**4. Neutrophil extracellular traps in parasitic infections**

*Salmonella typhimurium*, a Gram-negative bacterium, induces the release of NETs, and some of their components, such as histones (H2), have bactericidal activity, whereas others, such as

*Shigella flexneri*, a Gram-negative bacterium, induces the release of NETs. *S. flexneri* is trapped by NETs and killed via the neutrophil elastase; virulence factors such as IcsA and IpaB are

*Staphylococcus aureus* is some Gram-positive bacteria that cause sepsis. The role of NETs in controlling a *S. aureus* infection could be through the antimicrobial proteins associated to these, the bactericidal effect of H2 histones, the antimicrobial action of the cathelicidin LL-37, and neutrophil proteases that decrease the secretion of the alpha-toxin (α-toxin). The virulence factors LukGH and PVL help to induce the release of NETs. The *S. aureus*-induced

*Staphylococcus epidermidis* belongs to the group of coagulase-negative straphylococci. It is a quite common colonizer of healthy mice and human skin. It is a part of "normal" skin flora and plays a beneficial role in cutaneous niche. However, in immunocompromised patients, there is a high risk of developing infection mainly due to catheters use in hospitals. The exoprotein of *S. epidermidis*, the delta-toxin, PMSs (Phenol-Soluble Moduline-gamma) cooperates with host antimicrobial peptides to help kill pathogens of the group A of Streptococcus (GAS). In 2010, Cogen et al. [31] reported that the exoprotein phenol-soluble-moduline -gamma (PSMs) (δ-toxin) can induce NETs formation. The authors demonstrated a direct binding of δ-toxin to

*Streptococcus* spp. are Gram-positive bacteria that include non-pathogenic commensal strains and highly virulent pathogenic strains. The pathogenic strains express virulent factors that allow them to evade the immune system. *Streptococcus pneumoniae* infection leads to pneumonia and invasive diseases such as meningitis and bacteremia, whereas *Streptococcus pyogenes* is the major causative agent of Severe Group A Streptococcal Infections. *S. pneumoniae* and *S. pyogenes* induce the formation of NETs. However, these bacteria have evolved mechanisms that allow them to modulate the formation of NETs. Neutrophils, on the other hand, have evolved a NETs release mechanism in response to *Streptococcus*-derived virulence factors. The *S. pyogenes* virulent factor M1 decreases the induction of NETs while conferring bacterial resistance to be killed by NETs. The *S. pyogenes*-derived M1 exotoxin induces the formation of NETs, by associating with fibrinogen and forming a complex that stimulates neutrophils.

In summary, this review shows that in response to bacterial stimuli, neutrophils get activated and form NETs that may trap and kill invading bacteria. Besides the "classical" way of clearing pathogens by phagocytosis and intracellular exposure to bactericidal compounds, this novel mechanism of neutrophil extracellular killing plays an important role in primary host defense. Moreover, knowledge on the mechanisms of bacterial adaptation to evade the immune system could be used in the medical practice. For instance, DNases inhibitors can be used as potential therapeutics, to prevent degradation of NETs by Group A Streptococcus DNases. In the future, therapeutics aimed at the maintenance of NETs could be used to help clear bacterial infections.

elastase, can degrade virulence factors, as in the case of the alpha toxin [7, 29].

release of NETs is an NADPH oxidase-independent process [30].

Formation of NETs contributes to the pathogen elimination [32].

degraded by the neutrophil elastase [7].

32 Role of Neutrophils in Disease Pathogenesis

LL-37, CRAMP, hBD2, hBD3, as well as DNA.

Neutrophil extracellular traps have been broadly studied in regard to bacteria. The role of NETs against protozoa, however, has just recently been analyzed. Protozoa can induce NETs in neutrophils and macrophages, and knowledge on the mechanisms at play is just emerging.

In 2011, Abdi Abdallah [33] reported that human neutrophils produce NETs in response to stimulation with *Plasmodium falciparum* trophozoites, *Leishmania braziliensis*, and *Toxoplasma gondii* tachyzoites. *In vitro* experiments have demonstrated the presence of NETs upon bovine neutrophils stimulation with *Eimeria bovis* sporozoites, in human neutrophils after stimulation with promastigotes of *Leishmania donovani, Leishmania major, Leishmania chagasi, or L. amazonensis* amastigotes. A brief description of the mechanism involved in protozoa-induced NETs formation is next described.

*Toxoplasma gondii* is an obligated intracellular parasite that causes toxoplasmosis in immunocompromised individuals. In immunocompetent individuals, however, the immune system usually keeps the parasite from causing illness. *Toxoplasma gondii* tachyzoites induce the release of NETs by activating the MEK-ERK signaling pathway. NETs can trap *Toxoplasma gondii* tachyzoites, eliminating about 25% of them as parasite trapping avoids their dissemination [34].

