Neutrophil extracellular traps: casting the NET over pathogenesis

Florian Wartha1,2, Katharina Beiter1,2, Staffan Normark1,2 and Birgitta Henriques-Normark1,2

Neutrophil extracellular traps (NETs) are considered to be part of the human innate immunity because they trap and kill pathogens. NETs are formed by activated neutrophils and consist of a DNA backbone with embedded antimicrobial peptides and enzymes. They are involved in host defense during pneumococcal pneumonia, streptococcal necrotizing fasciitis, appendicitis and insemination. Recently, bacterial virulence factors that counteract NETs have been identied. These include the degradation of the NET-backbone by DNases enabling the liberation of bacteria from NETs, as well as capsule formation, which reduces bacterial trapping. Furthermore, pathogens can resist NET-mediated killing by adding positive charge to their cell surface.
Addresses 1 Department of Bacteriology, Swedish Institute for Infectious Disease Control Nobelsvag 18, SE-171 82 Solna, Solna, Sweden 2 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobelsvag 16, SE-171 77 Stockholm, Sweden Corresponding author: Henriques-Normark, Birgitta (Birgitta.Henriques@smi.ki.se) Current Opinion in Microbiology 2007, 10:5256 This review comes from a themed issue on Host-microbe interactions: bacteria Edited by Pamela Small and Gisou van der Goot Available online 8th January 2007 1369-5274/$ see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.12.005

neutrophil granules. The nuclear constituents are chromatin DNA and histones. DNA is the major structural component of NETs and it provides the backbone on which the proteinaceous effectors reside. Hence, treatment with DNase results in disintegration of the NETs. Membranes, membrane proteins and cytoplasmic markers such as annexin I, actin and tubulin are absent from NETs. The structure, as determined by Brinkmann et al. [3] using electron microscopy, involves brous DNA stretches with a diameter ranging between 15 nm and 17 nm and globular protein domains of around 25 nm that might aggregate to larger threads with diameters of up to 50 nm. These proteins provide the network with antimicrobial activity. For a more detailed overview of the proteinaceous part of the NET structure, see Table 1.

NET induction and formation

Brinkmann et al. [3] showed that NETs are made by neutrophils activated with interleukin-8 (IL-8), phorbol myristate acetate (PMA) or lipopolysaccharide, but not by nave cells. However, not much is known about exact induction pathways (for an overview of known stimuli leading to NET formation, see Figure 1b). Martinelli et al. [4] found, using a microarray approach, that mature neutrophils, in contrast to immature neutrophils, strongly express interferon target genes. Their functional in vitro studies suggested that interferons (types I and II) prime mature neutrophils, enabling the subsequent formation of NETs after stimulation with complement factor 5a (C5a). Without the priming step by interferons, no NETs were formed after stimulation with C5a; however, no priming step is needed to induce NET-formation when neutrophils are stimulated with PMA, lipopolysaccharide or IL-8 [3]. Furthermore, Urban et al. [5] showed that distinctive receptors such as the Fcg receptors and pathogen pattern-recognition receptors (Toll-like receptors or dectin) might be involved. It has been debated in the literature whether NETs are formed in an active, controlled process. Brinkmann et al. [3] showed that NETs are not the result of leakage during cellular disintegration. Also, the process of NET formation occurs too rapidly to be caused by apoptosis, because both Brinkmann et al. [3] and Buchanan et al. [6] found that neutrophils release NETs as early as 10 min after activation. However, the possibility remains that NET formation is an early event in the neutrophil programmed cell death. An interesting nding by Schirmer et al. [7] might give a hint as to why neutrophils are able to form NETs. The
www.sciencedirect.com

Introduction
Polymorphonuclear leukocytes (neutrophils) are important players in the rst line of defense against invading microbial pathogens. Their role in phagocytic uptake and intracellular killing of pathogens has been well described previously [1,2]. In 2004 neutrophils were shown to form neutrophil extracellular traps (NETs) that bind, disarm and kill pathogens extracellularly [3]. Five aspects of NETs are covered below: NET structure; NET induction and formation; the role of NETs in disease; escaping from NETs; and other extracellular means for bacterial trapping.

