants and the high mobility group box 1) and pro‐inflammatory cytokines, such as tumour necrosis factor‐α (TNF‐α), or interleukin‐1β (IL‐1β). Release of these components results in Toll‐like receptor (TLR) activation with an effect after 1–2 h . As this phase ensues, subacute cytokines including IL‐6, IL‐8 as well as IL‐12 and IL‐18, chemokines and leukocyte migratory factors drive an exaggerated activation of PMN leukocytes, and the increased production of reactive oxygen species (ROS) plays important roles in the process . It is also widely accepted that the initial pro‐inflammatory phase switches to a later anti‐inflammatory phase with extended anti‐inflammatory cytokine release to facilitate regenerative processes; however, the pro‐inflammatory and anti‐inflammatory forces may ultimately reinforce each other, creating a state of increasingly destructive immunologic dissonance . Cytokine signals are crucial in the inflammatory cascade by promoting the interactions of PMN leukocytes with endothelial cells through the up‐regulation of adhesion molecules, PMN degranulation, respiratory burst, lipid mediator synthesis  and enhanced migration through the endothelium. Via these reactions, the soluble mediators alter the microvascular homeostasis [21, 22] and blood flow, which have been associated with multiple organ failure . Of the cytokines, members of the low molecular weight chemokine family play a fundamental part in these events by virtue of their ability to attract and stimulate leukocytes . These mediators mutually and strictly regulate the expression level and generation of each via epigenetic regulation that propagate the commencement of repair mechanisms, although numerous cytokines are reported to be aberrantly regulated in association with more complicated clinical outcomes [25, 26].
While phagocytosis and degranulation usually take minutes to occur after being exposed to the inflammatory signal, NETosis is a more protracted event, takes place from 2–3 h up to 8 h from activation [27, 28]. About 20–60% of isolated human neutrophils typically release NETs 2–4 h after stimulation with microbes or chemicals . However, they were able to respond within minutes when activated by LPS‐stimulated platelets under conditions of flow . These studies suggest that NET formation might be more characteristic for the subacute/late phase of post‐injury inflammation and probably more inherent to the senescent PMN population. It is hoped that future studies will identify which factors determine the selection between these alternative antimicrobial activities and whether these processes can coexist in the same cell (Figure 1).
3. Mechanisms of NETosis
As members of the first‐line defence of the immune system, neutrophils are well known to interact with other cell types and active cellular crosstalk is followed by release of inflammatory mediators, stimuli‐specific receptor‐activation and homing. NET formation is described to occur in a particularly versatile manner under different pathophysiological conditions, and the complexity is just the beginning to be explored. We are yet to clarify which factors are required to prevent NET formation of a neutrophil and whether this alternative pro‐inflammatory function of the cells can co‐exist with the classical responses of the same cell. The current view of the role of surrounding cells, soluble mediators and intracellular elements is overviewed below.
3.1. Structure and function of NETs
NETosis has been described as a process in which activated neutrophils extrude a chromatin‐fibre‐based meshwork encompassing their own granules and antimicrobial enzymes, such as neutrophil elastase, cathepsin G, α‐defensines and MPO . Mass spectrometry results have revealed a series of additional protein components from various types of granules . The extrinsic and intrinsic factors contributing to NET formation are summarised in Figure 2.
These structures represent an important strategy to immobilise and kill invading microorganisms and are considered to be evolutionarily conserved, since they target both Gram‐negative and Gram‐positive bacteria, viruses and fungi . Besides humans, the phenomenon was proven to be present in insects, various vertebrates including fishes and even in plants [32–36]. The NET scaffold consists of chromatin components with a diameter of 15–17 nm and the connected proteins and microparticles. To date, nuclear DNA and histones are observed to represent the major NET constituents . The exact mechanism through which the genetic material is ejected from the cell and decorated by antimicrobial factors is still not well understood. Nonetheless, it is considered to be an active process, where the cells undergo an apoptosis‐like process with peptidylarginiedeamilase 4 (PAD‐4)‐mediated DNA decondensation, membrane disintegration and chromatin realignment , and the role of ROS formation in the process seems to be inevitable, but the mechanism remains controversial .
