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Review Article NEUTROPHIL EXTRACELLULAR TRAPS IN SEPSIS Gabriela Camicia,* Roberto Pozner,† and Gabriela de Larran˜aga* *Laboratorio de Hemostasia y Trombosis, Hospital de Infecciosas F. J. Mun˜iz; and † Laboratorio de Trombosis Experimental, Instituto de Medicina Experimental, Academia Nacional de MedicinaYCONICET, Buenos Aires, Argentina Received 8 Apr 2014; first review completed 23 Apr 2014; accepted in final form 2 Jun 2014 ABSTRACT—Sepsis is the leading cause of death in critically ill patients in intensive care units. Early recognition of sepsis and proper therapy are essential to reduce patient mortality. Moreover, treatment options for this deleterious inflammatory response to infection are limited. Neutrophils play an essential role in the innate immune response, providing the first line of host defense. It has recently been shown that these cells can trap and kill microorganisms by releasing neutrophil extracellular traps (NETs) composed of chromatin and antimicrobial proteins. Although the beneficial role of NETs during infections has been demonstrated, there is increasing evidence that NETs and their components contribute to the pathogenesis of several diseases, including sepsis. The aim of this review was to summarize the current evidence implicating NETs, as well as their components, in the development of sepsis and to discuss their potential use as novel therapeutic targets and as prognostic markers in septic patients. KEYWORDS—Sepsis, neutrophils, neutrophil extracellular traps, infection, innate immunity, inflammation

INTRODUCTION

pathophysiological role of NETs is controversial. This review summarizes current knowledge about the relationship between NETs and sepsis. Specifically, we focus on the possible implication of NETs and/or its constituents in sepsis and their potential use as prognostic markers in sepsis.

Despite relevant advances in critical care management and in understanding of the molecular basis of sepsis and its associated complications, septic syndrome remains a serious problem worldwide. It is estimated that 18 million cases of sepsis occur each year in the world, with mortality rates ranging from 30% to 50% (1Y3). In the United States, approximately $24.3 billion is spent per year on the costs of hospitalization for septic patients (4). Sepsis can be defined as a deleterious, nonresolving host inflammatory response to infection that leads to organ failure (5). Different host and microbial factors, including the genetics and any coexisting illness of the patient, the pathogenic virulence, and the size of the inocula, are involved in the physiological response to sepsis (6, 7). The relationships among these factors lead to either resolution of the disease or death. Neutrophils are the first line of innate immune defense against infectious agents. In addition to these cells_ ability to eliminate pathogens by phagocytosis and/or degranulation, it has recently been demonstrated that neutrophils can bind to and kill a wide range of microorganisms by forming neutrophil extracellular traps (NETs) (8). This novel mechanism consists of the release of antimicrobial proteins anchored to a network of chromatin by certain activated neutrophils. However, growing evidence demonstrates that although the release of NETs is important as an antimicrobial mechanism, the formation of these extracellular structures contributes to the pathogenesis of several diseases (9, 10). Regarding sepsis, the

SEPSIS Septic patients are an extremely heterogeneous mixture of cases, with different genetic backgrounds, associated comorbidities, causative microorganisms (with diverse virulence and loads), and anatomic sites of infection (6). In addition to the heterogeneous etiologies of sepsis, in cases of sterile inflammation, it has been discovered that a sepsis-like condition can also develop (as in polytrauma, acute pancreatitis, ischemia/ reperfusion injury or hemorrhagic shock) (11, 12). For many years, septic syndrome was considered to be an uncontrolled inflammatory response (13). The fact that several proinflammatory cytokines, such as interleukin 1 beta (IL-1") and tumor necrosis factor ! (TNF-!), are at increased concentrations in septic patients, together with the fact that inoculation with these cytokines in animal models mimics many features of sepsis, led to the concept of sepsis as a cytokine storm (14). Nevertheless, several clinical trials of diverse anticytokine and anti-inflammatory drugs in septic patients showed no clinical effectiveness or, in certain cases, increased mortality rates (14). Currently, it is well known that a proinflammatory response and an opposing anti-inflammatory response occur concomitantly in septic patients. During sepsis, activated leukocytes migrate from the bloodstream into inflamed tissues, accompanied by an exacerbated inflammatory response that has deleterious effects. This first phase, referred to as systemic inflammatory response syndrome, is characterized by the release of several proinflammatory mediators, including TNF-!, IL-1", IL-6, reactive oxygen species (ROS), nitric oxide, chemokines,

