Neutrophil extracellular traps in bacterial infections and evasion strategies

Neutrophils are innate immune cells that have a vital role in host defense systems. Neutrophil extracellular traps (NETs) are one of neutrophils’ defense mechanisms against pathogens. NETs comprise an ejected lattice of chromatin associated with histones, granular proteins, and cytosolic proteins. They are thought to be an efficient strategy to capture and/or kill bacteria and received intensive research interest in the recent years. However, soon after NETs were identified, it was observed that certain bacteria were able to evade NET entrapment through many different mechanisms. Here, we outline the recent progress of NETs in bacterial infections and the strategies employed by bacteria to evade or withstand NETs. Identifying the molecules and mechanisms that modulate NET release will improve our understanding of the functions of NETs in infections and provide new avenues for the prevention and treatment of bacterial diseases.


Introduction
Neutrophils are the most prevalent type of leukocytes circulating in the blood and are crucial for maintaining health.They differentiate from hematopoietic stem cells and are discharged from the bone marrow when terminally mature.They are the first cells to leave circulation and migrate to the infection site during an immunological challenge.There, they eliminate bacteria, communicate damage status to other immune cells, and are involved in the healing process (1).Much is known about the antimicrobial response of neutrophils.They are highly skilled in phagocytosis, which is their first vital mechanism for the eradication of disease-causing pathogens and elimination of dead cells and tissue debris (2,3).The second mechanism for eliminating pathogens is degranulation, which involves regulation of the immune system response via the release of various granules (4)(5)(6).
Approximately 20 years ago, a new mechanism by which neutrophils fight infections was termed neutrophil extracellular traps (NETs) (7).This involves the loosening of chromatin in the neutrophil nucleus and forming complexes with granular and cytoplasmic proteins, which are then released into the extracellular environment.There, NETs encapsulate and kill microorganisms, and block their dissemination (7,8).

NETs
NETs are a special form of programmed cell death, which is distinct from other cell death forms (apoptosis, necroptosis, and pyroptosis).In general, the complex process of NET formation begins with the recognition of microorganism, triggering the activation of the NETs pathway.This, in turn, leads to the breakdown of the nuclear membrane and the release of decondensed nuclear DNA into the cytoplasm.As this DNA mingles with cellular components including the histones, granules and cytoplasmic proteins, the process culminates in the rupture of the plasma membrane and the ultimate release of the NET structure into the extracellular space.Simultaneously, these traps are capable of degrading virulent bacterial factors (9)(10)(11).

NET components
The composition of NETs varies depending on the stimuli, and is primarily made up of DNA and histone proteins (H1, H2A, H2B, H3, and H4), which are originated from the nucleus followed by granular proteins that are normally stored in distinctive neutrophil granules in the cytoplasm of neutrophil such as neutrophil elastase (NE), myeloperoxidase (MPO), defensins, and cathepsin G from primary granules, lactoferrin, cathelicidins, and lysozyme C from secondary granules, and gelatinase from tertiary granules, as well as other proteins such as calprotectin and proteinase 3 are located in the cytoplasm of neutrophils (7,(52)(53)(54) (Supplementary Table S1).

Mechanisms of NETs formation
NETs were first reported in 2004 and were primarily described as a cell death process in activated neutrophil (7).When DNA leaks extracellularly, not all NET developments result in cell death, and not every neutrophil death necessarily results in NET formation."NETosis" is often employed throughout research for referring to NET formation, which can be categorized to as "lytic" and "nonlytic" forms.

Lytic mechanism
The "lytic" mechanism of NET formation is the interaction between stimuli and cell receptors, which triggers Raf-MEK-ERK signaling and activates the NADPH oxidase complex (55).This complex produces peroxide ions in neutrophils, increasing levels of cytosolic reactive oxygen species (ROS).MPO detects this rise and is often paired with other proteases to form the azurosome.MPO trigger the activation and translocation of NE from azurophilic granules to the nucleus, where elastase proteolytically processes histones to disrupt chromatin packaging (56,57).MPO then binds to chromatin and synergizes with NE to decondense chromatin independently of its enzymatic activity (57).The nucleus expands along with its chromatin, and cell lysis occurs because of gasdermin D, which can be activated by serine proteases in neutrophil inducing pore formation in the cell membrane (58).NETs are extruded into the extracellular space after membrane rupture and cell death within 3-8 h of neutrophil activation (9, 59).