NET structure
NETs are assembled from granular and nuclear constituents of neutrophils (see Figure 1a). The granule components are peptides and enzymes (e.g. elastase and myeloperoxidase) that are normally stored in distinctive
Current Opinion in Microbiology 2007, 10:5256

Neutrophil extracellular traps: casting the NET over pathogenesis Wartha et al. 53

Figure 1

Structure of neutrophil extracellular traps and stimuli leading to their formation. (a) NETs are extracellular fibrous structures consisting of a DNA-backbone, histones and neutrophil granular proteins (see inset). Microbes are trapped in NETs and killed by the high local concentration of antimicrobial components. Some pathogens such as S. pneumoniae and GAS express DNases that degrade the DNA-backbone of NETs, thereby liberating bacteria from entrapment. (b) Neutrophils form NETs in response to different stimuli, for example the LPS depicted here. The exact induction pathways are still unidentified. In NET formation nuclear DNA and granule proteins are released. Abbreviations: Abs, antibodies; IFN, interferon; FcgR, Fcg receptor; LPS, lipopolysaccharide; PRR, pattern-recognition receptor.

authors studied the nuclear lamina, which provides structural scaffolding for the nuclear envelope and consists largely of a lamin protein polymer. These data suggest that the ensemble (A, C, B1 and B2) of lamin interactions in a particular cell type affect the stability of the corresponding polymer. Decreased lamina stability in B2-rich neutrophils could facilitate the chromatin extrusion that occurs during NET formation. Furthermore, the specic chromatin structure of neutrophils, lacking distinctive protein markers (HP1a, HP1b, HP1g and monomethylated, dimethylated and trimethylated histone H3 lysine
www.sciencedirect.com

9 [H3K9]) might further facilitate the release of DNA and histones [8].

Role of NETs in disease

NETs interact with a variety of different pathogens. They capture both Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae and Group A streptococci [GAS]) and Gram-negative bacteria (Salmonella enterica serovar Typhimurium and Shigella exneri) as well as fungi (Candida albicans). By providing a high local concentration of antimicrobial proteins, NETs could disarm and kill
Current Opinion in Microbiology 2007, 10:5256

54 Host-microbe interactions: bacteria

Table 1 Proteins present in NETs and their cellular origins. Source of protein Nucleus Azurophilic (primary) granules Name Histones h1, h2a, h2b, h3, and h4 Neutrophil elastase Cathepsin G Myeloperoxidase (MPO) Bactericidal permeability increasing protein (BPI) Lactoferrin Gelatinase Peptidoglycan recognition proteins (PGRPs) [23]

keeping inammatory mediators from diffusing to adjacent, healthy tissue. Lee and Grinstein [14] argued that elastase, cathepsin G and other NET proteins, functioning as degrading enzymes, pose a risk for tissue damage. However, by binding to NETs, these enzymes are less likely to cause collateral damage after secretion. Nevertheless, NETs might have a deleterious effect on the host because of the leakage of nucleic acids, potentially playing a role during the development of autoimmune diseases like lupus erythematosus, a disease associated with autoimmune reactions to the hosts own DNA.
Escaping the NETs

