Netosis and NADPH Oxidase: at the Intersection of Host Defense, Inflammation, and Injury

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Almyroudis, N G., Grimm, M J., Davidson, B A., Röhm, M., Urban, C F. et al. (2013) NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury. Frontiers in Immunology, 4: 45 https://doi.org/10.3389/fimmu.2013.00045

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Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-80701 MINI REVIEW ARTICLE published: 01 March 2013 doi: 10.3389/ﬁmmu.2013.00045 NETosis and NADPH oxidase: at the intersection of host defense, inﬂammation, and injury

Nikolaos G. Almyroudis1,2, Melissa J. Grimm 2, Bruce A. Davidson 3,4,5, Marc Röhm 6, Constantin F. Urban 6 and Brahm H. Segal 1,2,7* 1 Division of Infectious Diseases, Department of Medicine, University at Buffalo School of Medicine, Buffalo, NY, USA 2 Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 3 Department of Anesthesiology, University at Buffalo School of Medicine, Buffalo, NY, USA 4 Department of Pathology and Anatomical Sciences, University at Buffalo School of Medicine, Buffalo, NY, USA 5 Veterans Administration of Western New York Healthcare System, Buffalo, NY, USA 6 Laboratory for Molecular Infection Medicine Sweden, Department of Clinical Microbiology, Umeå University, Umeå, Sweden 7 Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA

Edited by: Neutrophils are armed with both oxidant-dependent and -independent pathways for killing Marko Radic, University of Tennessee, pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate USA (NADPH) oxidase constitutes an emergency response to infectious threat and results in Reviewed by: the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation Heather Parker, University of Otago, Christchurch, New Zealand in neutrophils is linked to activation of granular proteases and generation of neutrophil Jinfang Ma, Johns Hopkins University, extracellular traps (NETs). NETosis involves the release of nuclear and granular components USA that can target extracellular pathogens. NETosis is activated during microbial threat and in *Correspondence: certain conditions mimicking sepsis, and can result in both augmented host defense and Brahm H. Segal, Division of Infectious inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not Diseases, Department of Medicine, Roswell Park Cancer Institute, only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Elm and Carlton Streets, Although there are significant gaps in knowledge regarding the specific contribution of Buffalo, NY 14263, USA. NETs to host defense, we speculate that the coordinated activation of NADPH oxidase e-mail: [email protected] and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury.

Keywords: NETs, NADPH oxidase, neutrophils, inﬂammation, injury

INTRODUCTION dying neutrophils may amplify microbial killing. Whereas neu- Neutrophils are armed with a broad repertoire of tools for killing trophil apoptosis leads to non-inflammatory physiological cell pathogens. Activation of the phagocyte nicotinamide adenine din- death, NETosis results in the extracellular release of proteases ucleotide phosphate (NADPH) oxidase constitutes an emergency and other injurious neutrophil constituents that can exacerbate response to infectious threat and results in the generation of inflammatory injury (Narasaraju et al., 2011; Caudrillier et al., antimicrobial reactive oxidant intermediates (ROIs). In addition 2012; Thomas et al., 2012). to oxidant-dependent host defense, neutrophils harbor proteases, We review the link between NETosis and NADPH oxidase antimicrobial peptides, lactoferrin, and other antimicrobial con- activation and their effects on host defense and modulation stituents that damage and kill microbes. Activation of NADPH of inflammation and injury. A greater understanding of these oxidase in neutrophils is linked to activation of intracellular gran- pathways may lead to novel therapeutic approaches to limit ular proteases and to generation of neutrophil extracellular traps inflammation-associated injury. (NETs). NETs are composed of an extracellular network of chromatin bound to granular and specific cytoplasmic proteins that HOW NEUTROPHILS DIE can target extracellular pathogens. One of the most important aspects of regulation of acute inflam- NETosis can be induced in vitro by conditions that lead to mation relates to its termination. Neutrophils recruited to sites of robust NADPH oxidase activation [e.g., stimulation with phor- microbial invasion or tissue injury are activated by microbial prod- bol myristate acetate (PMA)], and is triggered in vivo during states ucts (e.g., endotoxin and formylated peptides), damage-associated of emergency, such as infection and conditions mimicking sep- molecular patterns (DAMPs; Zhang et al., 2010), and cytokines sis, such as transfusion-associated acute lung injury (Caudrillier and chemokines within the inflammatory milieu. While this acute et al., 2012; Yipp et al., 2012). In this setting, NETosis in dead or inflammatory response is critical for host defense, subsequent