Non-lytic mechanism
The "non-lytic" or vital mechanism, involves increasing the concentration of cytosolic calcium through calcium ionophores, which activate the protein arginine deiminase 4 (PAD4) that leads to citrullates arginine histone residues, and reduces their positive charge.Histones then begin to lose their electrostatic attraction to DNA, and the chromatin expands along with the nucleus (37,60).This oxidase-independent mechanism occurs within minutes of gram-positive skin infections; the cells release NETs during crawling without lysis, which prevents systemic bacterial dissemination (13,61).However, vital NET formation might be more closely associated with infection than previously thought because soon after releasing NETs, neutrophils are still viable and can carry out additional host response processes, such as phagocytosis, chemotaxis, and microbial killing (62).In addition to non-lytic NETs with nuclear DNA release, NETs composed of mitochondrial DNA, NE, and are first described in GM-CSF-primed neutrophils stimulated in vitro with LPS or C5a (38, 63) since this process would not require neutrophil lysis, which would enable phagocytosis to continue (10).

Other forms of NETs
However, divergent views have persisted in this field.NETs can form in the absence of PAD4 and citrullinated histone 3 (64,65).It can also occur during leukotoxic hypercitrullination (LTH), defective mitophagy, and organ injury (66).Other types of NETs have also been described, including cloudy NETs, spiky NETs, aggregated NETs, and bicarbonate-induced aggregated NETs (67).Therefore, we cautiously take opinions about these studies into consideration 3 Effects of NETs in bacterial infections

Morphological effects
NETs have various effects on the morphology of bacteria, including physically trapping and immobilizing them, leading to changes in their morphology as they become entangled in the weblike structure of the NETs (7).Additionally, they can release DNA, enzymes, and antimicrobial proteins that can damage the cell walls of bacteria and/or membranes, leading to changes in their morphology as they become structurally compromised (68)(69)(70).Furthermore, NETs are involved in the disruption of bacterial biofilms, which can also lead to changes in the morphology of the bacteria (71).

Functional effects
Along with the impact of NETs on the shape of bacteria, they can kill and limit the growth of bacteria and prevent their spread in the environment.They possess antimicrobial properties with components such as DNA, histones, granules, and cytoplasmic proteins that have bactericidal effects (7,68,(72)(73)(74)(75).Interestingly, NETs play an important role in the defense against bacteria, even in the absence of microbicidal activity.These infections are ensnared but not eliminated by NETs (28, 76-79), indicating that NET-mediated microbial trapping alone plays a substantial role in immune defense.
As mentioned above, the formation of NETs and their effect on bacterial infection are mostly reported in vitro conditions.However, there is limited research describing the stimulation of NETs in response to bacterial infection in vivo.It is difficult to verify NETs in vivo because it calls for specialized technical knowledge.Besides, it is hard to assess the kind of stimulus, its dosage, and its exposure duration in vivo.Researchers may use genetically modified animal models to study the role of specific proteins or signaling pathways in the regulation of NET formation during bacterial infection (80).
Furthermore, the composition of NETs determines their efficiency.Mice exposed to low concentrations of cathepsin G are more susceptible to infection with the Gram-positive bacterium S. aureus (81), whereas those exposed to low concentrations of NE are more susceptible to Gram-negative bacteria such as E. coli, K. pneumoniae, and other Enterobacteriaceae (70,81).Additionally, the antimicrobial response of NETs is influenced by the environment in which they are developed; NETs under static conditions show limited bacterial killing, whereas those under dynamic conditions show enhanced bacterial trapping and reduced killing (78).
Although NETs protect the host against microbes, there are diseases and conditions that can interfere with the release of NETs.Some examples of diseases that can affect NET release or neutrophil function are autoimmune diseases, chronic inflammatory conditions and sepsis, which can alter the function of neutrophils and their ability to release NETs.In these conditions, the dysregulation of neutrophil function and NET release can contribute to the pathology of the disease and affect the body's ability to fight infections and maintain immune homeostasis (82,83).

Modulation of NET release in bacteria
As mentioned before, the release of NETs is essential for defense against pathogens; the evasion of NETs appears to be a widespread strategy to allow pathogen proliferation and dissemination and is currently a topic of intense research interest.Here, we review the current knowledge of evasion strategies used by bacteria to dysregulate NET formation and functions.

NET formation inhibition
Specific molecules and pathways that inhibit NET release.Thus, NET inhibition mechanisms in bacterial infections are summarized in this section of the review (Table 1).

