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Neutrophil Extracellular Traps Kill Bacteria

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Science  05 Mar 2004:
Vol. 303, Issue 5663, pp. 1532-1535
DOI: 10.1126/science.1092385

Abstract

Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.

In response to inflammatory stimuli, neutrophils migrate from the circulating blood to infected tissues, where they efficiently bind, engulf, and inactivate bacteria. Phagocytosed bacteria are killed rapidly by proteolytic enzymes, antimicrobial proteins, and reactive oxygen species (1, 2). Neutrophils also degranulate, releasing antimicrobial factors into the extracellular medium (3). Here, we show that neutrophils generate extracellular fibers, or neutrophil extracellular traps (NETs), which are structures composed of granule and nuclear constituents that disarm and kill bacteria extracellularly.

NETs were made by activated neutrophils. Although naïve cells were round with some membrane folds (Fig. 1, A and C), neutrophils stimulated with interleukin-8 (IL-8), phorbol myristate acetate (PMA), or lipopolysaccharide (LPS) became flat and formed membrane protrusions (Fig. 1B) as previously described (4). Surprisingly, we found that activated neutrophils but not naïve cells made prominent extracellular structures (arrows, Fig. 1, B and D). These fibers, or NETs, were very fragile, and specimens had to be washed and fixed carefully to preserve them. High-resolution scanning electron microscopy (SEM) showed that the NETs contained smooth stretches with a diameter of 15 to 17 nm (Fig. 1E, arrowheads) and globular domains of around 25 nm (Fig. 1E, arrows) that aggregated into larger threads with diameters of up to 50 nm. Analysis of cross sections of the NETs by transmission electron microscopy (TEM) revealed they were not surrounded by membranes (Fig. 1F).

Fig. 1.

Electron microscopical analysis of resting and activated neutrophils. (A) Resting neutrophils are round and devoid of fibers. (B) Upon stimulation with 25 nM PMA for 30 min, the cells flatten, make many membrane protrusions, and form fibers (NETs), arrows in (B) and (D). (C) TEM analysis of naïve neutrophils in suspension. (D) Ultrathin section of neutrophils stimulated in suspension with 10 ng of IL-8 for 45 min. Bars in (A) to (D) indicate 10 μm. The multilobular nuclei and different granules are clearly visible in both figures. The activated cells in (D) have many pseudopods and show NETs (arrow). (E) High-resolution SEM analysis of NETs that consist of smooth fibers (diameters of 15 to 17 nm, arrowheads) and globular domains (diameter around 25 nm, arrow). Globular complexes can be aggregated to thick bundles or fibers. (F) Ultrathin sections of NETs show that they are not membrane-bound. Neutrophils were stimulated as in (D). Bars in (E) and (F), 500 μm.

The composition of NETs was analyzed by immunofluorescence. NETs contained proteins from azurophilic (primary) granules (5, 6) such as neutrophil elastase (Fig. 2A), cathepsin G, and myeloperoxidase (table S1). Proteins from specific (secondary) granules and tertiary granules, such as lactoferrin and gelatinase, respectively, were also present (table S1). In contrast, CD63, a granule membrane protein, the cytoplasmic markers annexin I (7), actin, tubulin, and various other cytoplasmic proteins were excluded from NETs (table S1).

Fig. 2.

Immunostaining of NETs. Neutrophils were activated with 10 ng of IL-8 for 30 min and stained for neutrophil elastase (A), DNA (B), and the complex formed by H2A-H2B-DNA (C). Extracellular fibrous material is stained brightly. As expected, we found granular staining for neutrophil elastase (A) and nuclear staining for histones and DNA [(B) and (C)]. Samples were analyzed with the use of a Leica TCS-SP (Beusheim, Germany) confocal microscope. The images are projections of a z stack (original dimensions: x and y, 85.5 μm; z = 6.3 μm). Bar, 10 μm. (D) Immunodetection of histones (large gold particles, arrows) and neutrophil elastase (small gold particles, arrowheads) in ultrathin cryosections of neutrophils stimulated with IL-8 (10 ng, 1 hour). Bar, 200 nm. (E) Immuno-SEM, pseudocolored, of neutrophils treated as in (A) to (C). Overlay of images from secondary electron detector (red, topography) and backscattered electron detector (green, element sensitive, most back-scattered electrons from the site of gold binding). Bright yellow dots (arrows) show localization of 12-nm gold particles detecting neutrophil elastase. Bar, 200 nm.

DNA is a major structural component of NETs, because several DNA intercalating dyes stained NETs strongly (Fig. 2B) and a brief treatment with deoxyribonuclease (DNase) resulted in the disintegration of NETs (movie S1). Conversely, protease treatment left the DNA of the NETs intact (8). The NETs reacted with antibodies against histones H1, H2A, H2B, H3, and H4 (table S1) and against the H2A-H2B-DNA complex (9, 10) (Fig. 2C).