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termination of neutrophilic inflammation and transition to macrophages to phagocytose bacteria (Soehnlein et al., 2008a, inflammatory responses that mediate tissue repair (e.g., M2 or 2009a,b). alternative macrophage polarization; Sica and Mantovani, 2012) are necessary to limit tissue injury. GENERATION OF NETs Neutrophil homeostasis involves production and death of an Neutrophil extracellular trap generation was first described by extraordinarily large population of cells. The lifespan of circulat- Brinkmann et al. (2004), who showed that neutrophils release ing neutrophils has been estimated to be <1day;however,arecent granule proteins and chromatin that co-mingle in extracellular in vivo labeling study showed that the average estimated circulat- filaments that bind to and kill bacteria and degrade virulence ing human neutrophil lifespan was considerably longer (5.4 days; factors. NETosis progresses through stages that are distinct from Pillay et al., 2010). During infection and other major stressors, apoptosis and necrosis. The nuclei of neutrophils transition from granulopoiesis and circulating neutrophil counts can increase dra- hypersegmented lobes to a round, decondensed morphology. Pep- matically; these conditions also modulate neutrophil survival and tidylarginine deiminase 4 (PAD4) converts histone tail arginine death as well as the mode of death. residues to citrulline, leading to loss of positive charge and chro- Apoptosis is the default mode of neutrophil death. Apoptosis matin decondensation (Neeli et al., 2009; Wang et al., 2009; Li is stimulated by a number of factors [e.g., members of the tumor et al., 2010; Farley et al., 2012). Later, the nuclear envelope and necrosis factor (TNF) cytokine family], and is regulated by spe- the granule membranes break down, allowing mixing of these cific caspases and members of the Bcl-2 family of proteins (Croker components that are subsequently released as extracellular struc- et al., 2011; Geering et al., 2011). Factors that can delay neutrophil tures (Fuchs et al., 2007). Whereas NETosis has been considered to apoptosis include lipopolysaccharide (LPS), granulocyte colony- be an event following neutrophil death and breakdown of mem- stimulating factor (G-CSF), granulocyte-macrophage colony- branes, it is also possible for viable neutrophils to produce NETs stimulating factor (GM-CSF), and proinflammatory cytokines (Yousefi et al., 2009; Yipp et al., 2012). Yipp et al. (2012) showed (Coxon et al., 1999; Klein et al., 2001; Garlichs et al., 2004; Akagi that during experimental infection, anuclear neutrophils with et al., 2008; Jun et al., 2011). Neutrophils are likely commit- intact membranes formed NETs and were capable of migration ted to apoptotic death by their constitutive co-expression of and phagocytosis. cell-surface Fas and Fas ligand, an autocrine mechanism that NETosis can be triggered by exposure of neutrophils to PMA, is suppressed by proinflammatory cytokines (Liles et al., 1996). IL-8, LPS,interferon-gamma (IFN-γ ), bacteria and fungi and their In mice, phagocytosis of apoptotic neutrophils by macrophages products, and complement-mediated opsonization (Brinkmann reduces IL-23 production by these cells and downstream et al., 2004; Martinelli et al., 2004; Fuchs et al., 2007; Yamada et al., IL-17A production by T cells, thereby limiting granulopoiesis 2011; Saitoh