3.2. Post‐injury activators of NETosis
Studies aimed at describing the receptor‐ligand signalling pathways are fundamental in sterile NET formation revealed diverse and sometimes controversial mechanistic details. Endogenous ligands were described to bind to TLR (mainly TLR4 and TLR9), Fc receptors (e.g. FcRIIa) or cytokine receptors (such as IL‐17 R) accompanied by this process [38–40]. Complement receptor activation has also been reported to be implicated . Many sterile chemical stimuli were proven to induce NETosis in vitro without infection such as TNF‐alpha, IL‐8, interferon‐gamma, nicotine certain antibiotics or enhanced ROS generation produced by NADPH oxidases [1, 42–46].
As NETs consist of a significant amount of extracellular DNA as a scaffold, injury‐related NET formation may cause a further elevated DAMP concentration in the circulation, and therefore, it could result in more severe tissue damage [4, 48]. Mitochondrial DNA was subsequently demonstrated to be a trigger for NETosis after major trauma and demonstrated that the signalling was mediated through a TLR9‐dependent pathway, independent of the NADPH oxidase system . Our group demonstrated that NETs formed after trauma were almost exclusively composed of mtDNA . There has also been a relationship demonstrated in NETosis observed in systemic lupus erythematous (SLE) where NETs released were found to be highly enriched with oxidised mtDNA . Interestingly, this study also found that these NETs resulted in increased production of IFN I, which was dependent on STING pathway signalling. This perhaps suggests that mtDNA may play a role in driving autoimmunity in a rather novel and previously unstudied way.
3.3. Cell‐cell interactions as regulators of post‐injury NET formation
3.3.1. Interaction with platelets
There is growing evidence on the importance of neutrophil‐neutrophil crosstalk and communication with other cells related to NET formation. Platelets are far the most characterised players in NETosis as many platelet originated ligand/receptor pairs and soluble mediators perpetuate neutrophil activation . The proof‐of‐concept in vitro studies demonstrated that platelet activation is crucial as the initial step [29, 51]. Human neutrophils isolated from healthy volunteers underwent a robust NET formation in the presence of activated platelets treated with thrombin receptor‐activating peptide, while no NETosis occurred with the co‐incubation of resting platelets . In the same study, the early event of platelet‐platelet interaction was blocked with a glycoprotein IIb/IIIa inhibitor and resulted in reduced NET formation in a mice TRALI model . P‐selectin is suspected to largely be responsible for the ability to trigger sterile NET formation in human neutrophils , but other cell adhesion molecules found on platelets are demonstrated to play rather significant role as β2 integrin (CD18) [53, 54]. Among soluble mediators, chemokines (as CXCL4) and alarmins (as HMGB‐1) produced by platelets were observed to activate neutrophils to form NETs in vitro and in animal models [54, 55]; however, this feature of platelets is broadly connected to any kind of inflammatory response, and therefore, the direct or indirect contribution of this phenomenon is too limited to be predictable.
3.3.2. Endothelium‐neutrophil interactions
Circulating neutrophils tend to be quiescent and inactive, while their activation classically depends on their communication with endothelial cells. After neutrophil‐endothelial interaction, the cells can rapidly undergo degranulation, activation of their NADPH oxidase system and even NET formation [56, 57]. The importance of this interface is also supported by more recent studies, where endothelium‐produced matrix metalloproteinases induced NET formation followed by cytotoxicity and vessel dysfunction [58, 59].
3.4. Intracellular and molecular regulators of NETosis
Neutrophil extracellular trap formation is primarily dependent on histone abundance and alignment, activation of NADPH oxidase and MPO, interactions between platelets and neutrophils, expression of NET component proteins, and neutrophil autophagy.
3.4.1. The role of chromatin decondensation
Peptidylargininedeiminase 4 (PAD4)‐mediated chromatin decondensation, which occurs in the nucleus, is apparently a critical and initial step in NET formation. PAD4 is a nuclear enzyme that converts specific arginine residues to citrulline on histone tails . The release of NETs strongly depends on PAD4 activity  but was surprisingly found not to be essential in certain conditions . Neutrophils isolated from PAD4‐deficient mice were unable to citrullinate histones, decondense chromatin, and generate NETs . In fact, PAD inhibitors have demonstrated efficacy in a variety of immune pathologies [64, 65], supporting the importance of this pathway in NET formation.