Double immunostaining of ultrathin cryosections for TEM (Fig. 2D) confirmed the presence of neutrophil elastase (small gold particles, arrowheads) and H2A-H2B-DNA complexes (large gold particles, arrows) in NETs. Histone and neutrophil elastase staining was found on globular NET domains. Furthermore, immunostaining of SEM samples (Fig. 2E) corroborated the localization of neutrophil elastase to the globular domains of NETs. These data demonstrate that the structures visualized by different microscopy approaches (immunofluorescence, TEM, and SEM) are identical. NET formation was quantified in a fluorometer with the use of a DNA dye that is excluded from cells. Neutrophils release NETs as early as 10 min after activation, and the release depends on the dose of the activator (fig. S1).

Several lines of evidence indicate that neutrophils make NETs actively: (i) Stimuli that induce NETs do not promote the release of the cytoplasmic marker lactate dehydrogenase (LDH), and activated cells exclude vital dyes for at least two hours after stimulation (8). (ii) Stimuli such as IL-8 and LPS, which prolong the life of neutrophils (11), can induce NETs efficiently. (iii) Incubation with DNA intercalating dyes before neutrophil activation prevents NET formation but has no effect on the induction of apoptosis by staurosporine or tumor necrosis factor α (8). (iv) NETs are formed as early as 10 min after activation, a time course faster than apoptosis (fig. S1). (v) Time-lapse video microscopy (movie S2) shows that motile cells make NETs. Taken together, these data strongly indicate that NETs are not the result of leakage during cellular disintegration. We cannot exclude, however, the possibility that NET formation is an early event in the neutrophil program for cell death. Neutrophils are terminally differentiated cells that are programmed to die a few hours after they enter into circulation. Furthermore, isolated neutrophils are a heterogeneous population with respect to age, and a small portion of this “aged” subpopulation is expected to die. Neutrophils can undergo caspase-dependent (12) and -independent apoptosis in vitro (13), but the process that leads to neutrophil death in vivo is not known. It is conceivable that NET formation is an early event in cell death.

NETs associate with both Gram-positive (Staphylococcus aureus, shown in Fig. 3A) and Gram-negative pathogens (Salmonella typhimurium and Shigella flexneri, shown in Fig. 3, B and C, respectively). We have previously shown that neutrophil elastase degrades virulence factors of Gram-negative bacteria (14). Our finding that bacteria are trapped in NETs decorated with neutrophil elastase prompted us to test whether bacterial virulence factors were targeted extracellularly. Immunofluorescence staining of IpaB, a virulence factor of S. flexneri, was weaker in bacteria trapped in NETs compared to free Shigella (Fig. 3D, top left), although the bacteria and the NETs were clearly visible when DNA was stained. In contrast, when neutrophil protease activity was blocked by the secretory leukocyte proteinase inhibitor (SLPI), bacteria trapped in NETs contained high amounts of IpaB (Fig. 3D, bottom left). Interestingly, virulence factors from Gram-positive bacteria were also susceptible to neutrophil proteases. Lower amounts of the S. aureus virulence factor α toxin were found in NET-associated bacteria compared to that of free bacteria or when neutrophil proteases were blocked with SLPI (fig. S2). These results suggest that NETs can disarm a wide range of pathogens.

Fig. 3.

Gram-positive and Gram-negative bacteria associate with neutrophil fibers. SEM of S. aureus (A), S. typhimurium (B), and S. flexneri (C) trapped by NETs. Neutrophils were treated with 100 ng of IL-8 for 40 min before infection. Bar, 500 nm. (D) Immunofluorescence of neutrophils infected with S. flexneri stained for the virulence factor IpaB and DNA. IpaB is degraded by neutrophil elastase and is only detectable on the bacteria (arrows) when neutrophil elastase is blocked with SLPI. DNA staining shows NETs and bacteria (arrows). (E) Western blot showing that the virulence factors IcsA and IpaB but not OmpA were degraded by cytochalasin D–treated neutrophils incubated with S. flexneri. Lane 1, bacteria alone. Lane 2, bacteria incubated with cytochalasin D–treated neutrophils. (F) Extracellular bactericidal activity was greatly reduced in both S. flexneri and S. aureus infections after incubation with DNase, which dissociates NETs. (G) Extracellular bacterial killing by neutrophils was reduced by addition of antibodies against histones. Neutrophils were treated with cytochalasin D to prevent phagocytosis and infected with S. flexneri or S. aureus. In the presence of antibody against H2A, bacterial killing was abrogated.