Address reprint requests to Gabriela de Larran˜aga, PhD, Hemostasis and Thrombosis Laboratory, Hospital of Infectious Diseases BF. J. Mun˜iz,[ Uspallata 2272, C1282AEN, Buenos Aires, Argentina. E-mail: [email protected]. This work was supported by grants from the Ministerio de Salud del Gobierno de la Ciudad Auto´noma de Buenos Aires. DOI: 10.1097/SHK.0000000000000221 Copyright Ó 2014 by the Shock Society

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SHOCK OCTOBER 2014 and lipids, and by the upregulation of adhesion molecules (15). To counterbalance these proinflammatory mechanisms, certain cytokines with opposite functions are produced in a process called compensatory anti-inflammatory response syndrome, which is characterized by the expression and release of IL-10, IL-1 receptor antagonist, prostaglandins, and immunomodulatory hormones (15Y17). However, the balance often shifts toward inflammation, resulting in an early, dominant hyperinflammatory phase characterized by shock, fever, and hypermetabolism. In fact, early deaths in sepsis are usually due to the overwhelming hyperinflammatory response (18). Most patients survive because of the restoration of innate and adaptive immunity. However, if sepsis persists, the patient enters into a marked immunosuppressive state, and death occurs because of the inability of the patient to eliminate the primary infection and/or because of the development of secondary infections (18). A common complication in septic patients that increases the risk of death is thrombus formation. Coagulation abnormalities in sepsis range from a small decrease in the platelet count and subclinical prolongation of global clotting times to fulminant disseminated intravascular coagulation (DIC), characterized by simultaneous widespread microvascular thrombosis and profuse bleeding from various sites. Septic patients with severe forms of DIC may present with thromboembolic disease or less clinically apparent microvascular fibrin deposition (19). In fact, an early diagnosis and appropriate therapeutic interventions in the first hours of sepsis are crucial for patient survival (20, 21). Increasing complications of sepsis can lead to severe sepsis and to septic shock (22). The severity of organ dysfunction is an important determinant of prognosis in sepsis, and the cardiovascular and respiratory systems are the most commonly affected (23). The brain, kidneys, and liver are also often affected (16, 23). Unfortunately, it is currently not possible to promptly establish the etiology and progression of sepsis in order to choose a therapeutic strategy. Many patients cannot eradicate their infections and develop secondary hospital-acquired infections (24, 25). Moreover, multiple studies have suggested that patients who survive to hospital discharge after sepsis remain at increased risk of death in the following months or years (26). In a postmortem study, it was recently shown that approximately 80% of septic patients had unresolved septic foci at death (27). In addition, patients who survive early sepsis but remain in the intensive care unit (ICU) show evidence of immunosuppression (28). Indeed, a new perspective has emerged in light of recent reports indicating that the blood leukocytes of septic patients show reduced responsiveness to pathogens (28, 29). Therefore, early intervention, together with the development of new therapies that improve patient immunocompetence, could lead to faster resolution of the primary infection and to the prevention of secondary infections, improving the outcome of septic patients (18). THE FUNCTION OF NEUTROPHILS IN SEPSIS Neutrophils play an essential role in the innate immune system (30). These cells constitute approximately 70% of the