et al., 2012; Yipp et al., 2012). Activated platelets can (Stark et al., 2005). induce NETosisin neutrophils during bacterial sepsis, thereby cap- NETosis and necrosis of neutrophils are induced by differ- turing microbes and promoting their clearance (Clark et al., 2007; ent stimuli and have morphologically distinct features. Following Phillipson and Kubes, 2011; McDonald et al., 2012). The propor- toxin-induced necrosis, nuclear lobes in neutrophils lose their tion of activated neutrophils undergoing NETosis, the rapidity of hypersegmented structure, but the nuclear envelope and granules NET generation, and the dependence on specific signaling path- remain intact (Fuchs et al., 2007). Nuclear decondensation and ways vary based on the stimulus (Hakkim et al., 2011). The Raf– breakdown of the nuclear and granular membranes are unique MEK–ERK pathway is an upstream activator of NADPH oxidase in features of NETosis (Fuchs et al., 2007). NETs are demonstrated neutrophils and is involved in NETosis (Hakkim et al., 2011). Raf– by immunofluorescence showing mixing of nuclear (e.g., DNA MEK–ERK also upregulates expression of Mcl-1, an anti-apoptotic and histones) and granular constituents [e.g., neutrophil elas- protein, suggesting that Raf–MEK–ERK may inhibit apoptosis to tase (NE)] on the extracellular surface of neutrophils (Brinkmann allow for NETosis (Hakkim et al., 2011). Several other signaling et al., 2010). pathways can modulate NETosis. Inhibition of autophagy prevents How neutrophils die likely affects their clearance and cross- chromatin decondensation in neutrophils and abrogates NETosis signaling to monocytes/macrophages. At sites of inflammation, (Remijsen et al., 2011). Mammalian target of rapamycin (mTOR) neutrophils undergo spontaneous apoptosis (Savill et al., 1989). mediates LPS-stimulated NET formation by post-transcriptional In addition, neutrophil apoptosis can be induced by macrophages control of expression of hypoxia-inducible factor-1 alpha (HIF- releasing death receptor ligands, such as TNF-α and Fas lig- 1α; McInturff et al., 2012). IFN-γ produced by neutrophils can sti- and (Brown and Savill, 1999; Yamashita et al., 1999; Renshaw mulate NETosis through an autocrine/paracrine process (Yamada et al., 2000). Macrophages recognize and ingest apoptotic neu- et al., 2011). trophils (Savill et al., 1989). Phosphatidylserine products are In addition, specific neutrophil components that are NET con- externalized by neutrophils early during apoptosis and stimu- stituents are also required for NET generation. NE is required late phagocytosis of neutrophils by macrophages (efferocytosis), for NET generation in pneumonia in mice (Papayannopou- thus promoting resolution of inflammation (Frasch et al., 2011). los et al., 2010). In this model, NE traffics from primary In contrast, NETotic neutrophils display phosphatidylserine only granules to the nucleus where, together with myeloperoxidase after plasma membrane rupture (Fuchs et al., 2007). In addi- (MPO), it degrades histones and promotes chromatin deconden- tion, release of primary neutrophil granular proteins can recruit sation. MPO is also located in neutrophil primary granules and circulating monocytes to the site of inflammation, stimulate converts hydrogen peroxide to hypohalous acid, which has potent macrophages to produce cytokines, and enhance the ability of antimicrobial properties. MPO deficiency in humans leads to

