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Novel role of DNA in neutrophil extracellular traps
TIMI-1198; No. of Pages 2
Spotlight
Novel role of DNA in neutrophil extracellular traps Christoph Georg Baums1 and Maren von Ko¨ckritz-Blickwede2 1
Institute for Bacteriology and Mycology, Centre for Infectious Diseases, College of Veterinary Medicine, University Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany 2 Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bu¨nteweg 17, 30559 Hannover, Germany
Neutrophil extracellular traps (NETs) have been shown to play a crucial role in health and disease. In a recent paper in PLoS Pathogens, Halverson et al. demonstrate that the DNA backbone of NETs contributes to its antibacterial activity and serves as signal for entrapped microbes to employ immune evasion strategies. In 2004, the formation of neutrophil extracellular traps (NETs) was discovered by Brinkmann et al. as a novel host innate immune defense mechanism against infections [1]. This discovery altered the fundamental conception of the innate immune defense mechanism of phagocytes against pathogenic microbes in a very fascinating way. Whereas it was known for a long time that neutrophils engulf pathogens and induce subsequent killing in specialized compartments called phagolysosomes, the discovery of NETs revealed an additional mechanism. The cells release an extracellular trap consisting of histones and DNA associated with antimicrobial peptides and proteases. Importantly, NETs have been shown to entrap (Figure 1) and kill several microbes as bacteria [1]. But how do NETs mediate antimicrobial activity against entrapped bacteria? In the first description on NETs, the authors described histones as a major antibacterial factor in NETs [1]. Later on, the antimicrobial peptide LL-37, a cationic alpha-helical cathelicidin [2], was widely discussed as killing agent in NETs. Histones as well as cationic antimicrobial peptides exhibit their antimicrobial activity based on electrostatic interactions with the bacterial membranes and subsequent pore-forming activity [1,2]. But importantly, when bound to DNA, the antimicrobial activity of those cationic peptides is reduced leading to less efficient killing of bacteria [3]. Thus, additional factors may mediate the antibacterial activity of NETs. Here, we want to highlight a publication by Halverson et al. [4]. The authors show that DNA acts as antimicrobial component of NETs and contributes to killing of Pseudomonas aeruginosa. First, using microscopy they demonstrate that NETs are formed upon in vivo infection of mice. In addition to the presence of NETs, the authors also identified chemotactic active as well as phagocytic neutrophils in infected tissue, suggesting that multiple clearing mechanisms are employed in vivo to fight against P. aeruginosa. However, whether the individual Corresponding author: von Ko¨ckritz-Blickwede, M. ([email protected]). Keywords: neutrophils; antimicrobial activity; DNA; Pseudomonas aeruginosa; Staphylococcus aureus. 0966-842X/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2015.04.003
NET-releasing neutrophil is dying [1] or remains viable and chemotactic active as previously shown [5] remains to be clarified. As a next step, the authors used three independent readout systems with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils to characterize the antimicrobial activity of NETs in vitro [4]: quantification of surviving colony forming units (cfu) in the presence or absence of NETs after nuclease digestion, flow cytometry analysis of propidium iodide (PI)-stained bacteria as marker for membrane integrity, and measurement of luminescence from chromosomally-tagged luminescent P. aeruginosa. As a new finding, bacterial survival in the presence of NETs or DNA was restored when NETs or DNA were pretreated with excess Mg2+ or alkaline phosphatase, which saturates the cation chelating ability of DNA or cleaves 50 phosphates of the DNA backbone, respectively. As these treatments did not deteriorate the NET architecture, the authors conclude that the ion chelating activity of DNA is crucial for the antimicrobial activity of NETs against P. aeruginosa. Comparing the susceptibilities of P. aeruginosa, Escherichia coli, and Staphylococcus aureus to histone and DNA killing, P. aeruginosa was detected as the most susceptible to DNA killing, while S. aureus was the most tolerant. Interestingly, these data differ from the species-dependent susceptibility to NETs, since S. aureus shows higher susceptibility to NET-mediated killing compared to P. aeruginosa [4]. Thus, it may be hypothesized that other NETassociated factors besides DNA contribute to antimicrobial activity of NETs against S. aureus. However, in general, the overall