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DNA is an antimicrobial component of neutrophil extracellular traps.

Halverson TW, Wilton M, Poon KK, Petri B, Lewenza S - PLoS Pathog. (2015)

Bottom Line: During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing.Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA.Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

View Article: PubMed Central - PubMed

Affiliation: University of Calgary, Snyder Institute for Chronic Diseases, Department of Microbiology, Immunology and Infectious Diseases, Calgary, Alberta, Canada.

ABSTRACT
Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

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Quantification of NETosis in human neutrophils ex vivo and mouse neutrophils during an infection.(A) Human neutrophils were stimulated with PMA, P. aeruginosa, E. coli and S. aureus and Sytox green fluorescence was measured as an indicator of DNA release by NETosis after 1 hr stimulation. Exogenous DNase was added as a control to confirm extracellular DNA presence in NETs, indicated by a plus sign (+). Asterisks denote a significant difference in extracellular DNA release between stimulated and unstimulated neutrophils (white bar) (**P<0.01, ***P<0.001). Each value shown is an average from 6 replicates with error bars representing the standard error. (B) The total number of NETs in uninfected mice, or infected mice with P. aeruginosa PAO1 or S. aureus and (C) the total NET area quantified. # denotes a significant difference in NET area and number in P. aeruginosa PAO1 infected mice compared to the uninfected control (#P<0.05). Asterisks denote a significant difference in NET area and number in mice infected with P. aeruginosa compared to S. aureus infected mice (***P<0.001).
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ppat.1004593.g002: Quantification of NETosis in human neutrophils ex vivo and mouse neutrophils during an infection.(A) Human neutrophils were stimulated with PMA, P. aeruginosa, E. coli and S. aureus and Sytox green fluorescence was measured as an indicator of DNA release by NETosis after 1 hr stimulation. Exogenous DNase was added as a control to confirm extracellular DNA presence in NETs, indicated by a plus sign (+). Asterisks denote a significant difference in extracellular DNA release between stimulated and unstimulated neutrophils (white bar) (**P<0.01, ***P<0.001). Each value shown is an average from 6 replicates with error bars representing the standard error. (B) The total number of NETs in uninfected mice, or infected mice with P. aeruginosa PAO1 or S. aureus and (C) the total NET area quantified. # denotes a significant difference in NET area and number in P. aeruginosa PAO1 infected mice compared to the uninfected control (#P<0.05). Asterisks denote a significant difference in NET area and number in mice infected with P. aeruginosa compared to S. aureus infected mice (***P<0.001).

Mentions: Given that P. aeruginosa induces the production of NETs in vitro and in vivo (Fig. 1), we sought to compare the relative abilities of P. aeruginosa, S. aureus, E. coli and the chemical inducer PMA to elicit NETosis. In the presence of purified human neutrophils, PMA, E. coli and S. aureus induced significantly more NET formation, relative to P. aeruginosa within 1 hour of coincubation (Fig. 2A). However, at later time points (3h), P. aeruginosa elicited the formation of similar amounts of NET structures. Furthermore, quantification of the number of NETs and NET-area using the skin infection model confirmed that P. aeruginosa weakly induces NETosis relative to S. aureus in vivo (Fig. 2B and C).


DNA is an antimicrobial component of neutrophil extracellular traps.

Halverson TW, Wilton M, Poon KK, Petri B, Lewenza S - PLoS Pathog. (2015)

Quantification of NETosis in human neutrophils ex vivo and mouse neutrophils during an infection.(A) Human neutrophils were stimulated with PMA, P. aeruginosa, E. coli and S. aureus and Sytox green fluorescence was measured as an indicator of DNA release by NETosis after 1 hr stimulation. Exogenous DNase was added as a control to confirm extracellular DNA presence in NETs, indicated by a plus sign (+). Asterisks denote a significant difference in extracellular DNA release between stimulated and unstimulated neutrophils (white bar) (**P<0.01, ***P<0.001). Each value shown is an average from 6 replicates with error bars representing the standard error. (B) The total number of NETs in uninfected mice, or infected mice with P. aeruginosa PAO1 or S. aureus and (C) the total NET area quantified. # denotes a significant difference in NET area and number in P. aeruginosa PAO1 infected mice compared to the uninfected control (#P<0.05). Asterisks denote a significant difference in NET area and number in mice infected with P. aeruginosa compared to S. aureus infected mice (***P<0.001).
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Related In: Results  -  Collection

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ppat.1004593.g002: Quantification of NETosis in human neutrophils ex vivo and mouse neutrophils during an infection.(A) Human neutrophils were stimulated with PMA, P. aeruginosa, E. coli and S. aureus and Sytox green fluorescence was measured as an indicator of DNA release by NETosis after 1 hr stimulation. Exogenous DNase was added as a control to confirm extracellular DNA presence in NETs, indicated by a plus sign (+). Asterisks denote a significant difference in extracellular DNA release between stimulated and unstimulated neutrophils (white bar) (**P<0.01, ***P<0.001). Each value shown is an average from 6 replicates with error bars representing the standard error. (B) The total number of NETs in uninfected mice, or infected mice with P. aeruginosa PAO1 or S. aureus and (C) the total NET area quantified. # denotes a significant difference in NET area and number in P. aeruginosa PAO1 infected mice compared to the uninfected control (#P<0.05). Asterisks denote a significant difference in NET area and number in mice infected with P. aeruginosa compared to S. aureus infected mice (***P<0.001).
Mentions: Given that P. aeruginosa induces the production of NETs in vitro and in vivo (Fig. 1), we sought to compare the relative abilities of P. aeruginosa, S. aureus, E. coli and the chemical inducer PMA to elicit NETosis. In the presence of purified human neutrophils, PMA, E. coli and S. aureus induced significantly more NET formation, relative to P. aeruginosa within 1 hour of coincubation (Fig. 2A). However, at later time points (3h), P. aeruginosa elicited the formation of similar amounts of NET structures. Furthermore, quantification of the number of NETs and NET-area using the skin infection model confirmed that P. aeruginosa weakly induces NETosis relative to S. aureus in vivo (Fig. 2B and C).

Bottom Line: During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing.Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA.Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

View Article: PubMed Central - PubMed

Affiliation: University of Calgary, Snyder Institute for Chronic Diseases, Department of Microbiology, Immunology and Infectious Diseases, Calgary, Alberta, Canada.

ABSTRACT
Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

Show MeSH
Related in: MedlinePlus