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failure to generate NETs following stimulation with PMA or oxidase-competent neutrophils in vitro (Fuchs et al., 2007; Bianchi Candida (Metzler et al., 2011). et al., 2009). Neutrophil extracellular traps are also regulated by inhibiting Neutrophil NADPH oxidase can also modulate apopto- pathways. SerpinB1 is an inhibitor of the neutrophil serine pro- sis. NADPH oxidase stimulates phagocytosis-induced apoptosis teases. SerpinB1-deficient mice develop increased inflammation, (Coxon et al., 1996). TNF-α and Fas ligand can both induce tissue injury, and mortality following bacterial and viral infec- apoptosis in neutrophils, but through distinct signaling pathways; tion (Benarafa et al., 2007; Gong et al., 2011). SerpinB1 restricted NADPH oxidase was required for TNF-α-stimulated, but not Fas NETosis through a pathway that involves translocation of Ser- ligand-stimulated, apoptosis (Geering et al., 2011). Accelerating pinB1 from the cytoplasm to the nucleus early during NETosis neutrophil death and clearance are likely to be important modes by (Farley et al., 2012). Curiously, recombinant SerpinB1 also inhib- which NADPH oxidase limits acute inflammation. Potentially, the ited NETosis through mechanisms that are unclear (Farley et al., intensity or kinetics of ROI generation may modulate the balance 2012). MUNC13-4, a member of the MUNC13 family of pro- between apoptosis versus NETosis. teins, is involved in exocytosis of lytic granules in cytotoxic T lymphocytes, and its deficiency leads to familial hemophago- NADPH OXIDASE AND NETs IN HOST DEFENSE cytic lymphohistiocytosis (Feldmann et al., 2003). In neutrophils, NADPH oxidase can potentially target pathogens through a multi- MUNC13-4 mediates ROI generation, exocytosis of primary step process: direct antimicrobial effect of ROIs; intracellular granules, and phagolysosomal maturation, but also inhibits activation of proteases that can target phagocytized pathogens; NETosis (Monfregola et al., 2012). Finally, serum endonuclease and generation of NETs that can attack extracellular pathogens. DNase1 promotes degradation of NETs (Hakkim et al., 2010). NETs can mediate host defense by trapping microbes thereby lim- Pathways that inhibit NETosis may limit neutrophil-mediated iting their spread and by exposing them to high concentrations injury. of several antimicrobial products. NET constituents can target different pathogens. For example, NE can degrade certain viru- RELATIONSHIP BETWEEN NADPH OXIDASE AND NETosis lence factors of pathogens (Belaaouaj et al., 2000; Weinrauch et al., The phagocyte NADPH oxidase is comprised of a membrane- 2002). Calprotectin is a NET constituent that mediates nutritional bound cytochrome consisting of gp91phox (phagocyte oxidase) immunity by sequestering divalent metal ions and targets Candida and p22phox. Upon activation, the cytoplasmic subunits, p47phox, and Aspergillus species (Urban et al., 2009; Bianchi et al., 2011). p67phox, and p40phox and rac translocate to the cytochrome. However, the contribution of NET generation to host defense in NADPH is oxidized to NADP+, and electrons are transported vivo is difficult to determine because there are no genetic defects down a reducing potential gradient that terminates when oxygen that selectively disable NETosis while leaving all other immune accepts an electron and is converted to superoxide anion. Neu- pathways intact. Therefore, it remains elusive whether the major trophil NADPH oxidase activation occurs in response to microbes role of NETs is to trap versus directly kill pathogens in vivo.In and to stimuli that can mimic infectious threat, such as formy- addition, the biological activity of individual cellular components lated peptides, opsonized particles, integrin-dependent adhesion released into NETs is undetermined. With these gaps in knowl- (Mocsai et al., 2002; Graham et al., 2007), and to activation of edge, the host defense contribution of NETosisversus that achieved specific pathogen recognition receptors, such as dectin-1, which by intracellular killing by intact neutrophils remains unsettled recognizes fungal cell wall-associated beta-glucans (Gantner et al., (Nauseef, 2012). 2003; Goodridge et al., 2011). Specific pathogens may target NETs or exploit NET products Chronic granulomatous disease (CGD), an inherited disorder to enhance bacterial invasion. For example, nuclease expression of NADPH oxidase, is characterized by recurrent life-threatening by Staphylococcus aureus degrades NETs and augments pathogen bacterial and fungal infections. Patients with only residual virulence in vivo (Berends et al., 2010). In addition, bacteria may NADPH oxidase activity in neutrophils have a better prognosis exploit NET products to enhance microbial virulence. Shigella than those with completely absent oxidase function (Kuhns et al., flexneri binds to cationic granular proteins expressed in NETs, 2010). CGD is also associated with severe inflammatory complica- which enhances bacterial adherence to and invasion of epithe- tions, such as Crohn’s-like inflammatory bowel disease (Marciano lial cells (Eilers et al., 2010). Paradoxically, excessive NETosis may et al., 2004; Segal et al., 2011). impair host defense in vivo. SerpinB1-deficient mice had increased Activation of NADPH oxidase in neutrophils is linked to lung neutrophil NET generation but defective bacterial clearance the release of cationic proteins from an anionic proteoglycan compared to wild type mice in Pseudomonas aeruginosa pneumo- matrix within primary granules (Reeves et al., 2002). In this nia (Farley et al., 2012). While SerpinB1 may be required for host model, the released neutrophil serine proteases become acti- defense independently of its role in NETosis, these results raise vated and can target phagocytized microbes. NADPH oxidase the potential for injury caused by excessive NETosis abrogating can also stimulate NETosis. PMA-stimulated NET generation antibacterial killing. requires NADPH oxidase, while MIP-2 induces NETosis indepen- Neutrophil elastase and MPO are located in neutrophil pri- dently of NADPH oxidase (Farley et al., 2012). In pneumococcal mary granules, are constituents of NETs, and are required for lung infection, NETosis of lung neutrophils was reduced, but NET generation in response to specific stimuli (described above). not eliminated, in NADPH oxidase-deficient mice (Yamada et al., Therefore, deficiencies in these pathways can provide some insight 2011). Neutrophils from CGD patients are defective in NETo- into the role of NET constituents in host defense. The neutrophil sis, and gene therapy results in restored NETosis in NADPH serine proteases, NE, cathepsin-G (CG), and proteinase 3 (PR3),

