**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:


The degree of the dysfunctional post‐injury inflammation is further complicated by the inva‐ sive 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 suscepti‐ bility 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 [12], and more recently, cell free‐DNA neutro‐ phil extracellular traps (cf‐DNA/NETs) were used in the prediction of mortality in a popula‐ tion of 32 patients with severe burn injury [13]. 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**

int/violence\_injury\_prevention/key\_facts/en/ Accessed 9 May 2016). Tissue injury, traumatic shock and subsequent resuscitation and surgical interventions lead to localised and systemic inflammatory responses. Polymorphonuclear (neutrophil) granulocytes (PMNs) are an essen‐ tial 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 [1]. Since the initial description of their antibacterial function, a series of studies reported the existence of NETs in response to various types of sterile inflammations including traumatic injury [2–5]. The precise triggers, contributions and outcomes of NETs in trauma patients are not well understood. Given the significant clinical impact of sterile inflammation in these patients, understanding the role of NETosis may iden‐ tify novel biomarkers or therapeutic strategies to minimise post‐injury tissue damage and hyperinflammation. In this chapter, we summarise our current knowledge and existing gaps

Major trauma patients universally develop systemic inflammatory response syndrome (SIRS)

The degree of the dysfunctional post‐injury inflammation is further complicated by the inva‐ sive 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 suscepti‐ bility 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 [12], and more recently, cell free‐DNA neutro‐ phil extracellular traps (cf‐DNA/NETs) were used in the prediction of mortality in a popula‐ tion of 32 patients with severe burn injury [13]. These associations warrant further research

 L−1, or less than 4.0 × 109

 L–1 [6].

on post‐injury NET formation.

44 Role of Neutrophils in Disease Pathogenesis

**2. Post‐injury inflammation**

**2.1. Complications of post‐injury inflammation**

**a.** Temperature greater than 38°C or less than 36°C.

**d.** White blood cell count (WBC) greater than 12.0 × 109

**b.** Heart rate greater than 90 beats/min. **c.** Respiratory rate greater than 20/min.

criteria within 72 h of injury. SIRS is defined by the following criteria:

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 homeosta‐ sis 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 promi‐ nently 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.

**Figure 1.** Schematic figure about multiple functions of neutrophils in response to sterile inflammation, where CD11b, integrin alpha M; ICAM, intercellular adhesion molecule; IL‐8, interleukin‐8 (chemokine receptor ligand 8); CXCLs, chemokine ligands; CXCR, chemokine receptor; DAMP, damage associated molecular pattern; TLR, Toll like receptor; IL‐1, interleukin 1; PY2R, purinergic receptor; FPR, formyl peptide receptors; and TNFα, tumor necrosis factor‐alpha.

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 recep‐ tor (TLR) activation with an effect after 1–2 h [17]. 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 [18]. 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 [19]. 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 [20] 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 [23]. 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 [24]. 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 [2]. However, they were able to respond within minutes when activated by LPS‐stimulated platelets under conditions of flow [29]. 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 inflamma‐ tory 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‐inflam‐ matory 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**

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 recep‐ tor (TLR) activation with an effect after 1–2 h [17]. 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 [18]. 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 [19]. 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 [20] 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 [23]. 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 [24]. 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].

the same cell (**Figure 1**).

46 Role of Neutrophils in Disease Pathogenesis

**3. Mechanisms of NETosis**

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 [2]. However, they were able to respond within minutes when activated by LPS‐stimulated platelets under conditions of flow [29]. 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

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 inflamma‐ tory 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‐inflam‐ matory function of the cells can co‐exist with the classical responses of the same cell. The 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 [1]. Mass spectrometry results have revealed a series of additional protein components from various types of granules [30]. 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 microor‐ ganisms and are considered to be evolutionarily conserved, since they target both Gram‐neg‐ ative and Gram‐positive bacteria, viruses and fungi [31]. Besides humans, the phenomenon

**Figure 2.** (A) Representative image of neutrophils forming extracellular traps visualized by fluorescent microscopy (Nikon Diaphot 300 Inverted fluorescence & phase contrast microscope, 20× magnification) after staining the cells with Sytox Green DNA intercalating dye. (B) Schematic figure on the possible mechanism of NET formation, where DAMP, damage‐associated molecular pattern; IL‐8, interleukin 8; TNFα, tumor necrosis factor‐alpha; Raf, rapidly accelerated fibrosarcoma kinase; MEK, mitogen‐activated protein kinase; ERK, extracellular signal regulated kinase; NADPH, nicotinamide adenine dinucleotide phosphate.and PAD4, protein arginine deiminase 4.

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 [1]. 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 [37], and the role of ROS formation in the process seems to be inevitable, but the mechanism remains controversial [2].

#### **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 [41]. 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 sub‐ sequently 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 [39]. Our group demonstrated that NETs formed after trauma were almost exclusively composed of mtDNA [4]. 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 [49]. Interestingly, this study also found that these NETs resulted in increased production of IFN I, which was dependent on STING path‐ way 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 com‐ munication 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 [50]. 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‐incuba‐ tion of resting platelets [52]. 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 [52]. P‐selectin is suspected to largely be responsible for the ability to trigger sterile NET formation in human neutrophils [53], 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*

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 [1]. 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 [37], and the role of ROS formation in the process seems to be inevitable, but the mechanism remains

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 [41]. 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

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 sub‐ sequently 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 [39]. Our group demonstrated that NETs formed after trauma were almost exclusively composed of mtDNA [4]. 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 [49]. Interestingly, this study also found that these NETs resulted in increased production of IFN I, which was dependent on STING path‐ way signalling. This perhaps suggests that mtDNA may play a role in driving autoimmunity

There is growing evidence on the importance of neutrophil‐neutrophil crosstalk and com‐ munication 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 [50]. 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

controversial [2].

**3.2. Post‐injury activators of NETosis**

48 Role of Neutrophils in Disease Pathogenesis

NADPH oxidases [1, 42–46].

*3.3.1. Interaction with platelets*

in a rather novel and previously unstudied way.

**3.3. Cell‐cell interactions as regulators of post‐injury NET formation**

Circulating neutrophils tend to be quiescent and inactive, while their activation classically depends on their communication with endothelial cells. After neutrophil‐endothelial interac‐ tion, 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 forma‐ tion 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 neu‐ trophils, 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 [60]. The release of NETs strongly depends on PAD4 activity [61] but was surprisingly found not to be essen‐ tial in certain conditions [62]. Neutrophils isolated from PAD4‐deficient mice were unable to citrullinate histones, decondense chromatin, and generate NETs [63]. In fact, PAD inhibitors have demonstrated efficacy in a variety of immune pathologies [64, 65], supporting the impor‐ tance 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 path‐ way in PMA‐induced NET formation and their data suggest that the Raf‐MEK‐ERK pathway might be upstream of NADPH oxidase activation [66]. Other studies pointed out that phos‐ phorylation 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 corre‐ sponding response that is activated and several of them were found to facilitate profound inflammatory responses after binding endogenous ligands [67]. 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 [68]. TLR4 seems to be responsible for this kind of neutrophil activity in particular as many publi‐ cations demonstrated their interaction via HMGB‐1 [55], superoxide production [69], platelet activation [29] or IL‐1β [70]. 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 [71]. 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 [39], in liver ischemia/reperfusion injury [3] or due to surgical stress [72].
