**3. Role of NETs in health and diseases**

#### **3.1. The microbicidal activity of NETs**

The primary role of NETs is the antimicrobial activity, due to the cooperation of several mechanisms and components exposed at the high local concentrations in the NET fibers [55]. The pathogen spreading is limited by entrapment inside NET structure due to electrostatic interactions between the negatively charged DNA backbone and positively charged bacterial compounds localized on their cell surface [6]. Proteinaceous components of NETs are responsible for different types of NET antimicrobial activities. Proteases such as elastase, cathepsin G, and proteinase 3 are able to cleave virulence factors of *Yersinia enterocolitica*, *Shigella flexneri*, *Salmonella Typhimurium*, and other pathogens [4, 80]. The oxidative mechanisms of defense, e.g., the production of aggressive hypochlorous acid by myeloperoxidase, cause massive damages of NET‐entrapped pathogens with their membrane and protein oxidation [81, 82]. Histones, as well as antimicrobial peptides such as LL‐37 and BPI, also play an important role in pathogen elimination. Peptides derived from histones and LL‐37 take part in cell membrane permeabilization or bacterial cell lysis [83–85]. Moreover, NET‐associated factors can restrict nutrient supply for microbes, e.g., lactoferrin chelates iron and calprotectin sequesters zinc ions [79, 84].

#### **3.2. Pathogen escape from NETs**

Microorganisms that constantly compete with the host defense mechanisms for survival, elaborated also evasion strategies against toxic effects of NETs. The strategies can be divided into three groups, including: (1) an inactivation of NET components responsible for trapping and killing pathogens, (2) a suppression of NET formation and (3) development of resistance mechanisms against antimicrobial components of NETs.

The main NET component, DNA backbone is degraded by bacterial endonucleases, membrane‐ bound or released into the surrounding milieu. The group of microorganisms that produce such enzymes to avoid the killing activity of NETs includes *S. aureus* whose nuclease influences the bacterial survival and enhances its infectivity in a mouse respiratory tract infection model [86]. The same strategy, leading to decline NET integrity, is also adopted by other bacteria such as *Aeromonas hydrophila* [87], *Escherichia coli* [88], *Leptospira* sp. [89], *Neisseria gonorhoeae* [90], *Streptococcus agalactiae* [91], *Streptococcus pyogenes* [92, 93], *Streptococcus synguinis* [94], *Streptococcus suis* [95], *Vibrio cholerae* [96], and *Yersinia enterocolitica* [88]. *Streptococcus pneumoniae* uses cell‐associated endonuclease (EndA) to escape from local entrapment and promote bacterial spreading from lower airways to bloodstream during pneumonia [97]. Also, parasites such as *Leishmania infantum* use nuclease activity to resist the NET activity [98].

Moreover, the production of ROS involved in the initiation and progression of the main netosis pathway can be regulated by bacterial catalase activity in a self‐protection process [99].

Other interesting NET evasion strategies were proposed for meningococci [100], which apply the release of outer membrane vesicles for protection of bacteria from binding to NETs and express a high‐affinity zinc uptake receptor (ZnuD) to overcome possible ion sequestration by calprotectin, the NET component also known to be involved in *C. albicans* killing during netosis [101]. Moreover, a modification of meningococcal LPS with phosphoethanolamine protects bacteria from bactericidal activity of cathepsin G embedded into NET structures.

**3. Role of NETs in health and diseases**

The primary role of NETs is the antimicrobial activity, due to the cooperation of several mechanisms and components exposed at the high local concentrations in the NET fibers [55]. The pathogen spreading is limited by entrapment inside NET structure due to electrostatic interactions between the negatively charged DNA backbone and positively charged bacterial compounds localized on their cell surface [6]. Proteinaceous components of NETs are responsible for different types of NET antimicrobial activities. Proteases such as elastase, cathepsin G, and proteinase 3 are able to cleave virulence factors of *Yersinia enterocolitica*, *Shigella flexneri*, *Salmonella Typhimurium*, and other pathogens [4, 80]. The oxidative mechanisms of defense, e.g., the production of aggressive hypochlorous acid by myeloperoxidase, cause massive damages of NET‐entrapped pathogens with their membrane and protein oxidation [81, 82]. Histones, as well as antimicrobial peptides such as LL‐37 and BPI, also play an important role in pathogen elimination. Peptides derived from histones and LL‐37 take part in cell membrane permeabilization or bacterial cell lysis [83–85]. Moreover, NET‐associated factors can restrict nutrient supply for microbes, e.g., lactoferrin chelates iron and calprotectin sequesters zinc