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leukocytes in the peripheral blood. Neutrophils are terminally differentiated cells with a short lifespan that are recruited to the site of infection or inflammation for the containment and clearance of infectious agents, providing the first line of host defense (31). Upon activation, neutrophil apoptosis is suppressed, resulting in prolonged survival and activity. These qualities are often associated with unspecific damage to bystander tissues and the attraction of further inflammatory cells (32). During sepsis, neutrophil reprogramming occurs, resulting in impaired directed migration of neutrophils to infectious foci (33) and impaired neutrophil functions (17). These impairments result in the deleterious accumulation of neutrophils within vital organs, such as the lung and liver. Neutrophils_ migratory response in sepsis is modulated by a wide range of inflammatory mediators, which include bacterial products, cytokines, chemokines, lipids, and nitric oxide (33). Multiple mechanisms contribute to the sepsis-induced impairment of neutrophil migration; however, these processes are complex, and our understanding remains incomplete. As sepsis evolves, neutrophil gene expression is altered, leading to the suppression of proinflammatory and immunomodulatory genes and decreased production of ROS. In other words, in lethal cases, these key players in innate immunity are exhausted, the infection is not controlled, and the causative agent wins the war (34). NEUTROPHIL EXTRACELLULAR TRAPS Until recently, it was believed that neutrophils eliminate microorganisms through two mechanisms: phagocytosis, in which microbes are engulfed and killed by proteases and ROS in phagolysosomes, and degranulation, during which neutrophils release granules filled with antimicrobials and proteases into the nearby environment to destroy pathogens (31). However, in 2004, Brinkmann et al. (8) identified a novel antimicrobial mechanism used by activated neutrophils to capture and kill pathogens extracellularly. This mechanism consists of the release of web-like structures of DNA decorated with histones and antimicrobial proteins, known as NETs (Fig. 1). Microbes are immobilized in these traps, which contain a lethal concentration of antimicrobial agents. Neutrophil extracellular traps trap and kill a broad range of microorganisms, including gram-negative and gram-positive bacteria, fungi, viruses, and protozoa (35Y41). DNA is the major structural component of NETs (8). In addition, granule and cytoplasmic proteins, including neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, proteinase 3 (PR3), gelatinase, LL-37, lactoferrin, and calprotectin, among others (30, 42), as well as histones H1, H2A, H2B, H3, and H4, are embedded in the DNA backbone. The trapping within the DNA fibers promotes a physical containment of microbes (43), whereas the histones and granular proteins confer antimicrobial function to the NETs. It is well known that histones play a key role in chromatin architecture, forming the core around which DNA is wrapped. However, evidence for a new type of antimicrobial function is now reemerging (44), considering that histones kill bacteria more effectively than do common antimicrobials (44, 45). Regarding sepsis, it is known that histones

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FIG. 1. Neutrophil extracellular traps. In vitro stimulated neutrophils were stained with propidium iodide (red) and anti-NE (green). The NET formation was examined by confocal microscopy. Like a fly in a spider’s web, the microbes are stuck and can be further attacked by the antibacterial proteins that anchor the DNA. Courtesy of Dr Mirta Schattner, Laboratory of Experimental Thrombosis, Institute of Experimental MedicineYCONICET, National Academy of Medicine, Buenos Aires, Argentina.

have both antimicrobial actions and cytotoxic properties, as these proteins have been observed in the circulation and have been shown to contribute to death in septic mice (46). Concerning NET-associated proteases and peptides, it has been shown that NE and PR3 can inactivate and kill microbes by cleaving their virulence factors (8, 47). Moreover, at high concentrations, NE is considered a powerful antimicrobial factor because it is believed to penetrate and disrupt the bacterial membrane via its cationic charge (48). LL-37 is a cathelicidin that exhibits antimicrobial activity against a wide range of bacteria species and inhibitory activity against certain fungi and enveloped viruses (49). Neutrophil extracellular traps are also composed of factors that restrict the supply of vital nutrients to pathogens. In this regard, lactoferrins chelate iron and interfere with its uptake (42), and it has been demonstrated that NETs can help to eliminate pathogens such as fungal hyphae, which are too large to be removed by phagocytosis (37). Studies to characterize the antifungal factors within NETs led to the identification of calprotectin as the major antifungal protein acting against Candida albicans (50) and as an important agent against Aspergillus nidulans (51). It is noteworthy that certain microbes have evolved different strategies to evade NETs, such as the expression of DNases to degrade the DNA network (52, 53) or the modification of cell wall components (54). Several streptococcal species express and secrete DNase enzymes, and experimental evidence suggests that these enzymes increase bacterial infectiveness. For instance, it has been demonstrated that both S. pneumonia and group A streptococcus (GAS) DNases are capable of degrading NETs in vitro (52, 53). Group A streptococcus causes lifethreatening diseases in humans, including sepsis. It has been shown that the mutation-induced inhibition of the SdaD2 or Sda1 DNases within GAS increases their clearance by neutrophils (53, 55). In contrast, the induction of a nonnuclease GAS to express the Sda1-encoded DNase generates a hypervirulent form of Streptococcus (56). Similar results have been obtained for Staphylococcus aureus (57) and Streptococcus pneumoniae (52). However, DNases require divalent cations for their activ-

