**3. Neutrophil extracellular traps in bacterial infections**

Several mechanisms have been proposed to explain how NETs control bacterial infection. NETs bind to both Gram-negative and Gram-positive bacteria, precluding bacterial mobilization and dissemination, and some bacteria are killed extracellularly by NETs, due to their high content of serine proteases [17]. Some bacteria and their interaction with NETs are summarized as follows:

*Bordetella pertussis*, the causative agent of pertussis or whooping cough, is a Gram-negative aerobic bacterium that infects the respiratory tract and inhibits the host´s immune system by mean of its virulent factors, such as pertussis toxin, filamentous hemagglutinin, pertactin, fimbria, and tracheal cytotoxin. The pertussis toxin inhibits G protein coupling that regulates the adenylate cyclase-mediating conversion of ATP to cAMP. This event induces macrophages and neutrophils to convert the ATP to cAMP by intracellular eukaryotic calmodulin, causing disturbances in cellular signaling mechanisms and thus preventing phagocytosis and an efficient control of the pathogen. The formation of NETs induced by *B. pertussis* is NADPH oxidase dependent [18].

*Escherichia coli*, the causative bacteria of several pathologies, including bacterial sepsis, is a Gram-negative bacterium. NETs formation helps to control infection by trapping and killing the bacteria and avoiding dissemination to other organs. The proposed mechanisms for the formation of NETs depend on the bacteria strain and its pathogenesis. In the case of *E. coli*

involved in liver sepsis, the infection can be controlled by histones H2B or by activating the intravascular NETs release through the integrin lymphocyte function-associated antigen 1 (LFA-1) [19, 20].

release ETs. Although the molecular principles underlying the formation of ETs by mast cells [11], eosinophils [12], and monocytes/macrophages [13] are similar to those observed in neutrophils, there are some notable disparities. The most remarkable mechanism of ET formation has been described in eosinophils. In these cells, ETs are formed by both nuclear and mito-

Neutrophil extracellular traps are able to capture microorganisms trap microorganisms, killing them or not, this much depends on the type of pathogen involved. NETs are produced by the neutrophils of mice, humans, and some other animals, and can be induced by chemical compounds, bacteria, fungi, protozoa, and viruses. The role of NETs in viral infections is not

While some viruses are immobilized and inactivated by NETs, others such as HIV induce the production of an IL-10-like protein that inhibits the formation of NETs [15], and dengue virus inhibits PMA-induced formation of NETs. Interestingly, neutrophils seem to be arrested at the chromatin decondensation step, failing to liberate NETs, thus suggesting a metabolic-

Controversy surrounding neutrophil extracellular traps as a host defense mechanism makes it necessary to analyze how NETs limit the growth of various infectious agents, whereas, apparently, they have no effect on others. On the other hand, how NETs may cause damage

Several mechanisms have been proposed to explain how NETs control bacterial infection. NETs bind to both Gram-negative and Gram-positive bacteria, precluding bacterial mobilization and dissemination, and some bacteria are killed extracellularly by NETs, due to their high content of serine proteases [17]. Some bacteria and their interaction with NETs are

*Bordetella pertussis*, the causative agent of pertussis or whooping cough, is a Gram-negative aerobic bacterium that infects the respiratory tract and inhibits the host´s immune system by mean of its virulent factors, such as pertussis toxin, filamentous hemagglutinin, pertactin, fimbria, and tracheal cytotoxin. The pertussis toxin inhibits G protein coupling that regulates the adenylate cyclase-mediating conversion of ATP to cAMP. This event induces macrophages and neutrophils to convert the ATP to cAMP by intracellular eukaryotic calmodulin, causing disturbances in cellular signaling mechanisms and thus preventing phagocytosis and an efficient control of the pathogen. The formation of NETs induced by *B. pertussis* is NADPH

*Escherichia coli*, the causative bacteria of several pathologies, including bacterial sepsis, is a Gram-negative bacterium. NETs formation helps to control infection by trapping and killing the bacteria and avoiding dissemination to other organs. The proposed mechanisms for the formation of NETs depend on the bacteria strain and its pathogenesis. In the case of *E. coli*

chondrial DNAs, in a reactive oxygen species (ROS)-dependent manner.

yet clear. However, some viruses induce the release of NETs [14, 15].

related mechanism of NETs inhibition [16].

30 Role of Neutrophils in Disease Pathogenesis

summarized as follows:

oxidase dependent [18].

and autoimmune diseases also needs to be investigated.

**3. Neutrophil extracellular traps in bacterial infections**

*Klebsiella pneumoniae*, the common cause of pneumonia, is caused by this aerobic Gram-negative bacillus. The role of NETs in the killing of *K. pneumoniae* has been investigated; this bacterium is not sufficient to induce NETs in neutrophils *ex vivo*, but it is in the lungs of a murine model. Adenosine A2B receptor deficiency improves survival and enhances bacterial killing and clearance due to NETs formation [21]. In addition, TREM-1 also mediates NETs formation, leading to a bactericidal effect and the control of infection [22].

*Leptospira interrogans* is the causative agent of leptospirosis. The pathogen spirochetes Gramnegative belongs to the Leptospiraceae family and to the genus Leptospira. Leptospirosis is an emerging zoonotic disease, affecting animals and humans in the world, but most frequently in tropical and subtropical countries. This disease is associated with exposure of individuals to wild or farm animals. Scharrig et al. [23], demonstrated for the first time the induction of NETs in human *ex vivo* and murine *in vivo* models, when incubating human neutrophils with Leptospira interrogans LI-130 (LIC). This research group observed that the bacteria number, the pathogenicity, and viability were relevant factors for induction of NETs; however, the motility of bacteria was not. Entrapment of LIC in the NETs resulted in Leptospira death. Pathogenic, but not saprophytic, Leptospira exerted nuclease activity, thus degrading the DNA, concluding that formation of NETs was dependent on bacterial concentration, pathogenicity, and viability, but not motility, and that NETs could trap and kill *Leptospira interrogans* [23].

*Mannheimia haemolytica*, the causative agent of bovine respiratory disease complex (BRD), is a Gram-negative bacterium that induces a severe pleuropneumonia in bovine animals, where neutrophils play a key role in the pathogenesis. Extracellular traps are induced in neutrophils and macrophages exposed to the bacteria or to their virulent factor, leucotoxin (LKT) [24].

*Mycobacterium bovis*, the etiological agent of bovine tuberculosis, is a Gram-positive bacterium, with a worldwide distribution, easily transmitted to bovine animals and to humans. The extracellular traps formation has been demonstrated in neutrophils and macrophages. Neutrophils can sense the size of pathogens, and based on their size, neutrophils are induced to undergo necrosis, apoptosis, or NETosis [25].

*Mycobacterium tuberculosis* is the causative agent of tuberculosis. Ramos-Kichik et al. showed that both *M. tuberculosis* and *Mycobacterium canetti* can induce NETs, which trap but not kill these mycobacterial species [26]. On the other hand, the mycobacterium-derived early secretory antigenic target protein of 6 kDa (ESAT-6) can induce the formation of NETs in *M. tuberculosis*-infected neutrophils [27].

*Pseudomonas aeruginosa*, the causative agent of the cystic fibrosis lung disease, is a Gram-negative opportunistic bacterium. The formation of NETs in the context of *P. aeruginosa* is controversial, and evidence that NETs may have a major anti-*P. aeruginosa* activity must be clarified [28].

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

*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 degraded by the neutrophil elastase [7].

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

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

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

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.