Specic (secondary) granules Tertiary granules

bacteria, as has been shown for S. aureus, GAS and S. exneri [3,6]. Antimicrobial proteins include proteases such as neutrophil elastase that degrade virulence factors (IpaB of S. exneri and a toxin of S. aureus) [9], bactericidal permeability-increasing protein (BPI) and histones [3]. Also, Urban et al. [5] showed that both hyphae and yeast forms of the eukaryotic pathogen C. albicans were trapped and killed in NETs. By contrast, S. pneumoniae was shown to be trapped by NETs but to be relatively resistant to NET-mediated killing [10]. Wartha et al. [11] recently found that pneumococci resist killing as a result of a cooperative effect of polysaccharide capsule expression and lipoteichoic acid D-alanylation, resulting in the incorporation of positive charge to the surface, thereby repelling the positively charged antimicrobial peptides of NETs. Are NETs formed in vivo and do they play any role in disease? NETs have been found to be abundant at sites of infection. Brinkmann et al. [3] showed the occurrence of NETs in spontaneous human appendicitis and Shigellainduced experimental dysentery in rabbits, and Beiter et al. [10] showed the presence of NETs in a murine model of pneumococcal pneumonia, where NETs line the alveoli. Simultaneously, Buchanan et al. [6] showed the existence of NETs in a murine model of necrotizing fasciitis, induced by GAS. Recently, NETs were also found in bovine mastitis, where neutrophil phagocytosis and oxidative burst have been shown to be hampered by milk fat and proteins [12]. However, Lippolis et al. [12] found that bovine neutrophils form NETs after stimulation with PMA and ionomycin, even in the presence of milk. Finally, NETs have been detected in human preeclampsia, a severe pregnancy-related disorder, characterized by previously unexplainable high amounts of circulatory DNA of maternal origin. In this study by Gupta et al. [13], placenta-derived syncytiotrophoblast microparticles (STBM) and cytokines (e.g. IL-8) were independently able to activate neutrophils for NET-formation in vitro. Interestingly, NETs were able to entrap proinammatory STBM, thereby possibly controlling the immune response by
Current Opinion in Microbiology 2007, 10:5256

NETs constitute an important innate immune defense mechanism inuencing invasion of pathogens into the body. Therefore it is not surprising that virulence mechanisms have evolved to counteract NETs. One such mechanism includes DNases, which are expressed by many Gram-positive bacterial pathogens [6,10,15]. Recently, DNases of S. pneumoniae and GAS were shown to degrade the DNA-backbone of NETs, thereby promoting virulence (see Figure 1a). Beiter et al. [10] found that the pneumococcal surface nuclease EndA enabled pneumococci to degrade the DNA scaffold of NETs and then to escape. In a murine intranasal model, they demonstrated that escaping the NETs promoted the spread of pneumococci from the upper airways to the lungs and from the lungs into the bloodstream during pneumonia. Similarly, Sumby et al. [15] and Buchanan et al. [6] demonstrated DNase expression to be important for virulence in GAS M1 (spd, spd3 and sda) and GAS M49 (sda1), respectively. A triple-mutant strain of the chromosome-encoded and prophage-encoded extracellular DNases spd, spd3 and sda was signicantly less virulent than the wild type parental strain in two mouse models of invasive infection: intraperitoneal infection, as well as subcutaneous infection representing a model for necrotizing fasciitis [15]. Moreover, the DNase-decient strain was more sensitive to extracellular neutrophil killing in vitro. Buchanan et al. [6], using live-cell imaging, demonstrated that DNase-producing GAS were able to degrade the DNA-backbone of NETs. Interestingly, inhibition of DNase activity with G-actin reduced virulence in vitro and in vivo, suggesting a potential role of DNase-inhibitors for therapy. However, the use of DNase inhibitors as a potential drug to treat infections with DNase-producing pathogens conicts with the use of DNase in mucolytic therapy of patients with cystic brosis. The tenacious sputum, which obstructs the lungs of cystic brosis patients, has been found to be rich in DNA and elastase, and thus reminiscent of NETs. Therapy with inhaled DNase reduces the symptoms of cystic brosis and improves pulmonary
www.sciencedirect.com