Downregulation of NETstimulating phenotypes
Suppression of phenotypes that trigger NET release is a way to prevent their formation.Flagellum is the main bacterial component required to trigger maximal NET release, and flagellum-deficient bacteria remain seriously impaired in triggering NET formation (25).In addition, LasR-deficient P. aeruginosa strains harbor a limited capability to trigger neutrophil DNA release due to reduce the expression of elastase LasB and proteases LasA.The neutrophil NADPH oxidase pathway is required to decrease NET release caused by LasR-deficient strains, but it is not dependent on downstream quorum sensing pathways, LPS synthesis, or bacterial motility (84).

Inhibition of NET-triggered molecules
IL-8 is a prominent neutrophil chemoattractant and activator that induce NET formation (7).However, group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8 and reduces neutrophil production of extracellular traps (86).In addition, streptolysin O toxin (SLO) prevents the release of IL-8 and elastase from neutrophils, blocks NET formation, and inhibits NETs by dissolving cell membranes and fibrous extracellular DNA strands (87).

Suppression of NET-mediated receptors on neutrophil
Neutrophil surface receptors have been linked to suppression of NET formation.GAS expresses a high-molecular-weight hyaluronic acid capsule (HMW-HA).hSiglec-9 specifically binds to HMW-HA through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site.HMW-HA recognition by hSiglec-9 blocks the oxidative burst and limits NET formation, thereby promoting bacterial survival (88).Moreover, the sialylated capsular polysaccharide of Group B Streptococcus (GBS) interacts with neutrophil Siglec-9, dampening neutrophil responses in a Sia-and Siglec-9-dependent manner, causing a reduction in the neutrophil oxidative burst, diminished formation of NETs, and increased bacterial survival (91).In addition, b protein from GBS inhibits human leukocyte phagocytosis, oxidative burst, and extracellular trap formation by binding to hSiglec-5 in a Sia-independent manner (92).However, engagement of Siglec−14 by b−protein antagonizes the repressive effects of Siglec−5 by activating mitogen-activated protein kinase (MAPK) signaling (99).Moraxella catarrhalis limits ROS production by possibly binding to immunosuppressive Siglecs receptors (Siglec-5 and Siglec-9) and consequently suppressing NET generation (89).Furthermore, soluble Siglec-9 exhibited strong binding with a2-3-linked sialoglycoproteins adsorbed by P. aeruginosa.The interaction between P. aeruginosa (+Sias) and siglec-9 on neutrophils decreases the amount of ROS and the release of elastase, which in turn decreases the formation of NETs (85).Moreover, Acinetobacter baumannii inhibits the formation of NETs by suppressing the surface expression of CD11a in neutrophils, thereby escaping host immune responses and contributing to the development of A. baumannii infections (97).NET formation is related to the production of ROS, both cytosolic and mitochondrial (55,100).The adenylate cyclase toxin (ACT) of Bordetella pertussis inhibits formation of NETs by generating cyclic adenosine monophosphate (cAMP), consequently inhibiting oxidative burst (93).Bordetella parapertussis also expresses CyaA in a Bvg-regulated manner.This toxin is released into the extracellular space, which mediates the inhibition of ROS and reduces NET activation in human-derived neutrophils (94).
K. pneumoniae ST258 is a poor inducer of ROS generation, and consequently inhibits NET formation, suggesting that the polysaccharide part of LPS is responsible for this inhibition and results in increased bacterial survival (95).Burkholderia pseudomallei modifies the magnitude of NET formation via the action of its type 3 protein secretion system (T3SS), encoded by the bsa locus, and capsular polysaccharide I (CPS-I) encoded by the wcb operon, which attenuates NET release by inhibiting the NADPH oxidase pathway.B. pseudomallei mutants defective in the virulence-associated T3SS or CPS-I induced elevated levels of NETs.NET induction by these mutants is associated with increased bacterial killing (96).

Others
Wild-type p53-induced phosphatase 1 (Wip1) suppresses NET release in S. aureus in mice model.Inhibition of Wip1 significantly suppresses the activity of S. aureus and accelerates abscess healing in S. aureus-induced abscess model mice by enhancing NET formation (98).Moreover, uropathogenic E. coli secretes a multifunctional virulence factor called TcpC, which primarily inhibits NETs by serving as an E3 ligase, promoting the degradation of PAD4.TcpC not only inhibits the citrullination of chromatin histones, but also affects the transcription of related genes in the nucleus and represents an additional NET evasion function of this bacterial derived virulence factor (90).

