NET formation can occur independently of RIPK3 and MLKL signaling

Amini, Poorya; Stojkov, Darko; Wang, Xiaoliang; Wicki, Simone; Kaufmann, Thomas; Wong, Wendy Wei-Lynn; Simon, Hans‐Uwe; Yousefi, Shída

doi:10.1002/eji.201545615

Cited by 86 publications

(107 citation statements)

References 21 publications

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“…However, two recent studies on the role of necroptosis in NETs induction present contrasting evidence for and against the requirements of necroptosis (Amini et al, 2016; Desai et al, 2016). Our data strengthens the argument that necroptosis is a separate cell death signalling cascade that is not required by neutrophils to induce NETosis.…”

Section: Discussionmentioning

confidence: 98%

Diverse stimuli engage different neutrophil extracellular trap pathways

Kenny

Herzig

Krüger

et al. 2017

eLife

592

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Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence.DOI: http://dx.doi.org/10.7554/eLife.24437.001

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Section: Discussionmentioning

confidence: 98%

Diverse stimuli engage different neutrophil extracellular trap pathways

Kenny

Herzig

Krüger

et al. 2017

eLife

592

728

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“…Extracellular DNA was visualized by cell permeable fluorescent dyes, such as MitoSox Red or Syto 13 prior to fixation. SP‐D binding to the cell surface was analyzed by indirect immunofluorescence as previously described . Briefly, eosinophils pre‐incubated with either SP‐D or recombinant S‐nitrosothiol (SNO)‐SP‐D (5 μg/mL, both His‐Tagged), were seeded on glass coverslip, fixed with 4% paraformaldehyde and labeled by monoclonal anti‐His Ab (1:100; clone HIS.H8, ThermoFisher Scientific) overnight (4°C), and then with Alexa Fluor® 488 conjugated goat anti‐mouse secondary Ab (1:400; ThermoFisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

Yousefi

Sharma

Stojkov

et al. 2018

Journal of Leukocyte Biology

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The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O ), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein-D (SP-D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP-D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate-dependent manner. Combined allergic airway sensitization and O exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S-nitrosylation related de-oligomerisation of SP-D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP-D. Importantly, S-nitrosylation abolished the ability of SP-D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP-D and eosinophils is destroyed by the NO-rich oxidative lung tissue environment in asthma exacerbations.

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“…Accordingly, the problem of unspecific inhibition of various cell signaling pathways has been repeatedly brought up . Moreover, we and others have previously observed that slight modifications to experimental settings or conditions may lead to dissonant results . Therefore, the complexity of NETs formation mechanisms could be more easily studied if examined cells were amenable to genetic manipulations.…”

Section: Introductionmentioning

confidence: 99%

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

et al. 2018

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Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

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NET formation can occur independently of RIPK3 and MLKL signaling

Cited by 86 publications

References 21 publications

Diverse stimuli engage different neutrophil extracellular trap pathways

Diverse stimuli engage different neutrophil extracellular trap pathways

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

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