**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 [74] and psoriasis [75]. 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 [76] 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 con‐ stitutive NET formation [77]. Chronic inflammation is also characteristic in cardiovascular diseases and indeed, NETosis was found to contribute to the pathomechanism of deep vein thrombosis [78], acute myocardial ischemia/reperfusion in a mouse model [79], and NETs were observed to be localised in limb artherosclerotic plaques [80]. 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 [81]. 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 dur‐ ing the course pregnancy in healthy women when compared to matching non‐pregnant control donors [82]. 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 [83]. In the latter study, the authors identi‐ fied 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 [84]. 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 [85]. 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**

*3.4.3. Toll‐like receptors*

50 Role of Neutrophils in Disease Pathogenesis

due to surgical stress [72].

**4. Pathophysiology of post‐injury NETs**

**4.1. The role of NETs in sterile inflammation**

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

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 [74] and psoriasis [75]. 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 [76] 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 con‐ stitutive NET formation [77]. Chronic inflammation is also characteristic in cardiovascular diseases and indeed, NETosis was found to contribute to the pathomechanism of deep vein thrombosis [78], acute myocardial ischemia/reperfusion in a mouse model [79], and NETs were observed to be localised in limb artherosclerotic plaques [80]. 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 [81]. 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 dur‐ ing the course pregnancy in healthy women when compared to matching non‐pregnant control donors [82]. 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 [83]. In the latter study, the authors identi‐ fied a unique protein in the umbilical cord blood‐called neonatal NET‐inhibitory factor 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 inten‐ sive care unit population with systemic inflammatory response syndrome [87]. 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 [52]. 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 [88]. This result indi‐ cates 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 [48]. 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 [39].

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 [4], and other authors have found the same phenomenon under certain conditions [89]. 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 useful‐ ness 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 [91]. Juvenile‐onset systemic lupus erythe‐ matosus patients' isolated PMNs showed augmented NET formation, which was significantly modulated with HQ treatment [92]. N‐acetylcisteine (NAC), which is a commonly recom‐ mended supplement to treat various autoimmune symptoms, was described to inhibit NET release by PMA stimulated human neutrophils in a ROS‐dependent manner [93]. The appli‐ cation 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 [95]. Another FDA‐approved monoclonal antibody, Rituximab was also demonstrated to be protective against adverse NET formation in different human studies [96].

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 [97], and or PAD4‐mediated NET formation was described to be not essential against infection [62].

The direct inhibition of the granule and protein components of NETs is another way to manip‐ ulate NET formation. However, these are essential antimicrobial peptides and mediate impor‐ tant 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 for‐ mation [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 immu‐ nostimulatory 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) [52]. Human recombinant DNAse therapy has been used to good effect when nebulised in cystic fibrosis (CF) patients by enhancing sputum solubilisation [103]. 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 dramati‐ cally increased elastase activity [108]. Thus, the combined administration of DNase and spe‐ cific inhibitor could be useful to avoid the deleterious effects of excessive proteases. With such an emergent role of mtDNA in NETs associated with trauma [4] and more recently in SLE [49], the investigation of DNAse therapy in different inflammatory conditions including post‐injury inflammation would be very reasonable. Nevertheless, a long‐term DNase ther‐ apy presents side effects to patients [109] including dramatic increase in other antimicrobial activities [108] 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 [112].

#### *4.3.3. The clinical predictive value of NETs*

*4.3.1. Chemical inhibition of NETosis*

52 Role of Neutrophils in Disease Pathogenesis

formation in different human studies [96].

to be not essential against infection [62].

*4.3.2. The therapeutic effect of DNAse treatment*

[100–102].

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 [91]. Juvenile‐onset systemic lupus erythe‐ matosus patients' isolated PMNs showed augmented NET formation, which was significantly modulated with HQ treatment [92]. N‐acetylcisteine (NAC), which is a commonly recom‐ mended supplement to treat various autoimmune symptoms, was described to inhibit NET release by PMA stimulated human neutrophils in a ROS‐dependent manner [93]. The appli‐ cation 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 [95]. Another FDA‐approved monoclonal antibody, Rituximab was also demonstrated to be protective against adverse NET

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 [97], and or PAD4‐mediated NET formation was described

The direct inhibition of the granule and protein components of NETs is another way to manip‐ ulate NET formation. However, these are essential antimicrobial peptides and mediate impor‐ tant 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 for‐ mation [28, 98, 99], but recently more evidence revealed the opposite or conditional effect

The fact that extrachromosomal DNA and particularly mtDNA have such potent immu‐ nostimulatory 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) [52]. Human recombinant DNAse therapy has been used to good effect when nebulised in cystic fibrosis (CF) patients by enhancing sputum solubilisation [103]. 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]. 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 [12]. This ground breaking work was followed by other papers, such as the one of Altrichter and co‐workers who described that circulating free‐DNA neutro‐ phil extracellular traps (cf‐DNA/NETs) could be used in the prediction of mortality in a popu‐ lation of 32 patients with severe burn injury [13]. 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 [99]. However, in these cases, the dynamic profile of circulat‐ ing 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 [81].

Beyond blood‐based extracellular trap identification, Mohanty and co‐workers described a new approach to non‐invasive NET‐associated biomarker research, which showed the pres‐ ence of numerous neutrophils in morning saliva had undergone NETosis [113]. Tear fluid might also be informative. In a study conducted on dry eye disease (DED) patients and match‐ ing 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 [114]. A similar paper characterised the activated neutro‐ phil‐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 [115].

## **5. Final remarks**

In this chapter, we summarised the mechanism, regulation and clinical significance of neu‐ trophil granulocytes and the complex process of extracellular trap formation. The relevant lit‐ erature 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 acti‐ vated, non‐necrotic cells with intact cell membrane, such as flow cytometry‐cell‐sorting, sup‐ ported 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.

### **Author details**

Eszter Tuboly<sup>1</sup> , Gabrielle D. Briggs2 and Zsolt J. Balogh1,2\*


#### **References**


[4] McIlroy DJ, Jarnicki AG, Au GG, Lott N, Smith DW, Hansbro PM, Balogh ZJ. Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subse‐ quent surgery. Journal of Critical Care. 2014 Dec;**29**(6):1133.e1–e5

**5. Final remarks**

54 Role of Neutrophils in Disease Pathogenesis

**Author details**

Eszter Tuboly<sup>1</sup>

**References**

In this chapter, we summarised the mechanism, regulation and clinical significance of neu‐ trophil granulocytes and the complex process of extracellular trap formation. The relevant lit‐ erature 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 acti‐ vated, non‐necrotic cells with intact cell membrane, such as flow cytometry‐cell‐sorting, sup‐ ported 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

and Zsolt J. Balogh1,2\*

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2 University of Newcastle, Newcastle, Australia

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