Neutrophil extracellular traps: casting the NET over pathogenesis Wartha et al. 55

function. Therefore excessive NET formation might be counterproductive, preventing proper mechanical clearance of the airways [14]. However, Walker et al. [16] recently described actin and DNA laments in cystic brosis sputum as being a product of necrotic neutrophils. In contrast to NETs, these structures were not antimicrobial, but rather enhanced P. aeruginosa biolm formation and survival. Persistent P. aeruginosa infections are common among patients with cystic brosis and hence DNase, resolving the biolm substrate (actin and DNA laments), might be effective in cystic brosis treatment. Interestingly, NETs and DNases are not only involved in infectious diseases, but also during mammalian reproduction [17]. Insemination stimulates neutrophil migration into the female reproductive tract with the purpose of eliminating excess spermatozoa and microbial contaminants. An adverse effect is formation of the neutrophil spermatozoa cluster, affecting sperm motility and fertility. By an unknown mechanism, seminal plasma improves fertility of equine and swine spermatozoa inseminated in the presence of neutrophils. Alghandi et al. [17] found that neutrophil activation by spermatozoa results in formation of NETs and extensive sperm entrapment. Furthermore they showed that DNase present in the seminal plasma might free entrapped spermatozoa, to some extent explaining the fertility-promoting effect of seminal plasma.

[22] showed that SP-D is indeed involved in bacterial clearance during the early stages of infection.

Conclusion
Neutrophils are an important host defense against invading pathogens. It has, however, been an enigma as to how neutrophils mediate defense against encapsulated bacteria such as pneumococci, that in the absence of opsonization are not readily phagocytosed. NETs represent a novel mechanism by which neutrophils contribute to host defense. These traps are composed of DNA, histones and other antibacterial components, with the potential to conne, as well as kill bacteria and fungi. Recent data suggest that NETs are formed during infection, preventing the systemic spread of pathogens from local sites of infection. However, both the degree of trapping and of killing by NETs might be affected by bacterial surface structures. Another strategy of invasive streptococcal pathogens is to free themselves from entrapment by the production of surface-located endonucleases, thereby degrading the DNA scaffold of NETs. The benecial and possibly detrimental role of NETs in diseases involving inltrating neutrophils needs to be further investigated and the conditions required for NET-formation in vivo better dened.

Acknowledgement
We thank Arturo Zychlinsky for critically reading the manuscript and for his helpful comments. This work was supported by Marie Curie Early Stage Research Training Fellowships of the European Communitys 6th Framework Programme (called IMO-train and EIMID), the EU programme derbergs PREVIS in 6th Framework Programme, Torsten and Ragnar So foundation, Swedish Royal Academy of Sciences and the Swedish Research Council.

Other extracellular means for bacterial trapping

Connement of an infection to a local site in the body might be an important function of NETs. However, NETs are not the only means of entrapment. In insects, an innate immune mechanism named hemolymph coagulation has been described. The hemolymph coagulation is important for sealing wounds, trapping microbes and for blocking their entry [18]. Despite striking similarities between the clots formed in insects and mammalian neutrophil NETs, clots formed in Drosophila do not contain any substantial amount of DNA or antimicrobial activity that directly kill bacteria. Trapping mechanisms found in mammals include the salivary scavenger receptor gp340 and lung surfactant proteins. The oligomeric gp340 receptor aggregates streptococci and other bacteria at mucosal surfaces and might, in a similar way to NETs, generate extracellular networks that trap both pathogens and innate defense molecules in close proximity [19]. Surfactant proteins line the alveoli and prevent them from collapsing during expiration by lowering the surface tension. It was shown, however, that surfactant proteins A and D (SP-A and SP-D) bind to the pneumonia-causing bacteria S. pneumoniae, Klebsiella pneumoniae and P. aeruginosa, causing their aggregation [20,21]. With the help of a SP-D knockout mouse, Jounblat et al.
www.sciencedirect.com

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 2. 3. Mayer-Scholl A, Averhoff P, Zychlinsky A: How do neutrophils and pathogens interact? Curr Opin Microbiol 2004, 7:62-66. Nathan C: Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 2006, 6:173-182. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science 2004, 303:1532-1535. Martinelli S, Urosevic M, Daryadel A, Oberholzer PA, Baumann C, Fey MF, Dummer R, Simon HU, Youse S: Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. J Biol Chem 2004, 279:44123-44132. Urban CF, Reichard U, Brinkmann V, Zychlinsky A: Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 2006, 8:668-676.