Degradation of NET components
Studies investigating the production of NET-degrading molecules by bacteria have focused largely but not exclusively on the nucleases (Table 2; Figure 1).

Nuclease expression
DNA forms a NET framework that holds multiple enzymes in close proximity, sometimes allowing for synergistic interactions among them (68).DNA is hydrolyzed by nucleases that belong to a group of hydrolases, which are further classified as endonucleases and exonucleases.These enzymes are involve in replication or repair of genetic material to maintain chromosome (136).However, extracellular nucleases have been reported in some bacterial species for the degradation of NET scaffold DNA, enabling bacteria to evade the NET antimicrobial mechanism, promoting pathogenicity, and dissemination to other sites in the host.For example, nuclease (Nuc) (101, 102) and extracellular adherence protein (Eap) (103,104) are produced by S. aureus and interfere with the antimicrobial activity of NETs.These proteins help S. aureus to escape from NET-mediated killing, impede its removal, and increase infection-related mortality.Moreover, S. aureus escapes this defense by converting NETs to deoxyadenosine (dAdo) via the action of Nuc and adenosine synthase A (AdsA), which triggers caspase-3-mediated death of macrophages (105,106).Moreover, S. aureus synchronizes gene expression during skin infection through the ArlRS-MgrA regulatory system, which regulates nuclease expression.This cascade is required for both the appropriate structuring of the abscess and evasion of the host innate immune system, both of which are necessary for S. aureus virulence.In contrast, mutants lacking MgrA and ArlRS have reduced capacity to avoid NET function (107).In addition, S. aureus expresses the competence regulator (ComK) when exposed to ROS.ComK upregulates the expression of genes encoding the transport machinery for glucose and DNA uptake, providing extra nutrients to increase the fermentation possibility of bacteria that are unable to respire and a source of nucleotides to repair DNA damage from ROS. Bacteria may use competence-related genes to better withstand NETs, because NETs are a source of both nucleotides and ROS (108).
P. aeruginosa encodes an operon of two secreted enzymes, a DNase and a predicted alkaline phosphatase.DNase (eddB) contributes to the degradation of NET to defend P. aeruginosa against NET-mediated killing, whereas eddA has both alkaline phosphatase and phosphodiesterase (PDase) activities that do not cause DNA degradation similar to that of DNase, but its protective function is likely a result of removing cation-chelating phosphates from the extracellular DNA phosphodiester backbone (110).
GAS is a leading cause of severe invasive disease in humans, and has evolved numerous virulence factors that aid in blocking NET function through the expression and secretion of the extracellular nuclease Sda1, which is advantageous for promoting bacterial dissemination throughout the host organism and evasion of the host innate immune response (77, 112).Similarly, S. pyogenes nuclease A (SpnA), a cell wall-anchored DNase, shows a unique protein architecture and promotes survival in human blood and in neutrophil killing assays, enable for the destruction of NETs and is believed to be an important immune evasion mechanism (113).A pneumococcal nuclease (EndA) acts as a virulence determinant, counteracting host-mediated trapping by NETs, thereby promoting bacterial spread from local sites to the lungs and then to the blood stream (76).Furthermore, the competence-independent activity of EndA is important for the virulence of Streptococcus pneumoniae, which mediates the rapid degradation of extracellular DNA and NETs (114).In addition, the secretion of nucleotide sequenceindependent endodeoxyribonuclease TatD from S. pneumoniae is a novel potential extracellular DNase that plays a key role in evading NET-mediated bactericidal activity (115).
Streptococcus suis induces NET formation in porcine neutrophils and is entrapped, but not killed by NETs.The amount of NETs reduces over time due to the expression of nuclease A (SsnA), indicating that SsnA is a specific NET evasion factor in S. suis (116).In this sense, NET degradation is mediated not only by the known secreted SsnA but also by a putative endonuclease A of S. suis that is homologous to pneumococcal EndA (117).Actinobacillus pleuropneumoniae does not produce its own NET-degrading nucleases, but is hijacking other nucleases from host or from other co-infecting bacteria such as S. suis as a source for nicotinamide adenine dinucleotide (NAD) needed for efficient growth in the presence of NETs (137).Moreover, Streptococcus sanguinis utilizes cell surface nuclease with a cell-wall anchor domain, termed streptococcal wall-anchored nuclease (SWAN), and contributes to bacterial resistance against the bactericidal activity of NETs (119).Neisseria gonorrhoeae encodes a putatively secreted thermonuclease that is implicated in biofilm remodeling and degrades the NET matrix to help N. gonorrhoeae from killing by neutrophils (120).
Yersinia enterocolitica O:3, O:8 and O:9 are able to induce NETs in human blood-derived neutrophils, but the amount of NETs is reduced at a later time, suggesting that the degradation of NETs has occurred and postulates that Y. enterocolitica produces Ca 2+ /Mg 2 + -dependent NET-degrading nuclease (121).Vibrio cholera induces the formation of NETs upon contact with neutrophils, while V. cholerae in return to expresses two extracellular nucleases, Dns and Xds, in the presence of NETs, and rapidly degrades the DNA component of the NETs by the combined activity of the two nucleases (122).Leptospira spp. is able to induce NETs using human ex vivo and murine in vivo models, resulting in the release of NETs.However, Leptospira spp.exerts nuclease activity and  degrades DNA, resulting in a significantly reduced amount of NETs (123).In addition, the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) is involved in immune modulation; LAV has a nuclease activity and is demonstrated in the evasion of Leptospira from NETs (124).Additionally, surfaceexposed lipoproteins in Leptospira are important for modulating host immune responses, and most of Len family surface proteins exhibit nuclease activity and are linked to NETs degradation (125).Oral periodontopathogenic Prevotella intermedia produces two nucleases, NucA and NucD, which require Mg 2+ and Ca 2+ for their nuclease activity and contribute to NETs degradation (126).The fish pathogen Aeromonas hydrophila combats NETs using nuclease activity, while treatment of cells with b-glucan significantly protects NETs against bacterial degradation (127).Mycobacterium avium subsp.paratuberculosis encodes an extracellular nonspecific DNase that can destroy the NETs and promotes bacterial survival in vitro and in vivo (128).
Mycoplasma are the smallest bacteria that can infect and cause serious disorders in humans and various animal species (138,139).Nucleases are a crucial component of Mycoplasma pathogenesis, facilitating bacterial growth and persistence in the host by digesting host nucleic acids and producing free nucleotide precursors (140)(141)(142).Membrane-associated or secreted nucleases have been found in many Mycoplasma species (129,132,(143)(144)(145)(146), and are homologous to staphylococcal nuclease (101).Mycoplasma lipoproteins are a major determinants of NET release during the innate immune response (39).Interestingly, most of the described Mycoplasma nucleases are able to escape from NET entrapment and killing by digesting NET's DNA backbone, reducing structure stability, and enhancing NET elimination (129)(130)(131)133).