3.4.2. NADPH‐dependent ROS production, Raf‐MEK‐ERK pathway
Hakkim and co‐workers first described the importance of the Raf/MEK/ERK signalling pathway in PMA‐induced NET formation and their data suggest that the Raf‐MEK‐ERK pathway might be upstream of NADPH oxidase activation . Other studies pointed out that phosphorylation of ERK both in platelets and in neutrophils is also necessary for the formation of NETs mediated by activated platelets [52, 53].
3.4.3. Toll‐like receptors
Toll‐like receptors are classified according to the types of agonists that bind and the corresponding response that is activated and several of them were found to facilitate profound inflammatory responses after binding endogenous ligands . It was recently reported that neutrophil stimulation via TLR activation with various molecules leads to NET production. Further to this, the structure of the NETs is characteristic to the type of TLR stimulation . TLR4 seems to be responsible for this kind of neutrophil activity in particular as many publications demonstrated their interaction via HMGB‐1 , superoxide production , platelet activation  or IL‐1β . Oxidised low‐density lipoprotein, which has been implicated as an independent risk factor in various acute or chronic inflammatory diseases including SIRS, was also found to act as a NETosis trigger via TLRs . More recently, TLR9 has come into focus in NET research as mtDNA and other DAMPs that are recognised by TLR9 showed high potential to induce NETs in trauma patients , in liver ischemia/reperfusion injury  or due to surgical stress .
4. Pathophysiology of post‐injury NETs
4.1. The role of NETs in sterile inflammation
Recent discoveries have revealed the potential role of NETs in the pathogenesis of a wide range of non‐infectious diseases, in particular sterile chronic inflammatory conditions such as systemic lupus erythematous [38, 73], small vessel vasculitis  and psoriasis . In such conditions, both spontaneous NET formation and impaired NETosis were evident. Reduced ability of PMNs for to undergo NETosis was described in diabetes mellitus patients who were exposed to bacterial infections  that might be a possible explanation for why this population is more susceptible to life‐threatening infections. In another recent study conducted on diabetes patients, spontaneous release of isolated PMN NETs was increased, suggesting that a chronic pro‐inflammatory condition during hyperglycaemia favours constitutive NET formation . Chronic inflammation is also characteristic in cardiovascular diseases and indeed, NETosis was found to contribute to the pathomechanism of deep vein thrombosis , acute myocardial ischemia/reperfusion in a mouse model , and NETs were observed to be localised in limb artherosclerotic plaques . Furthermore, the content of plasma MPO‐DNA complexes was found to be associated with an increased risk of coronary stenosis in patients with severe coronary arthelosclerosis . Interestingly, healthy conditions but with an altered metabolic and oxygen consumption rate were also described to be associated with elevated NETosis of isolated PMNs. In a very recent paper, NET formation and neutrophil pro‐NETotic priming were found to be augmented during the course pregnancy in healthy women when compared to matching non‐pregnant control donors . What was found to be elevated in the mother, seemed to be blocked in the foetus, as newborn neutrophils isolated from umbilical cord blood on the day of delivery did not form NETs when stimulated . In the latter study, the authors identified a unique protein in the umbilical cord blood‐called neonatal NET‐inhibitory factor (nNIF) that would raise a very interesting question of a novel foetal adaptation mechanism and therapeutic approach. Acute injuries such as AKI and ALI were both described to be relevant pathologies to study increased NETosis in humans. NET biomarkers were present in transfusion‐related acute lung injury patients’ blood, and in fact, NETs were produced in vitro by primed human neutrophils when challenged with anti‐neutrophil alloantigen‐3a antibodies previously implicated in TRALI . In another human study, the cfDNA/NET content of 31 critically ill patient’s blood was in a significant positive correlation with the severity of acute kidney injury . This result encouraged the evaluation of serum (or plasma) NETs concentration as an early predictive biomarkers of complicated outcomes on the ICU.