4.

5.

6. 

Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, Feramisco J, Nizet V: DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr Biol 2006, 16:396-400. See annotation for [10]. Schirmer EC, Gerace L: The stability of the nuclear lamina polymer changes with the composition of lamin subtypes Current Opinion in Microbiology 2007, 10:5256

7.

56 Host-microbe interactions: bacteria

according to their individual binding strengths. J Biol Chem 2004, 279:42811-42817. 8. Lukasova E, Koristek Z, Falk M, Kozubek S, Grigoryev S, Kozubek M, Ondrej V, Kroupova I: Methylation of histones in myeloid leukemias as a potential marker of granulocyte abnormalities. J Leukoc Biol 2005, 77:100-111. Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A: Neutrophil elastase targets virulence factors of enterobacteria. Nature 2002, 417:91-94.

16. Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, Saavedra MT, Fessler MB, Malcolm KC, Vasil ML, Nick JA: Enhanced Pseudomonas aeruginosa biolm development mediated by human neutrophils. Infect Immun 2005, 73:3693-3701. 17. Alghamdi AS, Foster DN: Seminal DNase frees spermatozoa  entangled in neutrophil extracellular traps. Biol Reprod 2005, 73:1174-1181. A highly interesting nding that NETs are formed during insemination, and the fact that seminal DNase enables spermatozoa to free themselves. 18. Bidla G, Lindgren M, Theopold U, Dushay MS: Hemolymph coagulation and phenoloxidase in Drosophila larvae. Dev Comp Immunol 2005, 29:669-679. 19. Loimaranta V, Jakubovics NS, Hytonen J, Finne J, Jenkinson HF, Stromberg N: Fluid- or surface-phase human salivary scavenger protein gp340 exposes different bacterial recognition properties. Infect Immun 2005, 73:2245-2252. 20. Lim BL, Wang JY, Holmskov U, Hoppe HJ, Reid KB: Expression of the carbohydrate recognition domain of lung surfactant protein D and demonstration of its binding to lipopolysaccharides of Gram-negative bacteria. Biochem Biophys Res Commun 1994, 202:1674-1680. 21. Hartshorn KL, Crouch E, White MR, Colamussi ML, Kakkanatt A, Tauber B, Shepherd V, Sastry KN: Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria. Am J Physiol 1998, 274:L958-L969. 22. Jounblat R, Clark H, Eggleton P, Hawgood S, Andrew PW, Kadioglu A: The role of surfactant protein D in the colonisation of the respiratory tract and onset of bacteraemia during pneumococcal pneumonia. Respir Res 2005, 6:126. 23. Cho JH, Fraser IP, Fukase K, Kusumoto S, Fujimoto Y, Stahl GL, Ezekowitz RA: Human peptidoglycan recognition protein S is an effector of neutrophil-mediated innate immunity. Blood 2005, 106:2551-2558.

9.

10. Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A,  Henriques-Normark B: An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 2006, 16:401-407. These authors [6,10], simultaneously explain the mode of action of DNases as bacterial virulence factors by degradation of NETs. 11. Wartha F, Beiter K, Albiger B, Fernebro J, Zychlinsky A, Normark S, Henriques-Normark B: Capsule and D-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps. Cell Microbiol, in press. 12. Lippolis JD, Reinhardt TA, Goff JP, Horst RL: Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk. Vet Immunol Immunopathol 2006, 113:248-255. 13. Gupta AK, Hasler P, Holzgreve W, Gebhardt S, Hahn S: Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia. Hum Immunol 2005, 66:1146-1154. 14. Lee WL, Grinstein S: Immunology. The tangled webs that neutrophils weave. Science 2004, 303:1477-1478. 15. Sumby P, Barbian KD, Gardner DJ, Whitney AR, Welty DM, Long RD, Bailey JR, Parnell MJ, Hoe NP, Adams GG et al.: Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc Natl Acad Sci USA 2005, 102:1679-1684.

Current Opinion in Microbiology 2007, 10:5256

www.sciencedirect.com