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Extracellular Traps: Double-Edged Swords of Innate Immunity Mariana J. Kaplan and Marko Radic This information is current as J Immunol 2012; 189:2689-2695; ; of September 26, 2021. doi: 10.4049/jimmunol.1201719 http://www.jimmunol.org/content/189/6/2689 Downloaded from References This article cites 77 articles, 34 of which you can access for free at: http://www.jimmunol.org/content/189/6/2689.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Neutrophil Extracellular Traps: Double-Edged Swords of Innate Immunity Mariana J. Kaplan* and Marko Radic† Spectacular images of ejecting nuclear chro- has important chemotactic and immunostimulatory effects, as matin and bactericidal proteins, in response to mi- discussed below (1). crobes, were first reported in 2004. As externalized The mechanisms used by neutrophils to eliminate microbes chromatin could entangle bacteria, these structures were include , generation of reactive oxygen species named neutrophil extracellular traps (NETs). Subse- (ROS), and the release of microbicidal molecules from quent studies identified and sterile granules (degranulation). In 2004, another distinct antimi- conditions that stimulate NETs, as well as additional crobial activity was described: neutrophils extrude a meshwork cell types that release extracellular chromatin. The re- of chromatin fibers that are decorated with granule-derived Downloaded from lease of NETs is the most dramatic stage in a cell death antimicrobial peptides and enzymes such as neutrophil elas- process called NETosis. Experimental evidence suggests tase (Fig. 1), cathepsin G, and myeloperoxidase (MPO) (2). that NETs participate in pathogenesis of autoimmune These structures, called neutrophil extracellular traps (NETs), represent an important strategy to immobilize and kill in- and inflammatory disorders, with proposed involve- vading microorganisms. The NET scaffold consists of chro- ment in glomerulonephritis, chronic lung disease, sep- matin fibers with a diameter of 15–17 nm; DNA and histones http://www.jimmunol.org/ sis, and vascular disorders. Exaggerated NETosis or represent the major NET constituents (2). Mass spectrometry diminished NET clearance likely increases risk of auto- has identified various additional proteins associated with reactivity to NET components. The biological signifi- NETs, including components from various types of granules. cance of NETs is just beginning to be explored. A Although conserved proteins are found in extracellular trap more complete integration of NETosis within immu- fractions, it is still unclear whether the peptide composition of nology and pathophysiology will require better under- these structures may vary depending on the specific stimulus standing of NET properties associated with specific that promotes NET formation (3, 4).

disease states and microbial . This may lead The putative role of extracellular traps in host defense is by guest on September 26, 2021 to the identification of important therapeutic tar- exemplified by their conserved nature in various gets. The Journal of Immunology, 2012, 189: 2689– (5–8), insects (9) and even plants (10). Whereas the initial 2695. observation of NET formation placed the process within the context of innate immune responses to infections, recent evidence suggests that these structures also figure promi- eutrophils are the most abundant leukocytes in mam- nently at the center of various pathologic states. As such, mals and, as a first line of defense against microbes, NETs may function as double-edged swords, serving as ef- N they play crucial roles in innate immune responses. fective antimicrobial defenses, but also as putative sources of The homeostasis of neutrophils is maintained by balancing molecules with immune effector and proinflammatory roles their short lifespan in the circulation with their regulated that, in susceptible individuals, may promote tissue damage release from the bone marrow. Neutrophils have various types and . Furthermore, other (mast of granules, containing hundreds of proteins. These include cells and ) can form extracellular traps upon bactericidal proteins, some of them with important effects on stimulation, a process thus renamed as ETosis (11, 12). In innate and adaptive immune responses, such as a-defensins, the case of eosinophils, NET-like structures containing mi- the cathelicidin cationic antimicrobial protein of 18 tochondrial DNA and granular proteins have been proposed kDa, and lactoferrin, among others. LL-37, a proteolytic to contribute to host defense and to allergic responses (12, product of human cationic antimicrobial protein of 18 kDa, 13).

