**5. Neutrophil extracellular traps in fungus infection**

#### **5.1.** *Aspergillus fumigatus* **and** *Aspergillus nidulans*

The *Aspergullus fumigatus* species is isolated in 80% of invasive aspergillosis patients. Chronic granulomatosis disease patients, whose cells are not able to undergo respiratory burst, are highly susceptible to infection by fungus of the *Aspergillus* genus such as *A. nidulans*. This indicates the important role of the host respiratory burst, which is also involved in the formation of NETs.

Recent reports highlight the importance of glucosaminoglycans (GAG) in *A. fumigatus* virulence. GAG helps the formation of biofilms and purified soluble GAG induces NK cell-mediated apoptosis of neutrophils, *in vitro*.

Fungus resistance to neutrophil-mediated killing positively correlates with the amount of cell wall-associated GAG. Fungus GAG content functions as the analog of bacterial capside, enhancing resistance to NETs. Although the mechanism by which exopolysaccharides mediate resistance to NETs has not been defined, it is suggested that GAG may inhibit hyphae-NETs binding, perhaps due to the repulsion between the *Aspergillus* exopolysaccharide positive charges and the positive charges present in the NETs antimicrobial peptides and histones [44, 45]. *Aspergillus* induces respiratory burst through its glycosaminoglycans that activate the NAPDH oxidase system, yielding ROS and activating classic NETosis activation [44].

#### **5.2.** *Candida albicans*

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

*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,

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

*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

oxidase-dependent mechanism [41].

34 Role of Neutrophils in Disease Pathogenesis

shows a reduction of about 42% [41].

NETosis [42].

of infection [43].

In 2006, Urban et al. showed that NETs can kill *Candida albicans* in any of its two forms, yeast, which is the proliferating form, or the filamentous, which is the invasive and tissue destructive form. This was corroborated by means of electronic microscopy which showed NETs and *C. albicans* hyphae co-localization, which suggested that hyphae are trapped by NETs, thus controlling the infection [46, 47].

Experiments aimed at analyzing the effect that PMA-activated NETs have on *C. albicans* showed that 20–30% of fungus dies after exposure to NETs [46].

The analysis of the components present in the neutrophil granules that may be responsible for the killing of *Candida albicans* showed that histones are not accountable for this. It was determined that human Neutrophil Granular Extract (hNGE) is responsible for the fungus death, in a dose-dependent manner. These granules contain Bactericidal/permeability-increasing (BPI) protein lactoferrin, and defensins. It appears that the release of NETs is related to the microorganism cell wall composition; the binding of microorganisms by NETs is mediated by ionic forces and thus, the fact that the *Candida* wall contains numerous proteins with phosphodiester bonds with negative charges makes it likely that they bind the positive charges of proteins and histones present in NETs [48].

Kenno et al. analyzed the induction of NETs by *Candida albicans*, and they corroborated that the distinctive forms of *Candida albicans*, hyphae or yeast, may induce NETs. These authors found that hyphae induce higher amounts of NETs than the yeast form, after 4 hours of incubation. *Candida albicans* hyphae stimulate cells through autophagy but not ROS, whereas the yeast form induces NETs through autophagy and ROS. *C. albicans β-glycans* induce NETosis by an ROS-independent mechanism [49, 50].

#### **5.3. Cryptococcus neoformans**

In 2015, Rocha et al. described that the opportunistic fungus *Cryptococcus neoformans*, which possesses a glucuronoxylomannan (GMX)-containing capside, precludes this fungus to be phagocytosed by neutrophils. These authors also demonstrated that the acapsular strain of *Cryptococcus neoformans*, which harbor glucuronoxylomannogalactan (GMXgal), is capable of inducing NETs. In contrast, the capsular strain does not induce the release of NETs [51].

The release of NETs by the acapsular strain of *Cryptococcus neoformans* is dependent on ROS generation and the PAD4 enzyme. The capsular strain also inhibits PMA-induced NETs formation [51]. NETs release has also been observed in response to *Cryptococcus gattii* stimulation.

Analysis of *Cryptococcus neoformans* susceptibility to acapsular strain-induced NETs showed that NETs diminished colony-forming units (CFUs) by 80% in the capsular strain and by 54% in the case of the acapsular strain. For this, it is necessary that NETs contain MPO.

*Paracoccidioides brasiliensis* and *Paracoccidioides lutzii* are fungi of the *Paracoccidioides* genus that cause high mortality and morbidity by the systemic mycosis Paracoccidioidomycosis (PCM), mostly in Latin American countries. Della Coletta et al. [52] have investigated the role of neutrophil extracellular traps on these fungi, reporting the formation of NETs by the yeasts *P. brasiliensis* and *P. luttzii*.
