Neutrophils are the principal cells involved in host defense against microbial pathogen infections in the innate immune system (1, 2). Neutrophils also play a key role in the inflammatory response to injury (2, 3). Neutrophils are activated following injury, which is associated with an increased neutrophil accumulation in the damaged tissue or organ (4–7). Activated neutrophils infiltrate injured tissue following an injury-induced increase in the expression of adhesion molecules on endothelial cells and elevated local chemokine/cytokine levels (6–8). Severe injuries induce immune suppression, predisposing victims to postinjury complications (9–12). Neutrophils enter tissues and organs following an injury or during an infection and form neutrophil extracellular traps (NETs) (13–16). Neutrophil extracellular traps consist of neutrophil DNA, granular proteins, and several cytoplasmic proteins (17–19). Recent reports indicate that NETs may contribute to tissue damage and organ dysfunction (13, 15, 20, 21). This review provides a brief overview of the role of NETs in injury and discusses the potential mechanisms by which NETs are involved in organ function and immunity modulation following an injury. In addition, the proposed markers and therapeutic targets of NET-related injury will be discussed.
ROLE OF NEUTROPHILS IN TISSUE INJURY
Studies have shown that tissue or organ injury, including trauma and ischemia-reperfusion (IR)–induced injury, can induce a severe inflammatory response and increase the risk of development of sepsis (22, 23). Neutrophils play a pivotal role in this inflammatory component after injury. The processes of neutrophil-induced tissue injury include proinflammatory mediator release, oxygen free radical generation, proteases degranulation, and endothelial dysfunction (2, 24). Activated neutrophils can release a number of cytokines and chemokines (e.g., interleukin 8 [IL-8], macrophage inflammatory protein 1α, etc.) (25, 26), and proinflammatory mediators (e.g., IL-6, tumor necrosis factor α [TNF-α], IL-1β), and their secretion per se is regulated by immunoregulatory cytokines (27–29). These mediators and factors may participate in recruiting more neutrophils or other leukocytes to the site of infection or inflammation. Neutrophil activation can be induced by host mediators (e.g., IL-8, platelet-activating factor, and TNF-α) and pathogens (e.g., formylated peptide and lipopolysaccharide [LPS]) (28, 30–32).
Neutrophils can also fight against microorganisms by directly phagocytosing microbes or releasing cytotoxic molecules via degranulation (33, 34). Phagocytosis is the process by which neutrophils directly engulf and digest potential pathogens, as well as cellular debris. Internalized pathogens are contained in phagosomes, where antimicrobial proteins from cellular granules and reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase work together to create a toxic environment for invading pathogens (35, 36). Degranulation is the release of toxic ROS and antimicrobial granular proteins into the extracellular space. These granules contain antimicrobial proteins that fight infections in organs, including proteases, collagenases, lipoxygenases, phospholipases, and myeloperoxidase (MPO) (33, 37). Aside from phagocytosis and degranulation, neutrophils can also produce NETs during inflammation and infection (14, 17, 19, 38).
Extracellular DNA traps have been investigated in the context of neutrophil antimicrobial mechanisms, but not exclusively focusing on neutrophils, as other cells also release extracellular traps composed of DNA and antimicrobial proteins. Extracellular DNA traps can be generated by macrophages, eosinophils, and mast cells (14, 39, 40).
NET formation (NETosis)
Neutrophil extracellular traps contain granules and antimicrobial proteins, including histones, neutrophil elastase, MPO, pentraxin (PTX), lactoferrin, cathepsin G, and bactericidal permeability-increasing protein (17–19). Neutrophil extracellular trap formation, referred to as NETosis, was first described by Brinkmann and colleagues as a cell death pathway distinct from apoptosis and necrosis (18, 19). NETosis leads to the dissolution of the nuclear envelop and cytoplasmic granules, allowing chromatin to mix with granular antimicrobial proteins via the NADPH oxidase and RAf-MEK-ERK pathway (41). Subsequently, this mixture of granules, DNA, and histones is actively expelled from neutrophils into the extracellular environment, which limits the spreading of microbial pathogens. NETosis can be a protective process that sequesters microbes and prevents the spread of infection, but it can also be a pathological process that causes inflammation and serious tissue injury (42, 43). Although NETs play important roles in host defense by trapping bacteria or other pathogens, extensive formation of NETs with increased amounts of extracellular DNA may contribute to the perpetuation of inflammation and tissue damage (42, 44).