*Division of Rheumatology, Department of , University of Michigan School, 1150 W. Medical Center Drive, 5520 Medical Science Research Building-I, Medical School, Ann Arbor, MI 48109; and †Department of Microbiology, Immunol- Ann Arbor, MI 48109-5680. E-mail address: [email protected] ogy and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN Abbreviations used in this article: ANCA, anti-neutrophil cytoplasmic Ab; CGD, 38163 chronic granulomatous disease; EC, endothelial cell; MPO, myeloperoxidase; NET, Received for publication June 21, 2012. Accepted for publication July 27, 2012. neutrophil extracellular trap; PAD4, peptidylarginine deiminase 4; pDC, plasmacytoid ; PKC, protein kinase C; ROS, reactive oxygen species; SLE, systemic lupus This work was supported by the National Institutes of Health through Public Health erythematosus. Service Grant HL088419 (to M.J.K.) and by the Lupus Research Institute of New York (to M.R.). Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 Address correspondence and reprint requests to Dr. Mariana J. Kaplan, Division of Rheumatology, Department of Internal Medicine, University of Michigan Medical www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201719 2690 BRIEF REVIEWS: NETs AND IMMUNOLOGY

forming NETs upon stimulation. When comparing mouse and human neutrophils, murine cells make NETs more slowly and less efficiently and these structures have more compact ap- pearance than do human cells (5). Most studies of NETs in mice use bone marrow neutrophils, whereas the preferred source in is peripheral blood. This may account for some differences identified between these species.

Microbes and NETs An astounding variety of microbes induce NET formation (2, 29–35). NET-inducing stimuli include whole bacteria as well as cell surface components of Gram-positive and Gram- FIGURE 1. Representative images of NETs induced in vitro by LPS in negative bacterial lipoteichoic acid and LPS, as well as break- human neutrophils. NETs are visualized by costaining of neutrophil elastase down products of prokaryotic proteins, such as fMLP (18). (green) and nuclear material (DAPI, blue). Original magnification 340. Notable examples of bacteria that potently induce NETs in- clude Staphylococcus aureus (36, 37), Streptococcus sp. (29, 38), Two models for NET release have been proposed. In the first Haemophilus influenzae (30), Klebsiella pneumoniae (39), Lis- model, NETosis, a distinct form of active cell death, is char- teria monocytogenes (40), Mycobacterium tuberculosis (31), and Downloaded from acterized by release of decondensed chromatin and granular Shigella flexneri (2). Other examples include such as contents to the extracellular space. During NETosis, nuclear Yersinia (41) and members of the oral microbiome, including and granular membranes dissolve, and nuclear contents de- Porphyromonas gingivalis (33). condense into the cytoplasm. This is followed by plasma mem- Neutrophils from neonates, compared with adult neutro- brane rupture and release of chromatin decorated with granu- phils, are less capable of forming NETs in response to various lar proteins into the extracellular space (2, 14, 15). In contrast microbial stimuli, a phenomenon that may contribute to the http://www.jimmunol.org/ to apoptotic cells, netting neutrophils do not appear to dis- enhanced susceptibility to infections observed in this age group play “eat me” signals, and this may prevent their clearance (42). Containment of microbial pathogens to sites of initial by . Indeed, NETs are disassembled primarily by may be an important function of NETs. Indeed, nucleases (15). The second model involves a DNA/serine staphylococci and streptococci express virulence factors that protease extrusion mechanism from intact neutrophils, where degrade extracellular traps and may free the bacterium from mitochondrial DNA release is apparently not associated to NET chromatin (29). In cystic fibrosis, pathogens such as cell death (16). Furthermore, autophagy may contribute to Pseudomonas aeruginosa thrive in the lungs of affected patients

NETosis (17). and NETs accumulate, promoting chronic lung obstruction. by guest on September 26, 2021 Intact chromatin lattices appear to be required for the an- Clinical lung isolates from cystic fibrosis patients acquire a timicrobial function of NETs (2). In addition to micro- mucoid phenotype that makes P. aeruginosa less sensitive to organisms, NETosis is triggered by other various stimuli, in- NET inhibition (35). This suggests that microbes may develop cluding proinflammatory (TNF-a, IL-8), platelets, adaptations to resist NET-mediated growth suppression. activated endothelial cells (ECs), NO, monosodium urate Fungi known to elicit NETs include Aspergillus nidulans, crystals, and various (18–26). NETs are iden- Aspergillus fumigatus, and Candida albicans (43–45). Patients tified by various techniques. Fluorescence microscopy and with chronic granulomatous disease (CGD), who are deficient immunohistochemical analysisoftransmissionelectronmi- in the expression of a fully functional NADPH oxidase, are croscopy data may be preferred approaches to characterize unable to form NETs and often die of infection with A. NETosis. Importantly, fibrin may mimic