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ity, providing a potential point of attack by the host (42). Streptococcal nucleases such as Sda1, Spd1, and DNase D require both Mg2+ and Ca2+. Apparently, calcium- and zincchelating proteins, including S100, annexins, and others, may protect NETs from bacterial endonucleases by restricting the availability of divalent cations (42). As it is thought that the binding of NETs to their microbial targets may rely on electrostatic interactions between the anionic surface of most microbes and the positive charge of the NET proteins, pathogens may also avoid trapping by changing their surface charge or making a polysaccharide capsule, such as that formed by S. pneumoniae (54). The pneumococci dlt operon introduces a positive charge into the cell wall by the incorporation of D-alanine residues into lipoteichoic acids. In the absence of a capsule, inactivation of dltA enhances NETmediated killing, suggesting that a change in the surface charge may allow for clearance by NETs (54).

MECHANISMS LEADING TO NEUTROPHIL EXTRACELLULAR TRAP FORMATION Neutrophil extracellular trap formation is triggered in response to a wide variety of microorganisms (35Y41), as well as by inflammatory stimuli and chemical compounds, such as phorbol-12-myristate-13-acetate (8). To date, two major models describing the release of NETs have been proposed: a novel cell death program called NETosis (36, 58) and a DNA extrusion mechanism from live cells (59). NETosis is an active process that is distinct from apoptosis and necrosis, and it appears to be a major route of NET release. This process involves the decondensation of chromatin, rearrangement of the nuclear and granular architecture, mixing of chromatin with the antimicrobial granular content in the cytoplasm, and the subsequent release of granular proteins and peptides anchored to a chromatin network into the extracellular space after the rupture of the plasma membrane (36). The entire process of NET formation via this cell death mechanism takes approximately 2 or 3 h (Fig. 2). The process is irreversible and is dependent on ROS (36) production, which occurs through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, RAF-MEK-ERK, and p38 MAPK pathways (60Y62). However, the molecular mechanisms by which ROS drive this process remains poorly understood (51), and apparently, in the presence of some stimuli, the formation of NETs is not mediated by ROS (63, 64). It is known that the decondensation of chromatin is an essential step in NET formation (65). It has been proposed that this step is mediated by two enzymes stored in the azurophilic granules: NE and MPO. Upon neutrophil activation, NE translocates first to the nucleus, where it degrades H1 and the core histones, promoting extensive chromatin decondensation (66). Subsequently, MPO synergizes with NE in driving chromatin decondensation independent of its enzymatic activity (66, 67). Furthermore, histones are citrullinated by peptidyl arginine deiminase 4 (PAD4) (68), an enzyme that catalyzes the conversion of arginine to citrulline in the tail of histones H3 and H4 (69), resulting in the decondensation of chromatin (65).

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FIG. 2. NETosis. The formation of NETs followed a stepwise progression of chromatin decondensation, nuclear swelling, mixing of chromatin with the antimicrobial granular content in the cytoplasm, plasma membrane rupture, and the ultimate release of a chromatin network decorated with proteases and antimicrobial proteins.