Suicidal NETosis and vital NETosis
Recent study has reported that there are two different mechanisms by which NETs are formed, including a suicidal (lytic) NETosis and a vital (live cell) NETosis (45). Suicidal NETosis results from a developing membrane rupture and the loss of activating neutrophil functions, such as leukocyte phagocytosis, recruitment, and chemotaxis. The pathway of suicidal NETosis requires hours in the timing of NET release in response to phorbol 12 myristate 13 acetate (PMA) stimulation (46). In addition, MPO and neutrophil elastase mediate the chromatin decondensation leading to DNA and granule proteins mixing within the NET vacuole, then extruded out of a perforation in the plasma membrane (45). In vital NETosis, NET release is rapidly induced by LPS, and the pathogen-associated recognition occurs through host pattern recognition receptors. This rapid NETosis did not involve cell lysis and was mediated by platelets and Toll-like receptor 4 (TLR4) involved in the activation of polymorphonuclear neutrophils (47).
Activators of NETosis
Neutrophil extracellular traps are formed in response to biological and chemical stimuli, including IL-8, LPS, PMA, interferon γ, TNF-α, activated platelets, and interaction with endothelial cells, bacteria, fungi, and complement-mediated opsonization (17–19, 48), as well as through enhanced ROS generation by NADPH oxidase (49). Neutrophil extracellular traps and NET-like structures exist in many organisms, including humans (14, 50), mice, and other animals (39, 51, 52).
ROLE OF NETS IN INJURY
Neutrophil function and NETs are critical components involved in human immune defense. Although NETs are important, an excess of NETosis can lead to tissue damage (17, 44, 45, 53). Although NETs provide an important biological advantage for the host to protect against certain microbial infections, the generation of NETs may be a double-edged sword. For example, although NETs may promote the destruction of pathogens, the same pathways may also cause injury through an overexpression of NETosis (20, 53). Neutrophil extracellular trap constituents can damage epithelial and endothelial cells, which can exacerbate inflammation-induced organ injury (17, 44, 54, 55).
NETs and organ injury
It is well known that NET formation results in an extracellular release of proteases and other neutrophil constituents that can exacerbate injury (44). Several studies suggested that the magnitude of tissue damage and organ dysfunction are associated with the degree of NET formation (15–17, 21) (Table 1).
In human studies, NETs are present in the plasma of patients with acute lung injury and appear in the lung and plasma with transfusion-related acute lung injury (TRALI) (21). Serum NET levels were significantly increased immediately or 4 weeks after transplantation and were associated with an increased risk of transplantation-associated thrombotic microangiopathy (TA-TMA) (56). Neutrophil extracellular traps are also expressed in pulmonary capillaries and hepatic sinusoids during endotoxic shock (47).
In animal studies, NETs are found in the alveoli of mice experiencing antibody-mediated TRALI. Deoxyribonuclease 1 (DNase 1) inhalation prevented the accumulation of NETs in alveoli and improved arterial oxygen saturation after TRALI (15). A recent study has shown that NETs are detected in the reperfused hind limb skeletal muscle and thrombosed vessels of wild-type mice (13). When DNase was used as a therapeutic agent to degrade the NETs, the results showed that there was a marked decrease in the NET levels detected in the muscle fibers, perivascular space, and microvascular thrombi of the ischemic-reperfused hind limb following DNase treatment (13). In a mouse model of LPS-induced acute lung injury, NET formation appeared in the lung tissue, as well as bronchoalveolar lavage fluid. These findings reveal the important role of the protein components of NETs, particularly histones, which may lead to host cell cytotoxicity and be involved in lung tissue destruction. Histones and MPO are responsible for NET-mediated cytotoxicity (57). When coincubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induce NET generation and augment endothelial damage (16). Another study has shown that transmigrated neutrophils release NETs following a transient middle cerebral artery occlusion (MCAO) in mice. Furthermore, a blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas inhibition of neutrophil proteases released and decondensed DNA in the brain (58). Previous study also showed that the IR-related damage is decreased in TLR4 mutant mice (C3H/HeJ) compared with wild-type mice following MCAO injuries (59). In contrast, Enzmann and colleagues (60) reported that absence of polymorphonuclear leukocyte infiltration and NET formation in the infarcted brain tissues after transient MCAO.
