Toxoplasma gondii Triggers Release of Human and Mouse Neutrophil Extracellular Traps

Department of Microbiology and Immunology, Cornell University, Ithaca, New York, USA.
Infection and immunity (Impact Factor: 3.73). 11/2011; 80(2):768-77. DOI: 10.1128/IAI.05730-11
Source: PubMed


Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and
have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response
to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types
were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway
leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a
rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a
role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct
microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.

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    • "The novel paradigm of innate immunity is that, besides the classical functions of extracellular release of lytic enzymes and phagocytosis, PMN by means of their enzymatic activity , surface receptors and cytoskeleton (Neeli et al., 2009), also have the capacity to release their DNA in response to infectious stimuli (Brinkmann et al., 2004). Thus, at the site of infection PMNs form neutrophil extracellular traps (NETs) that have the ability to bind and kill pathogens outside the cell, such as bacteria (Fuchs et al., 2007; Pilsczek et al., 2010), protozoa (Guimarães-Costa et al., 2009; Wardini et al., 2010; Abdallah et al., 2012), or yeast (Urban et al., 2006, 2009). In addition, as reviewed by Brinkmann and Zychlinsky (2012), not only infectious agents, but also many physiological inducers of NET formation have been reported. "
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    ABSTRACT: Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1–3 h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4‘,6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p < 0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare.
    Journal of Reproductive Immunology 08/2014; 106. DOI:10.1016/j.jri.2014.08.003 · 2.82 Impact Factor
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    • "NETs also modulate protozoan infections, trapping and killing Leishmania amazonensis promastigotes [18] while the promastigotes of Leishmania donovani are captured but not killed [19]. The protozoan parasite Toxoplasma gondii induces human and mouse neutrophils to release NETs that have microbicidal effects [20]. Yet, a role for NETs during parasitic helminth infections has not been described. "
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    ABSTRACT: Neutrophils are multifaceted cells that are often the immune system’s first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.
    Microbes and Infection 06/2014; 16(6):502-511. DOI:10.1016/j.micinf.2014.02.012 · 2.86 Impact Factor
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    • "Overall, NET formation has been described as a novel form of cell death called ETosis which is distinct from apoptosis, autophagy and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase [17], [20]. Whilst most studies have focused on bacterial and fungal pathogens, few attention has been paid on effects of NETs on apicomplexan parasites [21], [22]. Thus, NET formation has been demonstrated for Plasmodium falciparum [23], E. bovis [24] and T. gondii [21], [22]. "
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    ABSTRACT: Besnoitia besnoiti infection in cattle is an important emerging protozoan disease in Europe causing economic losses and severe clinical signs, such as generalized dermatitis, orchitis, and vulvitis in affected animals. Neutrophil extracellular trap (NET) formation was recently demonstrated as an important effector mechanism of PMN acting against several invading pathogens. In the present study, interactions of bovine PMN with tachyzoites of B. besnoiti were investigated in this respect in vitro. For the demonstration and quantification of NETs, extracellular DNA was stained by Sytox Orange or Pico Green. Fluorescent illustrations as well as scanning electron microscopy analyses (SEM) showed PMN-promoted NET formation rapidly being induced upon contact with B. besnoiti tachyzoites. Co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of NET. Exposure of PMN to viable, UV attenuated and dead tachyzoites showed a significant induction of NET formation, but even tachyzoite homogenates significantly promoted NETs when compared to negative controls. NETs were abolished by DNase treatment and were reduced after PMN preincubation with NADPH oxidase-, NE- and MPO-inhibitors. Tachyzoite-triggered NET formation led to parasite entrapment as quantitative assays indicated that about one third of tachyzoites were immobilized in NETs. In consequence, tachyzoites were hampered from active invasion of host cells. Thus, transfer of tachyzoites, previously being confronted with PMN, to adequate host cells resulted in significantly reduced infection rates when compared to PMN-free infection controls. To our knowledge, we here report for the first time B. besnoiti-induced NET formation. Our results indicate that PMN-triggered extracellular traps may represent an important effector mechanism of the host early innate immune response against B. besnoiti which may lead to diminishment of initial parasite infection rates during the acute infection phase.
    PLoS ONE 03/2014; 9(3):e91415. DOI:10.1371/journal.pone.0091415 · 3.23 Impact Factor
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