Nevertheless, it has been recently observed that a small subset of neutrophils rapidly expels their chromatin through vesicular exocytosis of nuclear contents, yielding NETs and live cytoplasts, thus providing a rapid extracellular antimicrobial action while maintaining the capacity for phagocytosis (59, 70). This mechanism of NET release does not involve cell death and takes only few minutes after stimulation with S. aureus (59). Recently, the term Bvital NETosis[ was proposed to distinguish it from NETosis by cell lysis (71). In contrast to the cell deathYmediated mechanism of NET formation, which involves the activation of NADPH oxidase and decondensation of chromatin, resulting in a mixture of chromatin and antimicrobial proteins that are released into the extracellular space after the collapse of the plasma membrane, vital NETosis is a both Toll-like receptor 2 (TLR2)- and complement-dependent mechanism (70) that requires vesicular trafficking of DNA from within the nucleus to the extracellular space (59). In addition, the induction of NETs has been shown to occur within minutes in the presence of platelets and lipopolysaccharide (LPS) (72). This prompt mechanism of NET release is mediated by TLR4 on platelets that facilitate the activation of neutrophils, a strategy that has been implicated in sepsis-associated microvascular thrombosis (72), which will be discussed in detail later. NEUTROPHIL EXTRACELLULAR TRAPS: A TWO-FACED HOST DEFENSE MECHANISM The beneficial role of NETs during infections has been clearly shown, as deficiency in NET production (36, 60) or the destruction of NETs_ scaffold by DNases (57, 73) leads to increased susceptibility to infections. In addition to their antimicrobial function, NETs act as a physical barrier that prevents the spread of microbes, allowing the maintenance of a high concentration of antimicrobial agents in a restricted area (43, 74). The discovery of NETs by Dr Zychlinsky_s group in 2004 was a landmark finding in the field of immunology because it established a novel immune mechanism against infectious agents (8). However, there is growing evidence demonstrating that the formation of NETs in an uncontrolled manner or in the wrong place can be deleterious to the host. In this regard, it is well known that NETs and their components can damage tissues and activate inflammatory cells, contributing to the pa-

thology of several diseases, such as sepsis (72, 75), asthma (76), cystic fibrosis (77), thrombosis (78), atherosclerosis (79), systemic lupus erythematosus (80), psoriasis (81), and smallvessel vasculitis (82). Under severe pathological conditions, such as sepsis after major trauma (83), large amounts of NETs are released. As the structural backbone of NETs is composed of chromatin, the enzyme responsible for its clearance is human DNase (8, 83). In this context, if aberrant amounts of NETs cannot be sufficiently degraded by extracellular DNases within the bloodstream, these NETs could occlude capillaries, impair the microcirculation, damage tissues, and promote inflammation (84). Moreover, upon degradation by extracellular DNases, excessively accumulated NETs release histones and proteases that may provoke severe tissue damage (84). Patients with aberrantly elevated NET levels and/or with impaired DNase production or function may suffer from NET-associated tissue damage. Thus, NETs are a double-edged sword. On the one hand, NETs function as a valuable antimicrobial defense mechanism. On the other hand, NETs lead to organ failure and even death if the regulatory mechanisms fail or are absent. NETS AND SEPSIS Activation of the coagulation cascade is a crucial step in the events related to sepsis, preventing, through fibrin deposition, the dissemination of microorganisms (85, 86). However, the activation of coagulation and subsequent microthrombus formation may lead to DIC, resulting in multiple organ dysfunction (87). Recently, a novel role for neutrophils, linking inflammation with thrombosis via NETs, has been discovered. Neutrophil extracellular traps stimulate platelet adhesion and coagulation (78). Platelets, which are primarily involved in homeostasis, are decreased in number in septic patients. Moreover, thrombocytopenia is associated with sepsis severity and mortality (88). New data on NET formation have recently revealed a new and fascinating interaction between neutrophils and platelets. Clark et al. (72) suggested that platelets function as a barometer in the blood, becoming activated only during serious infections to stimulate neutrophils to release NETs. In a series of elegant experiments, the authors demonstrated that during severe sepsis platelets can trigger the activation of neutrophils sequestered in the microvasculature in a TLR4-dependent manner, leading to