Recent studies suggest that NETs are related to thrombosis and can damage the endothelium of blood vessels (51, 61, 62). Neutrophil extracellular trap–induced thrombosis may play an important role in the pathogenesis of sepsis with reduced blood flow. Neutrophil extracellular trap production requires platelet-neutrophil interactions and can be inhibited by platelet depletion or disruption of integrin-mediated platelet-neutrophil binding. During sepsis, NET release increases bacterial trapping by 4-fold. Blocking NET formation reduces the capture of circulating bacteria during sepsis, resulting in an increased dissemination to distant organs (51, 61, 62). Neutrophil extracellular trap constituents can activate platelets and promote an excessive coagulopathy and thrombosis, resulting in endothelial cell injury and organ damage (63).
NETs-related mediators and signaling pathways in injury
Neutrophil extracellular traps play an important role in both host defense and organ injury. Neutrophil extracellular trap formation is primarily dependent on histone levels, activation of NADPH oxidase and MPO, interactions between platelets and neutrophils, expression of NET component proteins, and neutrophil autophagy (15, 17, 18). The signaling molecules in NET formation include peptidylarginine deiminase 4 (PAD4) (64), Raf-MEK-ERK (41), nitric oxide (NO) (65), TLR4 (13), high mobility group box 1 (HMGB1) (66), and mammalian target of rapamycin (mTOR) (67) (Fig. 1).
Peptidylarginine deiminase 4
Chromatin decondensation, which occurs in the nucleus, is a critical step in NET formation. Peptidylarginine deiminase 4 is a nuclear enzyme that converts specific arginine residues to citrulline on histone tails (histone H3). The release of NETs depends on PAD4 activity (68, 69). Peptidylarginine deiminase 4 is activated by inflammatory stimuli, which is dependent on cell surface and cytoskeleton signaling (70). Neutrophils cannot release NETs in PAD4-mutant mice (64). Previous studies have shown that PAD4-mediated chromatin decondensation in neutrophils is critical in the pathogenesis of venous thrombosis (71).
The Raf-MEK-ERK signaling pathway is a common pathway through which different stimuli can induce NETosis. Previous studies have shown that the Raf/MEK/ERK signaling pathway is critical for PMA-induced NET formation (71, 72). Interruption of MEK signaling reduces NET formation in platelet activation. Because ROS production is essential to NET formation, and the Raf-MEK-ERK pathway is the upstream of NADPH oxidase, it is involved in NET formation. In a molecular biology experiment, it was demonstrated that the Raf-MEK-ERK pathway appears to be involved in NET formation through the activation of NADPH oxidase and upregulation of antiapoptotic proteins (41).
Nitric oxide–mediated NETs are important for nuclear and mitochondrial DNA, as well as for proteolytic enzymes. Previous studies have shown that augmenting NO with enzymatic free radical generation results in the release of NETs. These NETs are made up of mitochondrial and nuclear DNA and release proinflammatory cytokines (65, 73).
Toll-like receptor 4
Previous study showed that LPS could stimulate HMGB1 expression and contribute to NET formation. High mobility group box 1 can induce NET formation both in vitro and in vivo through a TLR4-dependent mechanism (13, 74). In thrombosis, NETs are reported to have potential roles in clot formation via the TLR4 pathway (71, 75). Deletion of TLR4 from platelets dramatically reduces the formation of NETs (71, 75). Recently, it was shown that platelet TLR4 expression was essential for NET formation during endotoxic shock (47). In sepsis, TLR4-activated platelets induce platelet-neutrophil interactions, resulting in NET formation in blood vessels, especially in pulmonary capillaries and liver sinusoids (47).
High mobility group box 1
High mobility group box 1 may contribute to neutrophil-mediated tissue damage and organ dysfunction during acute inflammatory processes. High mobility group box 1 also plays a beneficial role in microbial eradication through its proinflammatory action and modulation of neutrophil chemotaxis (76, 77). Previous studies have shown that HMGB1 can potentiate NET formation, suggesting a novel mechanism by which HMGB1 may enhance host defense against bacterial infection and contribute to inflammatory processes (74, 78).
Mammalian target of rapamycin
Mammalian target of rapamycin kinase may play an essential role in NET release by regulating autophagy (79, 80). Previous studies have shown that pharmacological inhibition of the mTOR pathway may enhance the rate of NET release. The release of mTOR-dependent NET is sensitive to inhibition of respiratory burst or blockade of cytoskeletal dynamics. The mTOR pathway is implicated in coordinating intracellular signaling of neutrophil activation related to NETosis (79, 80). Recently, mTOR was shown to regulate LPS-induced NET release by posttranscriptional control of hypoxia-inducible factor 1 (80).