4.2. Pathophysiology of trauma‐related NET formation
The potential role of NETs in the mechanical injury driven inflammatory response has recently been proposed [47, 86]. Similarly, the presence of NETs was demonstrated in a mixed intensive care unit population with systemic inflammatory response syndrome . NETs have also been implicated in the pathogenesis of acute lung injury and in sterile transfusion‐related acute lung injury, which are often antecedents of MOF . Recently, Grimberg‐Peters et al. published that neutrophils isolated from severely injured patients (days 1–2 after trauma) showed markedly elevated NET formation after pharmacological activation, and this effect was successfully attenuated by the treatment with hyperbaric oxygen . This result indicates the potential importance of oxido‐reductive burst in NETosis after traumatic injury, and it is well established that in such conditions, NET formation is generally NADPH oxidase‐dependent . However, the exact molecular mechanism behind is not fully understood, as indicated in a study by Itagakai and co‐worker, where human PMNs from young and elderly trauma patients formed NETs in a great number, via TLR9 activation, but independently from NADPH oxidase activation .
Moreover, the DAMP release after trauma might be fundamental in further promoting NET production. Besides its role in sterile inflammation, mitochondrial DNA may have another pivotal role in worsening the inflammatory response, via NET formation. Our recent data show NETs observed after injury and subsequent surgery can be composed of mitochondrial DNA , and other authors have found the same phenomenon under certain conditions . The exact molecular mechanism of mtDNA‐NET release is unclear; however, when a ROS production inhibitor (diphenyleneiodonium) was used, mitochondrial DNA‐NET formation was also blocked, and no DNA was released [89, 90].
4.3. NETs as therapeutic target for post‐injury inflammation
To date, the contribution of NET formation on the pathomechanism of a wide range of clinical conditions is evident, and there is emerging evidence about the potential therapeutic usefulness of pharmacological NET inhibition. While animal experiments and in vitro cell culture studies are promising, it is yet unknown if NET‐targeting therapies can be effective in clinical practice. As many protective physiological and pathophysiological processes require NET formation, the harm/benefit ratio of NET formation inhibition is unclear.
4.3.1. Chemical inhibition of NETosis
There are several drugs already used in clinical practice in autoimmune diseases that have potential for NETosis inhibition. Plaquenil Sulphate (hydroxychloroquine, HQ) is a disease‐modifying anti‐rheumatic drug, which inhibits prostaglandin and cytokine synthesis, and most of all induces a blockade in TLR signalling . Juvenile‐onset systemic lupus erythematosus patients’ isolated PMNs showed augmented NET formation, which was significantly modulated with HQ treatment . N‐acetylcisteine (NAC), which is a commonly recommended supplement to treat various autoimmune symptoms, was described to inhibit NET release by PMA stimulated human neutrophils in a ROS‐dependent manner . The application of NAC had similar effect in other recently published studies [70, 94], which supports the usage of other free radical scavengers as adjuvant therapy on the ICU trauma patients. Monoclonal antibodies such as the complement inhibitor Eculizumab might open up a new perspective in drug therapies targeting NETosis based on the findings that plasma NET markers of paroxysmal nocturnal haemoglobinuria patients with thrombosis history were significantly elevated than that of controls or patients without thrombosis history, while the Eculizumab treatment normalised the values to the control level . Another FDA‐approved monoclonal antibody, Rituximab was also demonstrated to be protective against adverse NET formation in different human studies .
The inhibition of histone decondensation via PAD4 targeting of the PMNs is another potential NET‐based therapeutic target, as PAD overexpression and upregulated enzyme activity have been observed in several diseases , and or PAD4‐mediated NET formation was described to be not essential against infection .
The direct inhibition of the granule and protein components of NETs is another way to manipulate NET formation. However, these are essential antimicrobial peptides and mediate important physiological pathways. Currently, the literature is conflicting as to whether MPO, NE and the other compounds connected to the NET scaffold are appropriate targets. In one study, MPO‐facilitated ROS‐generation was proven to be required for neutrophil extracellular trap formation in humans and pharmacological inhibition of MPO delays and reduces NET formation [28, 98, 99], but recently more evidence revealed the opposite or conditional effect [100–102].