Microorganisms that constantly compete with the host defense mechanisms for survival, elaborated also evasion strategies against toxic effects of NETs. The strategies can be divided into three groups, including: (1) an inactivation of NET components responsible for trapping and killing pathogens, (2) a suppression of NET formation and (3) development of resistance

The main NET component, DNA backbone is degraded by bacterial endonucleases, membrane‐ bound or released into the surrounding milieu. The group of microorganisms that produce such enzymes to avoid the killing activity of NETs includes *S. aureus* whose nuclease influences the bacterial survival and enhances its infectivity in a mouse respiratory tract infection model [86]. The same strategy, leading to decline NET integrity, is also adopted by other bacteria such as *Aeromonas hydrophila* [87], *Escherichia coli* [88], *Leptospira* sp. [89], *Neisseria gonorhoeae* [90], *Streptococcus agalactiae* [91], *Streptococcus pyogenes* [92, 93], *Streptococcus synguinis* [94], *Streptococcus suis* [95], *Vibrio cholerae* [96], and *Yersinia enterocolitica* [88]. *Streptococcus pneumoniae* uses cell‐associated endonuclease (EndA) to escape from local entrapment and promote bacterial spreading from lower airways to bloodstream during pneumonia [97]. Also, parasites such

Moreover, the production of ROS involved in the initiation and progression of the main netosis pathway can be regulated by bacterial catalase activity in a self‐protection process [99]. Other interesting NET evasion strategies were proposed for meningococci [100], which apply the release of outer membrane vesicles for protection of bacteria from binding to NETs and express a high‐affinity zinc uptake receptor (ZnuD) to overcome possible ion sequestration

**3.1. The microbicidal activity of NETs**

12 Role of Neutrophils in Disease Pathogenesis

ions [79, 84].

**3.2. Pathogen escape from NETs**

mechanisms against antimicrobial components of NETs.

as *Leishmania infantum* use nuclease activity to resist the NET activity [98].

The bactericidal activity of another NET component, cathelicidin LL‐37, can be abolished by its binding to the surface‐expressed M1 protein in *S. pyogenes* [102] or to surface exposed D‐alanylated lipoteichoic acid in *S. pyogenes* and *S. pneumoniae*, promoting bacteria survival within NETs [103, 104].

Moreover, *C. albicans* aspartic proteases, secreted during NET formation in response to fungal infection, are able to degrade and inactivate LL‐37 [105].

Many bacterial toxins are involved in induction of NETs but some of them are used by bacteria to regulate, in particular to inhibit NET formation [106]. *Bordetella pertussis* causing coughing syndrome adopts adenylate cyclase toxin (ACT) to suppress NET shaping [107]. ACT, after translocation into the host phagocyte, may influence the conversion of ATP to cyclic AMP, that in consequence prolongs neutrophil life span by inhibiting the oxidative burst, being one of the initial signals in NET production. This part of NET formation mechanism is also blocked by streptolysin O (SLO) produced by *S. pyogenes* [108].

In the defense against NET formation, microorganisms can also exploit host signaling as in the case of interleukine‐8 (IL‐8) production by epithelial cells in response to infection. This chemokine is responsible for neutrophil recruiting and amplification of NET release but *S. pyogenes* can produce a peptidase (SpyCEP) which inactivates IL‐8 and reduces NET formation [109].

A more complex strategy, used by *Pseudomonas aeruginosa* [110] or *S. agalactiae* [111], employs molecular mimicry with the acquisition of sialic acid motifs presented on the host cell surface which attenuate NET formation. A comparable, indirect mechanism suppressing NET release has been adopted by *Mycobacterium tuberculosis*. This microorganism that triggers NET release during the first stage of infection activates the production of anti‐inflammatory cytokine IL‐10 that inhibits TLR‐induced ROS production and suppresses further NET generation [112].