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are activated by lysosomal cysteine protease cathepsin C/dipeptidyl an excessive coagulopathy and thrombosis resulting in endothelial peptidase I (DPPI; Pham et al., 2004). Papillon–Lefèvre syndrome cell injury and organ damage. Recent studies have shown that is a rare autosomal recessive disease resulting from loss-of- NETosis can drive transfusion-related acute lung injury and that function mutations in the DPPI gene locus that is characterized by depleting NETs was protective (Caudrillier et al., 2012; Thomas palmoplantar hyperkeratosis, periodontitis leading to loss of teeth, et al., 2012). and severe bacterial infections, including liver abscesses (Almuneef NETosis has also been implicated in the pathogenesis of et al., 2003; Pham et al., 2004). MPO-deficient mice have defective autoimmune disorders, such as systemic lupus. Autoimmunity candidal killing in vivo (Brennan et al., 2001) and neutrophils from may be driven by a combination of factors related to aberrant MPO-deficient patients are impaired in the ability to limit growth NETosis, including direct damage to tissue, release of autoanti- of extracellular Candida albicans (Metzler et al., 2011). However, gens that stimulate immune complexes, complement activation, MPO deficiency in humans is usually asymptomatic, although and IFN-α release by plasmacytoid dendritic cells (Guiducci et al., severe candidal infections have been observed in patients with 2010; Hakkim et al., 2010; Meyer-Hoffert and Wiedow, 2010; co-existing diabetes (Cech et al., 1979a,b). Garcia-Romo et al., 2011; Lande et al., 2011; Villanueva et al., 2011; NADPH oxidase deficiency leads to a more severe phenotype Leffler et al., 2012). A subset of patients with systemic lupus have than Papillon–Lefèvre or MPO deficiency. For example, patients reduced ability to degrade NETs due to impaired serum DNase1 with CGD are at high risk for invasive aspergillosis and specific bac- activity, and have a higher incidence of lupus nephritis (Hakkim terial pathogens (e.g., Burkholderia cepacia, Serratia marcescens, et al., 2010). and Nocardia species; Winkelstein et al., 2000; van den Berg et al., While NETosis has, so far, been shown to enhance neutrophil- 2009). Consistent with these clinical observations, CGD mice mediated injury, the role of NADPH oxidase is more complex were highly susceptible to infection by Aspergillus and by B. cepa- and likely involves interplay of injurious and protective path- cia, while NE−/− × CG−/− mice and DPPI-deficient mice were ways. NET constituents such as neutrophil proteases generally resistant (Vethanayagam et al., 2011). lead to augmented inflammation and tissue injury (Adkison Additional studies point to specific host defense functions et al., 2002; Hu and Pham, 2005; Raptis et al., 2005; Akk et al., of neutrophil proteases that are distinct from NADPH oxidase. 2008; Soehnlein et al., 2008b). However, studies in NADPH Proteases in human neutrophils target Streptococcus pneumoniae oxidase-deficient mice point to a more complex interaction (Standish and Weiser, 2009) and protease-deficient mice have between NADPH oxidase and acute lung injury that is context- increased susceptibility to pneumococcal pneumonia (Hahn et al., dependent. NADPH oxidase worsened the severity of acute 2011). In contrast, neutrophil NADPH oxidase has variable effects lung injury following influenza virus challenge in mice (Imai on pneumococcal strains in vitro; CGD neutrophils have nor- et al., 2008). While wild type and NADPH oxidase-deficient mal killing of pneumococci that produce peroxide, but defective mice had similar levels of lung injury following LPS challenge killing of peroxide-deficient mutant strains (Pitt and Bernheimer, (Sato et al., 2002), NADPH oxidase-deficient mice had greater 1974; Shohet et al., 1974), suggesting that pathogen-derived lung neutrophil sequestration, but less lung injury compared reactive oxidants can complement the killing defect in CGD to wild type mice in E. coli sepsis (Gao et al., 2002). In con- neutrophils. trast, NADPH oxidase was protective in acid aspiration-induced Together, these observations in the clinic and in mouse mod- lung injury. NADPH oxidase-deficient mice had increased air- els support NADPH oxidase, neutrophil serine proteases, and way neutrophilic inflammation and acute lung injury (measured MPO having distinct functions in host defense. Interventions that as albumin leak), but less injury per recovered neutrophil, com- deplete NETs in vivo (e.g., histone blocking antibody and DNase1; pared to wild type mice (Segal et al., 2007; Davidson et al., Caudrillier et al., 2012) may be useful to delineate specific host 2013). In addition, NADPH oxidase was required for opti- defense functions of NETs. mal activation of Nrf2, an ROI-inducible transcriptional factor that stimulates cytoprotective and anti-inflammatory responses NADPH OXIDASE AND NETs HAVE DISTINCT EFFECTS ON (Davidson et al., 2013). Thus, NADPH oxidase likely has a dual INFLAMMATION AND INJURY effect on inflammation-induced lung injury. While the immedi- The generation of NETs can be a double-edged sword. On the ate effects of NADPH oxidase activation leads to ROI generation one hand, they may promote pathogen killing. However, the and possibly NETosis that are predicted to be injurious, NADPH same pathways that control microbial infection can also cause oxidase can also protect against injury by limiting neutrophilic injury through a number of mechanisms. NET constituents can inflammation and by inducing cytoprotective pathways, such as damage epithelial and endothelial cells, which can exacerbate Nrf2. inflammation-induced organ injury (Saffarzadeh et al., 2012). The interaction between NETs, platelets, and coagulation further illus- CONCLUSION trate the concept of the dual host defense and injurious potential In response to infectious threat, neutrophils go to war. NADPH of NETs. Platelet-derived defensins have intrinsic antimicrobial oxidase is rapidly activated by specific microbial stimuli, lead- activities and can stimulate NETosis (Kraemer et al., 2011). NET ing to ROI generation. NADPH oxidase can activate neu- constituents (NE, CG, and histones) can activate platelets and pro- trophil granular proteases within the phagolysosome, thereby mote coagulation leading to intravascular thrombus growth that targeting phagocytized pathogens. In addition, NADPH oxi- restrict tissue bacterial invasion (Fuchs et al., 2010, 2011; Mass- dase stimulates NETosis, a process that targets extracellular berg et al., 2010); however, these same mechanisms may promote pathogens. While important for host defense, NETs are