We corroborated that extracellular proteases degrade bacterial virulence factors by inhibiting neutrophil phagocytosis. This was accomplished by incubating activated neutrophils with cytochalasin D. In the presence of cytochalasin D, an inhibitor of actin polymerization, NETs persisted and phagocytosis was blocked. We infected these neutrophils that have NETs but cannot phagocytose with S. flexneri. Extracellular neutrophil elastase, like purified elastase (14), degraded the virulence factors IcsA and IpaB but not the control OmpA, an outer membrane protein (Fig. 3E). This confirms that neutrophil elastase presented in NETs actively targets bacterial virulence factors.

Activated neutrophils incubated with cytochalasin D after formation of the NETs can kill about 30% of a S. flexneri or S. aureus inoculum (Fig. 3F, without DNase). We propose the hypothesis that the NET structure is necessary for this extracellular bactericidal activity. Indeed, when NETs were dismantled with DNase (movie S1), the killing of bacteria was negligible (Fig. 3F). In these experiments, the cultures were not washed after treatment with protease-free DNase, leaving the total protein concentration unchanged. Hence, these data strongly suggest that the fibrous structure of NETs is necessary for the sequestration and killing of bacteria by delivering a high local concentration of antimicrobial molecules to the bound microbes.

In an alternative approach to demonstrate the antibacterial activity of NETs, we showed that a monoclonal antibody against the H2A-H2B-DNA complex abrogated S. flexneri and S. aureus killing in infections of neutrophils pretreated with cytochalasin D after NET formation (Fig. 3G). An isotype control antibody had no effect on killing. The factors responsible for bacterial killing are likely to include granule proteins like bactericidal permeability increasing protein (BPI) (table S1) and histones. The antimicrobial activity of histones (15), evolutionarily conserved proteins that bind DNA to form the nucleosome complex, and peptides derived from histones, is well established (16, 17) Indeed, purified H2A killed S. flexneri, S. typhimurium, and S. aureus cultures with concentrations as low as 2 μg/ml (140 nM) in 30 min (fig. S3). The concentration of H2A required to kill bacteria is low compared with other antimicrobial proteins (18).

To determine whether NETs are present in vivo, we analyzed samples from experimental shigellosis in rabbits and spontaneous appendicitis in humans. Staining of histological sections clearly showed extracellular fibrous material that contains NET components: histones (Fig. 4, A and F), DNA (Fig. 4, C and G), and neutrophil elastase (Fig. 4E). In vivo, NETs trap bacteria as shown by the localization of Shigella (Fig. 4B) to the NETs. These results indicate that NETs are abundant at inflammatory sites.

Fig. 4.

Analysis of tissue sections from experimental shigellosis in rabbits (A to D) and spontaneous human appendicitis (E to H). (A) Immunofluorescence staining of histones reveals nuclear and extracellular localization that largely overlaps with staining for DNA (C). (B) Staining with an antibody against Shigella-specific LPS. (D) The overlay indicates that numerous Shigellae are closely associated to fibrous material staining for histones and DNA. (E) Staining for neutrophil elastase in an area of neutrophil exudate in human spontaneous appendicitis reveals fibrous extracellular material that also stains for histone (F) and DNA (G). (H) Overlay of the images. The images are projections of confocal z stacks generated from sections of 5 to 6 μm thickness. Bar, 50 μm.

Neutrophils make NETs through an active mechanism that remains to be understood. NETs disarm pathogens with proteases such as neutrophil elastase. NETs also kill bacteria efficiently, and at least one of the NET components, histones, exerts antimicrobial activity at surprisingly low concentrations. These data correlate with previous findings showing that neutrophil degranulation releases antimicrobial factors extracellularly (3) and the observation that inflammatory exudates rich in neutrophils, like pus, contain DNA, which was not known to play an active role in antimicrobial defense. Also, these data are in accord with recent findings proposing that oxygen-independent mechanisms play an important role in the control of infections (19). The data presented here indicate that granule proteins and chromatin together form an extracellular structure that amplifies the effectiveness of its antimicrobial substances by ensuring a high local concentration. NETs degrade virulence factors and/or kill bacteria even before the microorganisms are engulfed by neutrophils. In addition to their antimicrobial properties, NETs may serve as a physical barrier that prevents further spread of bacteria. Moreover, sequestering the granule proteins into NETs may keep potentially noxious proteins like proteases from diffusing away and inducing damage in tissue adjacent to the site of inflammation (20). NETs might also have a deleterious effect on the host, because the exposure of extracellular histone complexes could play a role during the development of autoimmune diseases like lupus erythematosus.

Supporting Online Material

www.sciencemag.org/cgi/content/full/303/5663/1532/DC1

Materials and Methods

Figs. S1 to S3

Table S1

Movies S1 and S2

References and Notes

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