Also, viruses can apply this strategy of NET suppression, as demonstrated for HIV‐1 envelope glycoprotein [22]. Moreover, Dengue virus serotype‐2 can negatively affect NET formation by inhibiting glucose uptake in the ROS‐independent mechanism of netosis [113].

On the other hand, conidia *of Aspergillus fumigatus* expose hydrophobin (RodA) that suppresses the formation of NETs [114]. This process is also supported by the production of a positively charged exopolysaccharide—galactosaminogalactan that protects the microorganism from binding by NET components [115]. The polysaccharide capsule negatively modulating NET production that contributes to fungal disease severity was also observed in *Cryptococcus neoformans* infections [116].

Another way to subsist the antimicrobial activity of NETs is applied by *P. aeruginosa* in patients with chronic fibrosis where bacteria during its long‐term adaptation can form the resistant biofilm that protects the pathogen [117]. Moreover, *S. pneumoniae* and *Haemophilus influenzae* are even able to embed NETs into biofilm for self‐protection [118, 119]. Also, the extracellular matrix components of *C. albicans* biofilm alter its recognition by neutrophils and inhibit release of NETs [43].

All the above mechanisms developed by microorganisms to avoid killing by NETs confirm their ongoing adaptation to the sophisticated processes of host defense.

#### **3.3. Role of nets in noninfectious diseases**

Netosis is a process being under control of many mechanisms of activation, but NET fibers seem not to be a target or location specific, and in some cases, their release get out of the control. So, the process can be a double‐edged sword, acting also against the host cells. Therefore, NETs seem to play a significant role in several autoimmune disease and disorders, described in detail in others reviews [54, 120].

#### *3.3.1. Lung diseases*

A chronic inflammatory state of the lungs leads to the development of acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) [121–123]. The increased permeability of alveoli due to a mechanical ventilation or infection causes an activation of signaling involved in the release of proinflammatory factors by epithelial cells, and in consequence the massive migration and activation of neutrophils.

NET release can be also the trigger of sterile inflammatory state in the lung. Moreover, a lack of surfactant proteins makes a NET clearance difficult. The proteolytic enzymes contained in NETs damage epithelial cells, in consequence releasing more proinflammatory factors. This generates a self‐perpetuating mechanism of netosis activation [11, 124, 125].

A similar mechanism was observed in patients with cystic fibrosis (CF), a disease consisting in an increase in mucus viscosity, therefore hindering the clearance of mucus from the airways [126]. The presence of DNA in CF patient sputum increases a mucus viscosity, which correlates with the development of inflammation state and higher migration of neutrophils. The high viscosity of mucus makes it difficult to remove, generating good conditions for bacterial invasion [126, 127].

#### *3.3.2. Autoimmune disorders*

Autoimmune diseases including small vessel vasculitis (SVV), systemic lupus erythematosus (SLE), or rheumatoid arthritis (RA) seem to be also associated with uncontrolled release and ineffective clearance of NETs [128–130]. The high amount of NETs and free‐circulating DNA causes a production of antineutrophil cytoplasmic antibodies (ANCAs) against DNA and NET‐associated proteins such as MPO, cathepsin G, elastase, etc. Autoantibodies to citrullinated proteins (ACPA) seem to be a key pathologic factor in RA. The circulating complexes of antibodies‐DNA or antibodies‐NET proteins induce multiorgan inflammatory states, as well as inflammations of vessels [11, 13, 131, 132].

#### *3.3.3. Thromvbosis*

Deep vein thrombosis (DVT) is a next pathological state mediated by NETs. Neutrophils can be activated in veins by many different factors, including activated platelets, interleukins, proinflammatory cytokines, as well as von Willebrand factor (vWF), released by NET‐damaged endothelial cells. NETs, released inside veins, promote the formation of thrombi by binding of necessary blood cells and supporting of clot formation. The uncontrolled netosis can lead to massive DVT and consequently to multiple ischemia [11, 13, 133].