process of killing by NETs is controversial [6]. In good correlation to a previous study that used Live/Dead Bac-LightTM bacterial viability staining of entrapped S. aureus and microscopy [7], Halverson et al. [4] used flow cytometry of PI-stained bacteria to demonstrate that the membrane of P. aeruinosa entrapped in NETs is permeabilized. This results in a putative killing of the respective bacteria [4,7]. Using microbiological techniques that quantify surviving cfu in NETs, it has been additionally confirmed that surviving bacteria are reduced in the presence of NETs [1,4,7]. But per definition, we should avoid talking about a bactericidal activity of NETs, which is defined as 99.9% killing compared to initial inoculum [8], since this threshold is not met. Instead, the published phenotypes lead to the conclusion that NETs exhibit a bacteriostatic antimicrobial effect or a bacterial growth inhibition, eventually as a result of a partial killing of bacteria that are entrapped in NETs. Trends in Microbiology xx (2015) 1–2
1
TIMI-1198; No. of Pages 2
Spotlight
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
authors assumed that DNA was the most potent inducer of this bacterial host evasion response [4]. Overall, the work by Halverson et al. [4] highlights an intriguing novel antibacterial nature of DNA in NETs and reveals that DNA is also an inducer of bacterial NET evasion strategies. However, the in vivo relevance still remains to be determined, especially as there is increasing evidence that the formation of NETs does not only contribute to a protective effect for the host. Some bacteria have evolved efficient strategies to evade antimicrobial activity of NETs, and thereby contribute to chronic persistent infections [9]. Thus, it seems to depend on the pathogen and the place of infection if NETs have a protective or detrimental role for the host. Furthermore, since an excessive release of NETs has been associated with detrimental consequences for the host, e.g., autoimmune diseases [10], a fine balance between NET formation and NET degradation by the host itself seems to be essential for a final protective outcome of this host defense mechanism.
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TRENDS in Microbiology
Figure 1. Bacterial entrapment in neutrophil extracellular traps (NETs). Threedimensional confocal micrograph of rhodamine-labeled Staphylococcus aureus (strain Cowan, in red) entrapped by human neutrophil extracellular DNA, forming NETs as visualized by the blue DNA dye DAPI.
On top of the novel finding that DNA has antibacterial properties in NETs, Halverson et al. [4] show that P. aeruginosa responds to exposure to NETs by inducing expression of surface modifications to defend against DNA-induced membrane damage and killing. Following co-incubation with NETs produced by PMA-activated neutrophils, expression of the arn or spermidine synthesis genes in P. aeruginosa was strongly induced. Whereas the arn operon is required for the covalent addition of aminoarabinose to the phosphates of lipid A, the spermidine synthesis genes are required for production of the polycationic spermidine on the outer membrane surface. Thus, both factors stabilize the bacterial envelope and mediate resistance to antimicrobial peptides. Because the addition of excess Mg2+ and enzymatic treatment with DNAse or phosphatase blocked the function of those bacteria to produce factors in the presence of NETs, the
2
References 1 Brinkmann, V. et al. (2004) Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535 2 Neumann, A. et al. (2014) Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases. J. Innate Immun. 6, 860–868 3 Weiner, D.J. et al. (2003) The antimicrobial activity of the cathelicidin LL37 is inhibited by F-actin bundles and restored by gelsolin. Am. J. Respir. Cell Mol. Biol. 28, 738–745 4 Halverson, T.W. et al. (2015) DNA is an antimicrobial component of neutrophil extracellular traps. PLoS Pathog. 11, e1004593 5 Yipp, B.G. et al. (2012) Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med. 18, 1386–1393 6 Menegazzi, R. et al. (2012) Killing by neutrophil extracellular traps: fact or folklore? Blood 119, 1214–1216 7 Berends, E.T.M. et al. (2010) Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J. Innate Immun. 2, 576–586 8 Noviello, S. et al. (2003) Comparative activity of garenoxacin and other agents by susceptibility and time-kill testing against Staphylococcus aureus, Streptococcus pyogenes and respiratory pathogens. J. Antimicrob. Chemother. 52, 869–872 9 Short, K.R. et al. (2014) Antibodies mediate the formation of neutrophil extracellular traps (NETs) in the middle ear and facilitate secondary pneumococcal otitis media. Infect. Immun. 81, 364–370 10 Hakkim, A. et al. (2010) Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc. Natl. Acad. Sci. U.S.A. 107, 9813–9818