PAD4 secretion
Although DNA has been thoroughly examined, it is not the only NET component that is susceptible to degradation.PAD4 capable of deaminating arginine to citrulline and has been linked to NET formation in various context (147).PAD4 is involved in NETmediated bacterial trapping and killing.Histones with cationic residues interact with negatively charged bacterial membranes and disrupt them (148).Thus, the loss of the positive charge of histones can lower their antibacterial function due to PAD4 activity.For example, Porphyromonas gingivalis produces its own PAD, Porphyromonas peptidylarginine deiminase (PPAD), which citrullinates histone H3, thereby facilitating bacterial escape from NETs, whereas PAD-mutant P. gingivalis is more prone to NETmediated killing than its wild-type counterpart (134).Moreover, histone 3.1 displays bactericidal activity against P. aeruginosa.This bactericidal effect is reduced following citrullination by PAD4 or proteolysis by NE (111).

Degradation of NET-bound proteins
As previously mentioned, host defense peptides are key components of the innate immune system and act as the principal first line of defense against invading pathogens (149).The cysteine protease ApdS from S. suis cleaves cathelicidin LL-37 and impairs its ability to promote NETs formation and ROS production (118).K. pneumoniae affects the mobilization of primary granules and their components, which harbor proteins with more potent bactericidal properties and those related to NETs (135).Gingipain is a cysteine protease responsible for the virulence of P. gingivalis and is dependent on proteolytic activation of protease-activated receptor-2 (PAR-2).Intriguingly, P. gingivalis and purified Arg-specific gingipains (Rgp) induce NETs that not only lack bactericidal activity, but also stimulate the growth of bacterial species otherwise susceptible to killing in NETs.Taken together, gingipains play a dual role in NETs; they are potent direct inducers of NETs formation; however, their activity prevents P. gingivalis entrapment and subsequent killing (51).S. aureus produces a cell-wall-anchored protein known as fibronectinbinding protein B (FnBPB) that is the main histone receptor and bind to all types of histones.FnBPB provides a dual immuneevasion function that captures histones, and prevents them from reaching the bacterial membrane, and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone (109).