*Plasmodium falciparum*, an intracellular parasite, causes malaria. It is estimated that this parasite infects between 215 and 659 million humans per year, worldwide. Malaria is transmitted to humans by the bite of Anopheles mosquitoes. *P. falciparum* sporozoites develop into merozoites and enter into erythrocytes. Studies conducted in Nigerian children infected with *P. falciparum* showed NETs structures with trapped trophozoites, and in their blood, infected and non-infected erythrocytes were also observed [35–37].

*Eimeria bovis*. This parasite is the causative agent of enteritis in cattle, and NETs formed are released upon stimulation with *E. bovis* sporozoites. This parasite stage of *E. bovis* seems to be a better inducer of NETs than PMA. NETs have been shown to diminish infection by parasite immobilization and also by parasite killing, although to a lesser extent [38, 39].

Leishmania spp. These protozoal parasites are the causative agents of leishmaniosis, and the leishmaniosis model has been quite useful in studies on the role of NETs at the early stages of the disease. The promastigote has been identified as the main parasite stage as inducer of NETs. Promastigotes and amastigotes numbers diminish upon NETs release. Histones H2A and H2B are the main inducers of NETs, and these are highly toxic for the parasite. The promastigote form of the parasite can evade the NETs by means of its 3′ nucleotidase, enzyme that degrades the DNA, allowing *Leishmania* spp. to escape from being killed by NETs [40].

In 2015, Rochael et al. analyzed the role of reactive oxygen species, neutrophil elastase, myeloperoxidase, and the PAD4 enzyme in the formation of NETs by *L. amazonensis* promastigotes, in human cells. These authors observed that *Leishmania* promastigotes promote a redox disbalance in neutrophils. The exposure of neutrophils to H<sup>2</sup> O2 induces histone deamination mediated by PAD4, and the redox disbalance takes place independently of the parasite viability, thus suggesting that *Leishmani*a induces the production of ROS through an NADPH oxidase-dependent mechanism [41].

*Leishmania* as well as *Staphylococcus aureus* induces the release of NETs by an early and rapid mechanism, through an ROS-independent pathway, which is inhibited by an elastase inhibitor and, in contrast to classic NETosis, is not affected by chloramidina. PAD4 activity is only relevant during classic NETosis. Promastigotes viability after treatment of parasites with a NETs-rich supernatant, obtained from either the early and rapid or the classic pathways, shows a reduction of about 42% [41].

As previously described, the interaction of *Leishmania amazonensis* with human neutrophils leads to the release of NETs, which trap and kill the parasite. However, the signaling pathways leading to *Leishmania*-induced NETosis are still under study. However, it has been shown that PI3K, independently of protein kinase B, has a role in parasite-induced NETosis. The main PI3K isoforms involved are PI3Kγ and PI3Kδ. Activation of ERK downstream of PI3Kγ is necessary to trigger an ROS-dependent parasite-induced NETosis. Pharmacological inhibition of protein kinase C also significantly decreases parasite-induced NETs release. Intracellular calcium, regulated by PI3Kδ, represents an alternative ROS-independent pathway of NETosis stimulation by *L. amazonensis*. Finally, intracellular calcium mobilization and reactive oxygen species generation are the major regulators of parasite-induced NETosis. These results contribute to a better understanding of the signaling behind *Leishmania*-induced NETosis [42].

*Entamoeba histolytica*. This protozoan parasite causes amebiasis, amoebic colitis, and hepatic abscess. Since this parasite is too large to be phagocytosed, Avila et al. [43] analyzed the possibility that this parasite induces the formation of NETs. These authors demonstrated that the amoeba lipopeptidophosphoglycan induces NETs in a dose-dependent manner. NETs can be readily observed 15 min after stimulation; however, by 1 h at a 1:20 infection ratio, NETs occupy a whole microscopic field. NETs induction depends on trophozoite integrity; 30 min after contact with NETs, trophozoites show no changes in size or morphology, and this contact does not have any effect on viability or growth at any time of incubation. On the other hand, it was observed that *E. histolytica* is resistant to cathelicidin LL-37. Resistance to NETs exposure was also studied upon addition of a proteases inhibitor, resulting in that proteases are not responsible for trophozoite resistance to NETs. However, the use of ethylene glycolbis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a divalent anion chelant, had a deleterious effect in the growth of amoebas that were in contact with NETs, suggesting that trophozoites may have DNAse activity, responsible for its resistance to NETs [43].

Ávila et al. demonstrated that parasite growth could only take place in the absence of a calcium chelant, since enzymes such as trophozoite DNAsas require calcium. This provides an example of NETs inhibition by parasite-produced enzymes. *Entamoeba histolytica* is one of the main parasites that cause stomach diseases worldwide. It causes intestine and liver invasion, associated with the recruitment of large amounts of neutrophils at the early stages of infection [43].