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also implicated in the pathogenesis of a number of diseases, Future translational areas of research involve the application including acute organ injury and autoimmunity. Understanding of NETotic products as prognostic biomarkers for inflammatory mechanisms by which NADPH oxidase and its regulated pathways disorders (e.g., sepsis, autoimmunity). For example, in patients modulate inflammation and injury may identify novel therapeutic with sepsis, the degree of NETosis may correlate with a bet- approaches. ter outcome due to enhanced bacterial clearance or a worse There are several gaps in knowledge regarding NETosis. outcome reflecting inflammation-induced organ injury. Assays Although NETosis is induced by infection or conditions mimick- of NETotic markers (e.g., MPO–DNA complexes, NE) from ing infectious threat, we do not have a good understanding of the cell-free fluid (e.g., plasma) may provide a quantitative mea- molecular mechanisms that drive NETosis. NADPH oxidase can sure of NETosis from archived human samples that could be stimulate NETosis in certain settings and apoptosis in others; it is correlated with clinical outcome in a variety of diseases. This unclear how these dual effects are mediated. It is also unclear which knowledge may pave the way to therapeutic approaches to tar- pathways stimulate NETosisafter neutrophil death and breakdown get NETs, such as use of histone neutralizing antibody or of membranes versus NETosis in living neutrophils. Additional DNase1. questions relate to the relative contributions of NETosis versus intracellular killing of pathogens to host defense and whether the ACKNOWLEDGMENT major function of NETs is to prevent spread of versus killing of This work was supported by grants from the National Institutes of pathogens. Health: AI079253 (to Brahm H. Segal).

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