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FIG. 3. Advantages and disadvantages of NET formation in sepsis. The formation of NETs, induced by pathogens or activated platelets, is crucial for extracellular microbial trapping and killing. The excessive generation of NETs or alteration in their DNase-mediated clearance mechanism results in arterial/ venous thrombosis and endothelial and tissue damage.

the formation of NETs. These NETs trap bacteria within the vasculature, but at the expense of endothelial and tissue damage (72). The tissue injury is most likely caused by the release of proteases and by a reduction in sinusoidal perfusion of the blood, causing ischemic conditions. This mechanism increases the capacity of the innate immune system to ensnare and kill circulating bacteria but generates serious collateral damage. Moreover, thrombocytopenia during sepsis might be a consequence of an adherence of activated platelets to neutrophils during NET formation (89). In a mouse model of polymicrobial sepsis, it was observed that there was an inverse correlation between NET levels and platelets 24 h after sepsis induction. However, 24 h later, the amounts of both platelets and NETs decreased significantly. Because the authors found that the neutrophils were able to release NETs ex vivo at this time point, they speculated a lack of activated platelets 48 h after sepsis induction that may impair the production of enough NETs for sepsis control (89), but this hypothesis needs to be clarified. Furthermore, neutrophils derived from septic patients release tissue factorYbearing NETs (90). Tissue factor is a transmembrane protein that initiates the coagulation cascade and results in thrombin generation. Thus, NET-associated tissue factor could be implicated in the coagulopathy observed in septic patients (90). Moreover, a role for NETs in thrombus formation due to factor XII activation has been demonstrated in a mouse model of deep vein thrombosis (91). In addition, it has been suggested that NETs represent an additional scaffold for thrombi, providing a platform for platelet and red blood cell adhesion and promoting fibrin deposition (92).

Because histones are the most abundant proteins in NETs (50), these structures represent an important source of histones during sepsis. Histones associated with NETs or released after NET degradation may play an important role in stimulating coagulation. Histones interact with thrombomodulin and protein C, inhibiting thrombomodulinYprotein C activation, which leads to thrombin generation (93). Moreover, the histones present in NETs are able to trigger platelet activation, even individually. The stimulation of platelets with histones resulted not only in prothrombotic but also in proinflammatory and procoagulant responses, and the intracellular mechanisms included the ERK, Akt, p38, and nuclear factor .B pathways (94). In addition, it has been shown that extracellular histones, and mainly H4, are cytotoxic toward endothelial and epithelial cells (45, 46). The infusion of histones into mice mimicked the symptoms of sepsis, including neutrophil sequestration in the lungs, microvascular thrombosis, organ failure, and death (46). Moreover, antibodies against H4 reduced the mortality of mice in three different models of sepsis: injection of LPS, injection of TNF, and cecal ligation and puncture. The molecular mechanisms of histone-mediated cytotoxicity involve signaling through TLR2 and TLR4 (95). In brief, extracellular histones contribute to endothelial dysfunction, organ failure, and death during the septic process and are thus a promising molecular target for sepsis treatment. In line with this hypothesis, recent work has revealed that a nonanticoagulant heparin not only inhibited histone-mediated cytotoxicity in vitro but also improved survival in animal models of sepsis and sterile inflammation. However, the more exciting and clinically relevant