Other factors that influence NET formation
In addition to the factors described above, several proinflammatory mediators and complements, such as IL-8, TNF-α, interferon, and complement 5, can stimulate the formation of NETs via NADPH oxidase signaling (55).
NET-ASSOCIATED PREDICTIVE MARKERS IN INJURY
Previous studies have shown that circulating cell-free DNA (cf-DNA)/NET levels can be potentially used for calculating injury severity following trauma with sepsis (81–83). Clinical studies have also shown that cf-DNA/NET levels are important for predicting posttraumatic complications in the intensive care unit setting (83). Serum levels of cf-DNA/NETs are increased in trauma patients who subsequently develop sepsis (81). Cell-free DNA/NET levels in synovial fluid are also increased in patients with septic arthritis (82). Cell-free DNA/NET levels appear to be a valuable marker for the diagnosis of septic arthritis or periprosthetic infections. In an incremental treadmill test study, cf-DNA expression was shown to rapidly increase compared with other skeletal muscle damage markers, such as creatine kinase, uric acid, and C-reactive protein, suggesting that cf-DNA may be an important molecular marker in exercise physiology (84). In another clinical study, serum NET levels were significantly increased after TA-TMA (56). Increased NETs are an important risk factor for TA-TMA, suggesting that NET levels are a useful biomarker for TA-TMA.
DNase is naturally present in human blood (85) and produced as a defense mechanism associated with NETs. The expression of DNase is significantly increased in the early phase of sepsis after major trauma (81). DNase degrades NETs in a concentration-dependent manner, and levels of DNase have been suggested to be a potential biomarker of NET formation following injuries (81).
Pentraxin 3 is a key protein component of NETs (85, 86) and mainly acts as a soluble pattern recognition receptor in the innate immune response (87, 88). Pentraxin 3 forms a complex with some of the other components of NETs (89) and appears to be an important molecule involved in enhancing the actions of other NET component proteins (86, 89). The association of circulating PTX3 with NET component proteins in sepsis suggests that it may be a potential diagnostic target (89). The PTX3 protein may also contribute to cell-mediated immune defense in patients with ulcerative colitis and crypt abscess lesions (90).
POTENTIAL THERAPEUTIC TARGETS IN NET-RELATED INJURY
Reducing NET accumulation in tissues may be important for preventing or attenuating damage to the host. Interfering with NET structure and component proteins could reduce tissue injury following various insults. The potential targets in NET-associated injury are listed in Table 2.
DNase has a regulatory effect on NET formation in neutrophils and can degrade NETs. DNase levels are increased in early trauma-induced sepsis. Targeting NET components with DNase 1 is effective in protecting mice against TRALI (15). DNase treatment is used for patients with acute lung injury and cystic fibrosis (91). Intrapulmonary DNase is an effective therapy in cystic fibrosis, targeting the extracellular DNA that interferes with mucociliary clearance (91). Antiproteases are also used in therapy to dampen the activity of proteases, and the combined use of antiproteases and DNase may be potentially helpful in controlling NET-mediated lung damage (92). However, previous studies have shown that degradation of NETs may not be a good therapeutic strategy during active infection in a cecal-ligation-and-puncture mouse model of sepsis (93). The recombinant human DNase therapy increased circulating IL-6 levels and enhanced sepsis-related tissues damage after cecal ligation and puncture in mice (93). Thammavongsa et al. (94) have reported that Staphylococcus aureus can release nuclease and adenosine synthase during staphylococcal infections in mice, which convert NETs to deoxyadenosine and trigger the caspase 3–mediated death pathway of immune cells. The ability of S. aureus to cause degradation of NETs is important for the exclusion of macrophages from infection sites (94).
Histone toxicity originates from the integration of cell membranes, which results in a large inward surge of ion currents and calcium influx (95). Histone-induced NETs could induce surrounding cell death (57). An antihistone antibody may decrease histone-induced cytokine elevation, endothelial damage, coagulation activation, platelet aggregation, and NET formation in a trauma-associated lung injury model (96). The use of antihistone antibodies has been shown to be protective from NET-mediated lung damage (15). Thus, circulating histones are therapeutic targets for improving survival in patients after severe traumatic injury and are considered to be potential drug targets for vessels thrombosis (71).