NETs in scanning nidulans. Gene therapy to reconstitute NADPH oxidase electron microscopy (27). function in CGD patients was associated with decreased Kinetic studies in both human and mouse neutrophils have fungal load and a gain in capacity to form NETs (45). The been published. Whereas phagocytosis and degranulation NET component implicated in immune defense against A. usually take minutes to occur after microbe exposure, NETosis nidulans and C. albicans is calprotectin, a Zn2+ chelator and is a more protracted event (5, 14). It is unclear which factors heterodimer of the cytosolic S100A8 and S100A9 proteins determine the selection between these alternative antimicro- present in the NETs. Thus, NETs contain neutrophil proteins bial activities and whether these processes can coexist in the that serve specialized functions in fungal immunity and pro- same cell. About 20–60% of isolated human neutrophils vide high local inhibitor concentrations in close proximity to typically release NETs 2–4 h after stimulation with microbes pathogens (3, 45). or protein kinase C (PKC) activators; however, they respond Various protozoan parasites induce NETs (46–48). In vitro, within minutes when activated by LPS-stimulated platelets Leishmania amazonensis or its surface lipophosphoglycan in- under conditions of flow (22). The differences in predispo- duces NETs, whereas their disruption with DNase I improves sition to form NETs and the characteristics of these struc- survival of the (46). In the Toxoplasma gondii in- tures made by immature and mature neutrophils remain to fection model, the tachyzoite induces neutrophil influx into be determined. Priming with type I or type II IFNs and sub- the infected tissues and release of NETs that may limit sequent stimulation with C5a leads to NETosis in mature, pathogen viability. In children with , NETs have been but not immature, neutrophils (28). observed in fresh blood smears and correlate with specific Bone marrow-derived neutrophils from common murine profiles and with the development of anti-DNA strains used in immunology research appear equally capable of autoantibodies (48). The Journal of Immunology 2691

Given the diversity of microbes that elicit NETs, it is not surprising that, depending on the microbial agent, these lattices may have different effects on the spread and severity of an infection. Just how NETs contribute to immune defense is open to argument. It is important to distinguish microbial death mediated by NETs from failure to recover viable or- ganisms as a result of their entrapment. Histones, the most abundant proteins in NETs, have bactericidal properties. Proteolytic fragments of histones function as defensins in the innate (49). However, some bacteria may survive association with NETs without a loss of viability (50). For these, NETs may represent an important mechanism that limits their spread in the host. Whereas some pathogens are susceptible to containment or damage by NETs, others may have found ways to counteract these lattices or even to incorporate NET chromatin into their support structures (51). In a surprising twist of evolutionary adaptation, certain bacteria may even benefit from NETs. Downloaded from Colonizing H. influenzae often resists treatment owing to their ability to form biofilms that contain structural elements of NETs. Thus, although stimuli produced by H. influenzae can induce NETs, the bacterium avoids damage by these structures. Instead, its lipooligosaccharide coat tightly asso- ciates with NETs and, as result, these bacteria escape phago- http://www.jimmunol.org/ cytic uptake and clearance (30). Conflicting reports of bacterial killing by NET chromatin have prompted examination of bactericidal properties of in vitro-generated NETs. Purified NET chromatin alone is not very efficient at killing S. aureus, and MPO present in NETs provides the bactericidal activity needed to kill this pathogen in the presence of hydrogen peroxide (52). Other NET com-

ponents with antibacterial properties include LL-37, lacto- by guest on September 26, 2021 ferrin, neutrophil elastase, and proteinase-3. One added level of complexity in studies of the bactericidal and bacteriostatic properties of NETs is that some of the components of these structures, such as MPO, assist in the production of NETs and also directly promote pathogen degradation (53). Additionally, proteases that damage captured microbes also may have important roles in shaping the struc- ture of NET chromatin. Histones associated with NETs are “trimmed” by proteases, whereby they lose specific lengths of their amino termini. These histone “tails” have important roles in organizing the higher order structure of chromatin, and their cleavage may play a crucial role in producing the “beads on a string” structure of extended NET chromatin (2, 3). The role of NETs in fighting viral infections remains to be FIGURE 2. Putative stimuli and steps in NETosis. (A) Microbes and their fully characterized, although these web structures may promote products, immune complexes, autoantibodies, cytokines (IL-8, TNF, and lung injury following influenza infection (7, 54, 55). NETs type I and II IFNs), and other stimuli can induce NETosis. Binding via capture HIV and