Novel cell death program leads to neutrophil extracellular traps

Fuchs, Tobias A.; Abed, Ulrike Abu; Goosmann, Christian; Hurwitz, Robert; Schulze, Ilka; Wahn, V.; Weinrauch, Yvette; Brinkmann, Volker; Zychlinsky, Arturo

doi:10.1083/jcb.200606027

Cited by 2,685 publications

(2,820 citation statements)

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“…It has been shown that PMA‐induced NETosis is a ROS‐dependent process and is inhibited by the NADPH oxidase inhibitor, DPI 3. After DPI treatment (10 µM, 30 min), SYTOX Green‐positive cells were significantly reduced in comparison with the samples without DPI treatment (Fig.…”

Section: Resultsmentioning

confidence: 85%

“…NETosis is thought to be distinct from apoptosis or necrosis 3. To determine that SYTOX Green does not recognize apoptotic cells, apoptosis was induced on neutrophils by etoposide.…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, DPI, the NADPH oxidase inhibitor, namely a NET inhibitor 3, can partially but significantly reduce the number of SYTOX Green‐positive cells induced by PMA. This important finding suggests that the SYTOX Green‐positive cells surely include, but not all of them, neutrophils that formed NETs.…”

Section: Discussionmentioning

confidence: 99%

“…Neutrophils that are stimulated by PMA undergo NET formation and subsequent cell death. The death of PMA‐treated neutrophils followed by NET formation is different from typical necrosis or apoptosis 3.…”

Section: Introductionmentioning

confidence: 87%

“…To determine that the SYTOX Green‐positive cells include neutrophils that formed NETs, we present the following findings: 1 Co‐localization of MPO and plasma membrane‐appendant DNA of the SYTOX Green‐positive cells, 2 Inhibition by a NET inhibitor, diphenylene iodonium (DPI), and 3 No staining of SYTOX Green on neutrophils with etoposide‐induced early apoptosis. In addition, we further demonstrate the correlation of the flow cytometry‐based method with the established approach of image analysis of NETs and the applicability of the method for detection of NETs formed in vivo using a rat model of peritonitis.…”

Section: Introductionmentioning

confidence: 99%

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Measurement of NET formation in vitro and in vivo by flow cytometry

et al. 2017

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Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane‐impermeable DNA‐binding dye, SYTOX Green. SYTOX Green‐positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12‐myristate 13‐acetate (PMA). The number of SYTOX Green‐positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co‐localization of MPO and plasma membrane‐appendant DNA of SYTOX Green‐positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green‐positive cells induced by PMA. The collective evidence suggests that SYTOX Green‐positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well‐used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

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Section: Resultsmentioning

confidence: 85%

“…NETosis is thought to be distinct from apoptosis or necrosis 3. To determine that SYTOX Green does not recognize apoptotic cells, apoptosis was induced on neutrophils by etoposide.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 87%

Section: Introductionmentioning

confidence: 99%

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Measurement of NET formation in vitro and in vivo by flow cytometry

et al. 2017

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Spontaneous formation of neutrophil extracellular traps in serum‐free culture conditions

et al. 2017

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Neutrophils play a critical role in the innate immune response. Recently, a new neutrophilic biological defense mechanism, termed neutrophil extracellular traps ( NET s), has been attracting attention. Neutrophils have been observed to release both lysosomal enzymes and their nuclear contents, including unfolded chromatin, which together trap and inactivate bacteria. The environment in tissues where neutrophils act is thought to be different from that of the blood serum. In this study, we assessed the effect of serum on NET formation. We found that neutrophils spontaneously form NET s in serum‐free cultivation conditions at early times. These NET s functioned properly to trap bacteria. Furthermore, we demonstrated that reactive oxygen species play a critical role in the spontaneous formation of NET s. These results suggest that the serum condition must be considered in studies on neutrophils, including the formation and mechanism of action of NET s.

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Transcriptional dynamics of granulocytes in direct response to incubation with SARS‐CoV‐2

et al. 2022

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Severe coronavirus disease 2019 (COVID‐19) is characterized by acute respiratory distress syndrome and multiple organ dysfunction, in which the host immune response plays a pivotal role. Excessive neutrophil activation and subsequent superfluity of neutrophil extracellular traps (NETs) can lead to tissue damage, and several studies have shown the involvement of neutrophils in severe COVID‐19. However, the detailed responses of each neutrophil subset to SARS‐CoV‐2 infection has not been fully described. To explore this issue, we incubated normal‐density granulocytes (NDGs) and low‐density granulocytes (LDGs) with different viral titers of SARS‐CoV‐2. NDGs form NETs with chromatin fibers in response to SARS‐CoV‐2, whereas LDGs incubated with SARS‐CoV‐2 display a distinct morphology with condensed nuclei and moderate transcriptional changes. Based on these transcriptional changes, we suggest that AGO2 possibly plays a role in LDG regulation in response to SARS‐CoV‐2.

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Novel cell death program leads to neutrophil extracellular traps

Cited by 2,685 publications

References 38 publications

Measurement of NET formation in vitro and in vivo by flow cytometry

Measurement of NET formation in vitro and in vivo by flow cytometry

Spontaneous formation of neutrophil extracellular traps in serum‐free culture conditions

Transcriptional dynamics of granulocytes in direct response to incubation with SARS‐CoV‐2

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