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SHOCK OCTOBER 2014 finding was that all of these benefits occurred without increasing the risk of bleeding (96). Finally, the essential role of NETs in the early phase of infection was recently demonstrated in a murine model of polymicrobial sepsis. The depletion of NETs by DNase administration led to advanced sepsis progression, with temporarily increased mortality, enhanced bacterial dissemination, and more severe organ injury (89). Thus, NET depletion impedes the early immune response and deeply aggravates the pathology that follows polymicrobial sepsis. In summary, during sepsis, NETs benefit the host in the early phase of the infection by trapping and killing pathogens. However, during systemic infection, NETs or their components can damage tissues and endothelia, causing diffuse thrombosis that leads to DIC and acute organ injury, both of which increase mortality during sepsis (Fig. 3). Therefore, the issue of whether NET formation in sepsis is beneficial or detrimental remains to be elucidated. NETS AND THEIR COMPONENTS AS PROGNOSTIC MARKERS IN SEPSIS It has been reported that aberrantly elevated levels of NETs in the plasma may predict multiple organ dysfunction and sepsis in ICU patients (75). A pilot study in trauma patients showed that initially high values of circular free DNA derived from NETs (cf-DNA/NETs) correlated with an increased risk of secondary inflammation and sepsis. Consequently, patients with low cf-DNA/NET values would have a good prognosis. Furthermore, cf-DNA/NETs were reported to be a useful additional marker for the early diagnosis of septic arthritis or periprosthetic infections in knee joints in another pilot study (97). According to the authors, high cf-DNA/NET value seems to be a marker for intra-articular infections, with a detection time faster than that of microbiological diagnostics. Thus, the quantitation of cf-DNA/NETs in the plasma could be a valuable tool to predict the development of sepsis in this posttraumatic patient population when in the ICU. In addition, Xu et al. have shown that the concentration of the histone H3 is increased in an experimental monkey model of sepsis. This increase is accompanied by the onset of acute renal failure. The authors also found high levels of H3 in clinical samples from septic patients, raising the possibility of using extracellular histones as biomarkers of sepsis progression (46). Moreover, it has recently been shown that serum levels of citrullinated H3 are associated with sepsis severity and mortality in a mouse model of LPS-induced septic shock (98). Thus, citrullinated H3 could be another valuable serum predictor of a diagnosis of septic shock and, more importantly, of mortality in sepsis. PERSPECTIVES In the future, the early detection of septic syndrome, along with the development of new therapies that improve patients_ immunocompetence, will likely be the key step toward a better prognosis. A focus on the late mediators of septic injury or on delayed physiological consequences, such as tissue or endothelial damage, may be an effective approach. The recent

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finding that sepsis can cause immunosuppression raises the possibility of using immunostimulatory therapy for sepsis treatment. In addition, various genetic polymorphisms have been shown to be associated with increased susceptibility to sepsis and poor outcomes, providing new potential therapeutic targets and opening the way to customized treatment (99, 100). Delayed diagnosis and inappropriate initial treatment may play a pivotal role in sepsis mortality. Moreover, the heterogeneity of septic patients complicates the treatment of the syndrome. Many patients fail to have their initial infection eradicated and acquire secondary hospital infections. In this context, the discovery of NETs is emerging as a powerful additional tool for the early diagnosis of sepsis and as a promising new therapeutic target for its treatment. It seems clear that inhibiting NET formation would be an attractive strategy to prevent the deleterious effects of NETs or their components in septic patients. However, it is widely accepted that NETs play an essential role in trapping and killing microbes to prevent microbial dissemination. Accordingly, the administration of DNase in a murine model of polymicrobial sepsis demonstrated the importance of NETs in the early phase of sepsis, suggesting that a NET-depletion therapy in the early phase of sepsis would lead to a defective eradication of the microorganisms at the infection site, aggravating the pathology (89). Because NETs are beneficial in the early phase of sepsis and detrimental in systemic infection, the fine-tuning of NET formation throughout the course of sepsis would be the goal for the development of new NET-targeted sepsis therapies. Targeting NET-derived extracellular DNA by DNAse treatment or histones by antihistone antibodies or nonanticoagulant heparin could be an attractive strategy for preventing thrombosis and organ damage when NETs become deleterious in sepsis. As mentioned above, histones are implicated in endothelial damage, organ failure, and death in sepsis (46). It has been shown that the administration of antibodies against histones reduces mortality in mouse models of sepsis (95). Thus, histones constitute a hopeful molecular target for sepsis treatment. Moreover, administration of DNase I has been shown to prevent thrombus formation in murine models of deep vein thrombosis (101). It would be interesting to introduce a combined therapy with DNase and histones or protease inhibitors, which should offer a better compromise between tissue injury and NET removal. The anticoagulant heparin dismantles NETs and prevents histone-platelet interactions, thus likely decreasing NET-driven thrombosis. DNase I activity is enhanced in vitro by the presence of serine proteases, and this can be mimicked by heparin, as it displaces histones from chromatin and allows better accessibility for enzyme (102). Combining DNase I with heparin could further reduce the risk of thrombotic events and significantly reduce histone-mediated damage, but this could, at the same time, increase the risk of bleeding. In this context, it was very recently demonstrated that a nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding (96). These findings suggest that administration of nonanticoagulant heparin could be a novel and promising approach that may be further developed to treat patients