promote its elimination through MPO and TLRs, Fc receptors, or complement receptors have been implicated in in- a-defensin. This can develop an anti-NET response duction of NETs. (B) NETosis is initiated by binding of neutrophil surface mediated by IL-10 (56). receptors (R) to microbes or microbial breakdown products, inflammatory stimuli, or endogenous inducers. Binding to receptor(s) (exemplified in dia- Mechanisms of NETosis gram as R1 and R2) induces endoplasmic reticulum calcium store release and opening of membrane channels that lead to cytoplasmic calcium increases. Studies of NETosis are difficult to design and interpret. The Elevated calcium stimulates PKC activity, phosphorylation of gp91phox, and use of pharmacologic stimuli for NETosis only compounds the assembly of functional NADPH oxidase, leading to ROS and NO produc- problem. Despite these caveats, important insights have been tion. Morphological changes observed during NETosis include breakdown of gained into the mechanisms of NETosis (Fig. 2). The stimuli nuclear and granule membranes and the mixing of nuclear, granular, and cytoplasmic contents. Deimination and proteolytic cleavage of histones may that induce NETosis can be broadly classified into microbe- initiate before nuclear breakdown and contribute to chromatin decondensa- associated, inflammatory, or endogenous (“sterile”) triggers. tion. A rupture in the plasma membrane allows the release of extracellular Diverse neutrophil receptors can signal to induce NETosis, as chromatin traps. binding via TLRs, Fc receptors, or complement receptors has 2692 BRIEF REVIEWS: NETs AND IMMUNOLOGY been implicated in the induction of NETs (2, 24, 57). Cy- model explaining the externalization of putative modified tokine receptors likely play roles in NETosis signaling, as IL- nuclear autoantigens (such as DNA) and their access to the 8, TNF, and IFN-g can trigger this process (2, 18, 28). More immune system has been that apoptotic debris represents the studies are required to address the synergy or cross-talk be- primary source (63, 64). However, recent evidence that ex- tween different classes of neutrophil receptors in the induc- tracellular DNA is a common, and even necessary, process in tion of NETosis. naturally occurring immune responses mediated by NETosis The most frequently used compound to induce NETosis is has opened new possibilities of better understanding the PMA, a synthetic activator of the PKC family of enzymes. PKC mechanisms that lead to breaking of tolerance and the pro- is directly responsible for activation of NADPH oxidase and motion of autoimmunity. ROS production. Many of the natural NET inducers lead to Several of the molecules decorating the NETs (e.g., MPO, PKC activation, as elevated cytoplasmic calcium and its release dsDNA, histones) are autoantigens in systemic autoimmune from endoplasmic reticulum stores are direct consequences of diseases such as anti-neutrophil cytoplasmic Ab (ANCA)- receptor activation. The requirement for ROS production is positive vasculitis and systemic lupus erythematosus (SLE). consistent with the inhibition of NETosis by ROS scavengers Thus, various groups have proposed that aberrant NET for- such as N-acetylcysteine, diphenylene iodinium, or Trolox mation may be important in the generation of autoimmune (15, 58). How ROS contribute to NETosis is controversial. responses in predisposed individuals (24–26, 65). This hy- One possibility is that they directly promote the observed mor- pothesis is supported by the observation that initiation or phologic changes occurring in neutrophils during NETosis exacerbation of autoimmune responses often occurs follow- Downloaded from (39). A proposed alternative is that ROS serve to inactivate cas- ing microbial infections. Various autoantibodies can promote pases, thereby inhibiting apoptosis and instead inducing auto- the release of NETs containing antimicrobial proteins, includ- phagy, a process leading to dissolution of cellular membranes ing LL37 (24–26). This combination of DNA and LL37 is (17). These alternatives may not be mutually exclusive and a potent stimulus for plasmacytoid dendritic cells (pDCs) to they each may occur under different experimental conditions. synthesize type I IFNs. Type I IFNs have antimicrobial roles How NETosis signals are transduced is suggested by studies http://www.jimmunol.org/ phox but also potent immunostimulatory effects in autoimmune on Rac2. Phosphorylation of gp91 by PKC allows the diseases such as SLE and psoriasis (11, 24, 26, 65). Impor- cytosolic and membrane-bound subunits of NADPH oxidase tantly, neutrophils from patients with various autoimmune to assemble into a functional complex that generates ROS diseases appear more prone to NETose. However, isolated in and, in particular, superoxide ions (59). Whereas Rac1- vivo exposure to NETs may be insufficient to break tolerance, deficient neutrophils release similar quantities of NET chro- and additional factors (e.g., genetic influences, inflammatory matin as wild-type neutrophils in response to PMA or LPS, milieu) may need to be present to trigger full, sustained au- Rac2-deficient cells cannot form a functional enzyme complex toimmune responses, as recently suggested (66).