Spotlight
Novel role of DNA in neutrophil extracellular traps Christoph Georg Baums1 and Maren von Ko¨ckritz-Blickwede2 1
Institute for Bacteriology and Mycology, Centre for Infectious Diseases, College of Veterinary Medicine, University Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany 2 Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bu¨nteweg 17, 30559 Hannover, Germany
Neutrophil extracellular traps (NETs) have been shown to play a crucial role in health and disease. In a recent paper in PLoS Pathogens, Halverson et al. demonstrate that the DNA backbone of NETs contributes to its antibacterial activity and serves as signal for entrapped microbes to employ immune evasion strategies. In 2004, the formation of neutrophil extracellular traps (NETs) was discovered by Brinkmann et al. as a novel host innate immune defense mechanism against infections [1]. This discovery altered the fundamental conception of the innate immune defense mechanism of phagocytes against pathogenic microbes in a very fascinating way. Whereas it was known for a long time that neutrophils engulf pathogens and induce subsequent killing in specialized compartments called phagolysosomes, the discovery of NETs revealed an additional mechanism. The cells release an extracellular trap consisting of histones and DNA associated with antimicrobial peptides and proteases. Importantly, NETs have been shown to entrap (Figure 1) and kill several microbes as bacteria [1]. But how do NETs mediate antimicrobial activity against entrapped bacteria? In the first description on NETs, the authors described histones as a major antibacterial factor in NETs [1]. Later on, the antimicrobial peptide LL-37, a cationic alpha-helical cathelicidin [2], was widely discussed as killing agent in NETs. Histones as well as cationic antimicrobial peptides exhibit their antimicrobial activity based on electrostatic interactions with the bacterial membranes and subsequent pore-forming activity [1,2]. But importantly, when bound to DNA, the antimicrobial activity of those cationic peptides is reduced leading to less efficient killing of bacteria [3]. Thus, additional factors may mediate the antibacterial activity of NETs. Here, we want to highlight a publication by Halverson et al. [4]. The authors show that DNA acts as antimicrobial component of NETs and contributes to killing of Pseudomonas aeruginosa. First, using microscopy they demonstrate that NETs are formed upon in vivo infection of mice. In addition to the presence of NETs, the authors also identified chemotactic active as well as phagocytic neutrophils in infected tissue, suggesting that multiple clearing mechanisms are employed in vivo to fight against P. aeruginosa. However, whether the individual Corresponding author: von Ko¨ckritz-Blickwede, M. ([email protected]). Keywords: neutrophils; antimicrobial activity; DNA; Pseudomonas aeruginosa; Staphylococcus aureus. 0966-842X/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2015.04.003
NET-releasing neutrophil is dying [1] or remains viable and chemotactic active as previously shown [5] remains to be clarified. As a next step, the authors used three independent readout systems with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils to characterize the antimicrobial activity of NETs in vitro [4]: quantification of surviving colony forming units (cfu) in the presence or absence of NETs after nuclease digestion, flow cytometry analysis of propidium iodide (PI)-stained bacteria as marker for membrane integrity, and measurement of luminescence from chromosomally-tagged luminescent P. aeruginosa. As a new finding, bacterial survival in the presence of NETs or DNA was restored when NETs or DNA were pretreated with excess Mg2+ or alkaline phosphatase, which saturates the cation chelating ability of DNA or cleaves 50 phosphates of the DNA backbone, respectively. As these treatments did not deteriorate the NET architecture, the authors conclude that the ion chelating activity of DNA is crucial for the antimicrobial activity of NETs against P. aeruginosa. Comparing the susceptibilities of P. aeruginosa, Escherichia coli, and Staphylococcus aureus to histone and DNA killing, P. aeruginosa was detected as the most susceptible to DNA killing, while S. aureus was the most tolerant. Interestingly, these data differ from the species-dependent susceptibility to NETs, since S. aureus shows higher susceptibility to NET-mediated killing compared to P. aeruginosa [4]. Thus, it may be hypothesized that other NETassociated factors besides DNA contribute to antimicrobial activity of NETs against S. aureus. However, in general, the overall process of killing by NETs is controversial [6]. In