Peptidylarginine deiminase 4
Peptidylarginine deiminase 4 is an important histone-modifying enzyme, and inhibition of PAD4 has been shown to prevent NET formation (64, 97). In addition, histones are modified by PAD4 in the process of NET formation (71).
High mobility group box 1
Therapies against HMGB1 have been shown to be beneficial in neutrophil-associated inflammatory conditions, including acute lung injury, sepsis, and IR-induced tissue injury (76, 77, 98). High mobility group box 1 can induce NET formation through a TLR4-dependent pathway and provide a novel therapeutic target for NET-associated injuries (74).
Activation of NADPH oxidase is essential for NET formation. Protein kinase C (PKC) plays an important role in NET formation and is a mediator of NADPH oxidase activation (99). Phorbol 12 myristate 13 acetate–induced NET formation is dependent on ROS production through the activation of NADPH oxidase and PKC (41, 100). Neutrophil extracellular trap formation in response to PMA is dependent on PKC activation, with PKCβ being the predominant isoform responsible for NET formation. Inhibition of PKC completely inhibits PMA-induced NET formation (100), suggesting that modulation of NET production via PKC inhibition may offer a novel anti-inflammatory strategy (100). Previous studies have shown that the Raf-MEK-ERK signaling pathway, which is upstream of NADPH oxidase, is critical for NET formation. Drugs that target the Raf-MEK-ERK pathway may also potentially regulate NADPH oxidases and NET formation (41).
MPO and superoxide dismutase
Myeloperoxidase activity on NETs could contribute to tissue injury when NETs are released from neutrophils at sites of infection or inflammation (101). Previous studies have shown that excessive NET levels can cause alveolar-capillary damage in influenza pneumonia–induced acute lung injury. However, following anti-MPO antibody and superoxide dismutase inhibitor treatment, NET formation is decreased, and influenza pneumonitis–induced lung injury is attenuated (16).
Platelet-neutrophil interactions are important for inflammation and hemostasis (75). Previous studies have shown that platelet-induced NET formation is mediated by platelet TLR4 (47). Blockage of platelet-platelet interactions with a glycoprotein IIb/IIIa inhibitor can reduce NET formation and protect against TRALI (15). Accordingly, preventing platelet-neutrophil interactions may serve as a novel therapeutic target following injury.
Previous studies have shown that NO mediates NET release from human neutrophils through free radical generation. Nitric oxide–mediated formation of NETs is reduced by pretreating neutrophils with the NADPH oxidase inhibitor (diphenyleneiodonium [DPI]) and MPO inhibitor (ABAH: 4-aminobenzoic acid hydrazide) (65, 73). Surfactant protein D (SP-D) is a collectin with innate immune function that is capable of inhibiting NET formation. Therefore, SP-D may serve as another therapeutic candidate for regulating NETosis and NET clearance (101). Garcia et al. (102) suggested that targeting complement 5 (C5) activation may reduce neutrophil infiltration and NET formation in the airways and attenuate influenza virus A–induced lung injury. Thus, C5 may also be considered as therapeutic target for preventing NET-related injury.
Increasing evidence suggests that NETs play an important role in organ function following injury. The pathways involved in NET formation are complex. Reducing NET formation may protect against ischemic brain, limb, and muscle damage, as well as LPS-induced lung injury. Circulating DNase, cf-DNA, and NET levels may serve as markers of trauma and sepsis. Current evidence suggests that efforts in developing potential therapies for pathological NET formation in the setting of trauma or sepsis should focus on targeting DNase, histones, proteases, NADPH oxidase, and MPO. Additional experimental studies and clinical trials are warranted to elucidate the complex mechanisms involved in NET formation, as well as potential therapeutic targets. A better understanding of the underlying mechanisms responsible for the formation of NETs and their effects may provide new insight into how to improve organ function following injury.
In this article, we provide a mini overview regarding the role of NETs in organ injury and offer some perspectives on the future directions for this field.
(a) Neutrophil extracellular traps or NETs-related intermediates may be promising biomarkers for the early prediction of organ injury severity in future clinical applications.
(b) Neutrophil extracellular traps may serve as a target to develop new therapeutics in the trauma or sepsis in the future.
(c) Further clarify the complex mechanisms of NET formation and interactions through various experimental research and clinical studies.
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