and are impaired in NET formation (58). Further steps in by guest on September 26, 2021 Recent evidence also indicates that NETs can shape adaptive the conversion of superoxide to hydrogen peroxide and to immune responses, as these structures can prime T cells and perchloric acid are also essential for NET release. Superoxide reduce their activation threshold (67). The specific role of itself is not essential, as rescue of the NETosis pathway in CGD neutrophils was observed with addition of exogenous NETs in other components of adaptive immune responses peroxide. NETosis may also occur in certain conditions remains to be fully characterized. Below, we summarize recent through ROS-independent pathways (42, 60), which suggests discoveries on the potential role of NETs in the pathogenesis that specific stimuli and the surrounding inflammatory milieu of autoimmune diseases. may modulate how NETs are generated. Vasculitis. Small vessel vasculitis is a systemic autoimmune Histone deimination by peptidylarginine deiminase 4 (PAD4) disease characterized by necrotizing inflammation of small is a prominent posttranslational modification in NETs that is blood vessels. Many patients with this disease develop ANCAs induced by inflammatory or infectious stimuli (18, 61). The with specific reactivity against PR3 or MPO, which are con- conversion of certain arginine residues to citrullines in core sidered diagnostic of this disease. ANCAs induce NETosis, histones by PAD4 eliminates the positive charge from that whereas NET-derived material is detected in blood and in residue and may thereby weaken the binding of histones to kidney biopsies from a large proportion of small vessel vas- DNA. Because the PAD4 substrate arginines are part of his- culitis patients (25). Importantly, autoantigens recognized by tone tails that mediate interactions between nucleosomes, loss ANCAs have been found in the NET structure (25). The drug of positive charge may unravel the compact nature of nuclear propylthiouracil can induce ANCA vasculitis; this may occur chromatin and allow it to disperse in the form of NETs. through an effect on the conformation of NETs, leading to Analysis of neutrophils from mice deficient in PAD4 revealed their impaired degradation (68). These observations suggest that they were impaired in their capacity to form NETs in new mechanisms of ANCA generation and induction of or- vitro (54, 62). In vivo, PAD4-deficient mice had more severe gan damage in this autoimmune condition. bacterial infections, although the inability to form NETs did Psoriasis. Psoriasis is a common inflammatory disorder of the not affect viral titers and leukocyte infiltration rates during skin and other organs, where IL-17 may play prominent influenza infections (54). pathogenic roles. Neutrophils and mast cells were recently reported to represent the predominant cell types expressing NETs as sources of autoantigens and immunostimulatory proteins IL-17 in in psoriasis, and this cytokine is re- NETs may play crucial roles in the regulation of immune leased during ETosis (11). As such, externalization of IL-17 responses, protease-mediated effector functions, and inter- in the extracellular traps of neutrophils and mast cells may be cellular signal transduction. Until recently, the predominant central to psoriasis pathogenesis. The Journal of Immunology 2693

Systemic lupus erythematosus. SLE is an autoimmune syndrome is an antimicrobial component of platelets that can induce that affects many organs and is characterized by autoantibodies NETs. Platelet–neutrophil interactions leading to NETs may against DNA, chromatin, and DNA-associated proteins, includ- play important roles in ECs and tissue damage not only during ing NET components. Recent evidence points to an imbalance sepsis, but also in chronic inflammatory disorders (22). between NET formation and NET clearance in this disease NETs and atherosclerosis. Neutrophil-derived CRAMP (the mu- (24, 26, 65, 69). As such, patients with aberrantly elevated rine equivalent of LL37) is proatherogenic by recruiting in- NETosis, in conjunction with impaired degradation of these flammatory into arteries. CRAMP/DNA complexes structures by nucleases, may be particularly prone to NET- stimulatepDCsinplaquelesionsandcontributetogeneration associated tissue damage. Decreased NET degradation occurs of autoantibodies and aggravation of atherosclerosis in apoli- 2 2 in a subset of SLE patients, correlating with renal disease, and poprotein E / mice (75, 76). These observations suggest a secondary to DNase1 inhibitors and anti-NET Abs (69). Self putative role for NETs components in atheroma formation DNA in lupus immune complexes contains LL37, which that requires further investigation. triggers TLR9 in pDCs, and consequent IFN-a synthesis, and Pregnancy. In preeclampsia, a pregnancy-related inflammatory it protects nucleic acids from degradation by nucleases (26). and vasculopathic disorder, massive numbers of NETs have These DNA/antimicrobial complexes are released during been reported in vivo in the intervillious space, apparently NETosis, a process enhanced in SLE, and