good correlation to a previous study that used Live/Dead Bac-LightTM bacterial viability staining of entrapped S. aureus and microscopy [7], Halverson et al. [4] used flow cytometry of PI-stained bacteria to demonstrate that the membrane of P. aeruinosa entrapped in NETs is permeabilized. This results in a putative killing of the respective bacteria [4,7]. Using microbiological techniques that quantify surviving cfu in NETs, it has been additionally confirmed that surviving bacteria are reduced in the presence of NETs [1,4,7]. But per definition, we should avoid talking about a bactericidal activity of NETs, which is defined as 99.9% killing compared to initial inoculum [8], since this threshold is not met. Instead, the published phenotypes lead to the conclusion that NETs exhibit a bacteriostatic antimicrobial effect or a bacterial growth inhibition, eventually as a result of a partial killing of bacteria that are entrapped in NETs. Trends in Microbiology xx (2015) 1–2
1
TIMI-1198; No. of Pages 2
Spotlight
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
authors assumed that DNA was the most potent inducer of this bacterial host evasion response [4]. Overall, the work by Halverson et al. [4] highlights an intriguing novel antibacterial nature of DNA in NETs and reveals that DNA is also an inducer of bacterial NET evasion strategies. However, the in vivo relevance still remains to be determined, especially as there is increasing evidence that the formation of NETs does not only contribute to a protective effect for the host. Some bacteria have evolved efficient strategies to evade antimicrobial activity of NETs, and thereby contribute to chronic persistent infections [9]. Thus, it seems to depend on the pathogen and the place of infection if NETs have a protective or detrimental role for the host. Furthermore, since an excessive release of NETs has been associated with detrimental consequences for the host, e.g., autoimmune diseases [10], a fine balance between NET formation and NET degradation by the host itself seems to be essential for a final protective outcome of this host defense mechanism.
100.00 µm
Y
100.00 µm
X
TRENDS in Microbiology
Figure 1. Bacterial entrapment in neutrophil extracellular traps (NETs). Threedimensional confocal micrograph of rhodamine-labeled Staphylococcus aureus (strain Cowan, in red) entrapped by human neutrophil extracellular DNA, forming NETs as visualized by the blue DNA dye DAPI.
On top of the novel finding that DNA has antibacterial properties in NETs, Halverson et al. [4] show that P. aeruginosa responds to exposure to NETs by inducing expression of surface modifications to defend against DNA-induced membrane damage and killing. Following co-incubation with NETs produced by PMA-activated neutrophils, expression of the arn or spermidine synthesis genes in P. aeruginosa was strongly induced. Whereas the arn operon is required for the covalent addition of aminoarabinose to the phosphates of lipid A, the spermidine synthesis genes are required for production of the polycationic spermidine on the outer membrane surface. Thus, both factors stabilize the bacterial envelope and mediate resistance to antimicrobial peptides. Because the addition of excess Mg2+ and enzymatic treatment with DNAse or phosphatase blocked the function of those bacteria to produce factors in the presence of NETs, the
2
References 1 Brinkmann, V. et al. (2004) Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535 2 Neumann, A. et al. (2014) Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases. J. Innate Immun. 6, 860–868 3 Weiner, D.J. et al. (2003) The antimicrobial activity of the cathelicidin LL37 is inhibited by F-actin bundles and restored by gelsolin. Am. J. Respir. Cell Mol. Biol. 28, 738–745 4 Halverson, T.W. et al. (2015) DNA is an antimicrobial component of neutrophil extracellular traps. PLoS Pathog. 11, e1004593 5 Yipp, B.G. et al. (2012) Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med. 18, 1386–1393 6 Menegazzi, R. et al. (2012) Killing by neutrophil extracellular traps: fact or folklore? Blood 119, 1214–1216 7 Berends, E.T.M. et al. (2010) Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J. Innate Immun. 2, 576–586 8 Noviello, S. et al. (2003) Comparative activity of garenoxacin and other agents by susceptibility and time-kill testing against Staphylococcus aureus, Streptococcus pyogenes and respiratory pathogens. J. Antimicrob. Chemother. 52, 869–872 9 Short, K.R. et al. (2014) Antibodies mediate the formation of neutrophil extracellular traps (NETs) in the middle ear and facilitate secondary pneumococcal otitis media. Infect. Immun. 81, 364–370 10 Hakkim, A. et al. (2010) Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc. Natl. Acad. Sci. U.S.A. 107, 9813–9818