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suffering from sepsis. Moreover, they raise the possibility of using a combined therapy of DNase and nonanticoagulant heparin as a possible strategy to prevent tissue damage and thrombosis in sepsis when the mechanism of NET formation appears to be detrimental. Other suitable targets for treatment are PAD4 or NADPH oxidase, because both are essential for NET formation (68, 103, 104). In fact, an NADPH oxidase inhibitor improved influenza A virusYinduced lung inflammation in which excessive NETs were involved (105). Nevertheless, assuming that NET inhibition in late sepsis is safe, it will be very important to examine at which step NETosis would be arrested. Still, further and more extensive studies are needed to explore the therapeutic implication of targeting NETs in sepsis. Perhaps a combined therapeutic approach, derived from inhibiting selected host defense mechanisms when they become detrimental (such as NETs and/or their components) and adopting cell and molecular therapies that repair tissue damage and improve later immunosuppression, is likely the best pathway for the treatment of sepsis. Furthermore, an early diagnosis based on new targets and/or a reduction in the development of sepsis in high-risk patients, such as those who are undergoing risky surgical procedures, may improve the outcome of this devastating and lethal disease, which causes millions of deaths per year worldwide. REFERENCES 1. New hope for sepsis. Lancet 379(9825):1462, 2012. 2. Poli-de-Figueiredo LF: The ongoing challenge of sepsis in Latin America. Shock 34(1):1Y3, 2010. 3. Dombrovskiy VY, Martin AA, Sunderram J, Paz HL: Rapid increase in hospitalization and mortality rates for severe sepsis in the United States: a trend analysis from 1993 to 2003. Crit Care Med 35(5):1244Y1250, 2007. 4. Lagu T, Rothberg MB, Shieh MS, Pekow PS, Steingrub JS, Lindenauer PK: Hospitalizations, costs, and outcomes of severe sepsis in the United States 2003 to 2007. Crit Care Med 40(3):754Y761, 2012. 5. Vincent JL, Opal SM, Marshall JC, Tracey KJ: Sepsis definitions: time for change. Lancet 381(9868):774Y775, 2013. 6. Carlet J, Cohen J, Calandra T, Opal SM, Masur H: Sepsis: time to reconsider the concept. Crit Care Med 36(3):964Y966, 2008. 7. Riedemann NC, Guo RF, Ward PA: The enigma of sepsis. J Clin Invest 112(4):460Y467, 2003. 8. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science 303(5663):1532Y1535, 2004. 9. Kaplan MJ, Radic M: Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol 189(6):2689Y2695, 2012. 10. Camicia G, de Larranaga G: Neutrophil extracellular traps: a 2-faced host defense mechanism. Med Clin (Barc) 140(2):70Y75, 2013. 11. Chen GY, Nunez G: Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 10(12):826Y837, 2010. 12. Ward PA: New approaches to the study of sepsis. EMBO Mol Med 4(12): 1234Y1243, 2012. 13. Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 348(2):138Y150, 2003. 14. Hotchkiss RS, Monneret G, Payen D: Immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach. Lancet Infect Dis 13(3):260Y268, 2013. 15. Bone RC, Grodzin CJ, Balk RA: Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 112(1):235Y243, 1997. 16. Faix JD: Biomarkers of sepsis. Crit Rev Clin Lab Sci 50(1):23Y36, 2013. 17. Kovach MA, Standiford TJ: The function of neutrophils in sepsis. Curr Opin Infect Dis 25(3):321Y327, 2012. 18. Hotchkiss RS, Monneret G, Payen D: Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol 13(12): 862Y874, 2013.

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