4.3.2. The therapeutic effect of DNAse treatment
The fact that extrachromosomal DNA and particularly mtDNA have such potent immunostimulatory effects makes it an exciting and very rational target for immunomodulation therapy and silencing NET formation is one of the many possible trends. Whether nDNA or mtDNA are conjugated with NETs, both are readily digestible with DNAse. There is certainly good evidence to suggest that focally targeting NETs with DNAse have yielded a reduction in associated inflammatory lung damage in a mouse model of transfusion‐related acute lung injury (TRALI) . Human recombinant DNAse therapy has been used to good effect when nebulised in cystic fibrosis (CF) patients by enhancing sputum solubilisation . This effect may be beneficial to other conditions with excessive NETosis, as several studies have recently demonstrated that NETs and NET‐associated proteins are present in CF sputum [104–107]. However, there might be dangerous consequences if the extracellular DNA is not cleared up perfectly or if the freely floating pro‐inflammatory peptides have entered the bloodstream. Dubois and colleagues have demonstrated that DNase administration to CF sputum dramatically increased elastase activity . Thus, the combined administration of DNase and specific inhibitor could be useful to avoid the deleterious effects of excessive proteases. With such an emergent role of mtDNA in NETs associated with trauma  and more recently in SLE , the investigation of DNAse therapy in different inflammatory conditions including post‐injury inflammation would be very reasonable. Nevertheless, a long‐term DNase therapy presents side effects to patients  including dramatic increase in other antimicrobial activities  or further impedance of the immune system which makes the host susceptible to disseminated and lethal infections [110, 111]. The latter has notable consideration in the management of major trauma patient as 39.5% of trauma deaths occur in the hospital mainly due to nosocomial infections .
4.3.3. The clinical predictive value of NETs
The number of studies investigating the presence or the predictive value of NETs and NET components alongside extracellular DNA concentration as potential biomarkers in different human body fluids has grown significantly in recent years. Serum and plasma certainly are the most investigated materials, as being the natural habitat for PMNs, although it raises some concern whether activated NET‐forming PMNs are representative enough in the blood.
In cases of acute injuries, such as major trauma, quantification of NETs from blood seems to be a trustworthy biomarker for clinical prediction. Margraf and co‐workers published in 2008 that NETs quantities in plasma may predict multiple organ failure and sepsis on the ICU in patients after multiple trauma . This ground breaking work was followed by other papers, such as the one of Altrichter and co‐workers who described that circulating free‐DNA neutrophil extracellular traps (cf‐DNA/NETs) could be used in the prediction of mortality in a population of 32 patients with severe burn injury . Similarly, early diagnosis of septic arthritis by cfDNA/NETs measurement could guide the surgical team to rescue the joint by deciding to perform an immediate operation . However, in these cases, the dynamic profile of circulating neutrophils and NETs in the acute and subacute phase of inflammation should be taken into consideration when determining the optimal timing of biomarker measurement. It is also important to note that NET components, namely DNA complexes and elastase, may also accumulate in the blood during other programs of cell death, for example, during endothelial cell apoptosis or macrophage necrosis .
Beyond blood‐based extracellular trap identification, Mohanty and co‐workers described a new approach to non‐invasive NET‐associated biomarker research, which showed the presence of numerous neutrophils in morning saliva had undergone NETosis . Tear fluid might also be informative. In a study conducted on dry eye disease (DED) patients and matching controls, tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of extracellular DNA, histones, cathelicidin, and neutrophil elastase on the ocular surface was increased significantly . A similar paper characterised the activated neutrophil‐specific biomarkers in the tear fluid among ocular graft versus host disease patients, and a marked increase in both NE and MPO concentrations was evident .
5. Final remarks
In this chapter, we summarised the mechanism, regulation and clinical significance of neutrophil granulocytes and the complex process of extracellular trap formation. The relevant literature shows that a highly specialised population of neutrophils facilitate NET formation in response to infection and also sterile inflammation. Interest in the potential role of NETs in the posttraumatic injury setting and their possible role in the subsequent inflammatory response has gained significant attention lately. To date, the contribution of NET formation on the pathomechanism of a wide range of clinical conditions was proven to be inevitable and the observation of NETosis became more important in post‐injury clinical outcome prediction.
For the better understanding of the exact mechanistic details and the role of NETs in normal recovery and disease, improved methodology and quantification are urgently needed. The current techniques combine fluorescent microscopy or fluorescent intensity measurements and generally use DNA‐intercalating dyes, while taking the risk of visualising necrotic cells with dye permeable cell membrane. Antibody‐based techniques are required to detect activated, non‐necrotic cells with intact cell membrane, such as flow cytometry‐cell‐sorting, supported by microscopic imaging. Additionally, a consensus on the structural and behavioural definition of NET formation is essential for future NET research, due to their fragility, their highly dynamic nature and their morphological heterogeneity.