mediated in part by triggered by trophoblast microdebris and IL-8. A pathogenic autoantibodies present in serum from a subset of lupus role for NETs in preeclampsia and pregnancy outcomes patients (24, 26). NETs may also activate complement, thereby remains to be proven (19). Downloaded from amplifying disease (70). A subset of aberrant low-density gran- ulocytes present in SLE displays enhanced capacity to form Therapeutics NETs and kill ECs in a NET-dependent manner. Further- There is already some precedent in manipulating NETosis for more, netting lupus neutrophils are detected in blood and therapeutic purposes, as mentioned above in the case of CGD infiltrate affected organs, where they expose immunostimu- (45). Various ROS scavengers are effective at reducing the latory molecules (65). There is also preliminary evidence that release of NETs (15), and similar avenues may be available to http://www.jimmunol.org/ nuclear material that is externalized on NETs promotes auto- repress NETosis in vivo in chronic inflammatory disorders. formation in SLE (26). MPO inhibitors, such as 4-aminobenzoic acid hydrazide, and Felty’s syndrome. Felty’s syndrome is characterized as rheuma- various PAD4 inhibitors may have similar effects (39, 77). toid arthritis, , and neutropenia. In Felty’s syn- The role of colchicine or other drugs that destabilize the cy- drome, autoantibodies were found to be directed against toskeleton, a structure implicated in NETosis, should be ex- PAD4-deiminated histones and to induce NETosis (71). plored (78, 79). Inhibition of the effects of histones contained Netting neutrophils were found to be reactive with autoanti- in NETs may prove beneficial in various inflammatory con- bodies from Felty’s syndrome, SLE, and rheumatoid arthritis ditions or in EC damage. Clearly, a better understanding of by guest on September 26, 2021 patients, suggesting a common cellular mechanism in these how NETs are generated and whether NETs that form upon disorders. The role that NETs play more broadly in rheu- microbial exposure are distinct from those that form under matoid arthritis pathogenesis remains to be determined. “sterile” conditions, as exist in autoimmune or vasculopathic . Gout is characterized by acute neutrophilic joint in- disorders, may allow the development of compounds that flammation triggered by inflammatory responses to uric acid selectively target the deleterious aspects triggered by these crystals. Activation of neutrophils in gout is associated with the lattices. formation of proinflammatory NETs, a phenomenon that was linked to both autophagy and IL-1b release (23). Conclusions Additional studies are required to assess the exact role of The biological significance of NETs is just beginning to be aberrant NETosis in induction and perpetuation of the diseases explored. In addition to playing a central part in antimicrobial mentioned above and in other chronic inflammatory diseases. innate immunity, NETs are generated upon noninfectious stimuli in various clinical settings. In acute or chronic in- NETs in vascular damage and atherothrombosis flammatory disorders, aberrantly enhanced NET formation EC activation can elicit NETosis, and, in turn, NETs induce and/or decreased NET degradation may play key roles in vascular damage. Furthermore, netting neutrophils may play initiation and perpetuation of autoimmune responses and important roles in the promotion of atherosclerosis, throm- organ damage. Various areas pertinent to NET need to bosis, and other vascular complications (21, 65). be better defined, including the molecular mechanisms gov- NETs and thrombosis. NETs can provide a stimulus and scaffold erning their generation and the downstream pathways leading for thrombus formation by promoting platelet and RBC ad- to their formation. At this point, it is unclear whether all NETs hesion and by concentrating effector proteins and coagulation are created equal and whether traps from different disease factors involved in clotting (72–74). Thrombus-resident neu- conditions or species will be phenotypically and functionally trophils are indispensable for deep venous thrombosis propa- identical. Identification of endogenous stimuli leading to gation by binding factor XII and supporting its activation NETosis has been incomplete and needs to be better under- through NETosis. Interestingly, the anticoagulant heparin stood. For example, a more accurate distinction between the can remove histones from NET chromatin fibers, leading to effects of NET trapping and NET killing on different types of their destabilization, suggesting an additional mechanism by microbes and a better understanding of the kinetics of NET which this compound is antithrombotic (72–74). Ligation of release in different tissues or in the bloodstream are needed. TLR4 by LPS in narrow vessels induces platelet binding to Better characterizing formation of NETs and their role in adherent neutrophils, promoting NETosis. Human b-defensin1 biological processes in vivo remains a priority. Unraveling the 2694 BRIEF REVIEWS: NETs AND IMMUNOLOGY

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