NET resistance mechanisms
In addition to inhibition of NET release and the degradation of NETs components, certain bacteria are resistant to the antimicrobial activity of NETs (Table 3).

Capsule
Capsule is a surface structure of the organism and plays a critical role in virulence, principally by interfering with host clearance mechanisms.The thickness of the pneumococcal capsule plays a crucial role in determining the extent of NET formation and may contribute to pneumonia severity (161,162).Interestingly, the electrostatic charge of the capsule helps evade NET-mediated killing by repelling interactions with antimicrobial proteins in the DNA framework.In this context, S. pneumoniae contains a dlt operon that mediates the incorporation of d-alanine residues into lipoteichoic acid (LTA), thereby introducing a positive charge that reduces trapping by NETs in vitro (150).Kingella kingae polysaccharide capsule and exopolysaccharide function distinctly to promote neutrophil evasion.The K. kingae polysaccharide capsule prevents ROS production and neutrophil association, whereas the K. kingae exopolysaccharide reduces neutrophil phagocytosis and sensitivity to antimicrobial peptides (152).In addition, the hyaluronic acid capsule and M protein of the GAS serotype M1T1 promote resistance to human cathelicidin LL-37, which may be an important contributor to the NET-resistance phenotype (155).

Biofilm formation
Microbial biofilm communities are microbes embedded in a matrix of self-produced polysaccharides and other molecules, such as lipids, proteins, and nucleic acids that can bind to various surfaces, which complicates the immunological and therapeutic response (163).Biofilm formation by P. aeruginosa is a major cause of bacterial keratitis and is facilitated by bacterial Psl exopolysaccharide and T3SS.NETs are stimulated by high expression of T3SS and form a barrier, known as dead-zone, confining bacteria to the external corneal environment, preventing them from spreading to the brain.Once formed, ocular biofilms advance eye pathology because they are resistant to neutrophil killing and antibiotics (71).Furthermore, P. aeruginosa acquires resistance to NET-mediated killing in cystic fibrosis airways.This resistance correlates with the development of excess exopolysaccharide production (which characterizes the mucoid phenotype) (74).In addition, studies conducted in vitro have demonstrated that P. aeruginosa can profit from the released DNA of NETs and incorporate it into the extracellular matrix, potentially strengthening the matrix's resistance to antibiotics and host antimicrobial peptides (164)(165)(166)(167).
Clinical isolates of H. influenzae share a common tendency to produce biofilms, and the factors that facilitate biofilm formation include the expression of certain lipooligosaccharide (LOS) glycoforms (168-170), type IV pili (171), and double-stranded DNA release (172).Nontypeable Haemophilus influenzae (NTHi) promotes resistance to killing within NETs structures through LOS moieties that promote biofilm formation (23,157).Methicillinresistant S. aureus (MRSA) biofilms rapidly skew neutrophils toward NETs formation through the combined activity of the Panton-Valentine leukocidin (PVL) and g-hemolysin AB, which are important for biofilm-mediated neutrophil killing.Through this response, S. aureus can persist because the antimicrobial activity of the released NETs is ineffective in eradicating bacterial biofilm (12).S. aureus nuclease-mediated NET degradation promotes the persistence of biofilm bacteria entrapped in NETs (173).S. suis serotype 2 induces NETs release and can be captured by the NETs, whereas biofilm formation inhibit NETs release through the biofilm extracellular matrix and enables survival by allowing the pathogen to persist and resist the host immune system (160).In addition, pneumococci produce biofilms in vivo and observe web-or net-like structures surrounded by dense matrix material, which is intertwined with the formation of NETs (151).

Outer membrane vesicles
OMVs are nanostructures released by pathogenic and nonpathogenic bacteria in vivo and in vitro and can act as decoys for NET capture (174).Ocular keratitis is often associated with P. aeruginosa infection.Neutrophils release NETs in response to both cytotoxic and invasive clinical isolates of P. aeruginosa.Cytotoxic strains are less prone to NET capture than invasive strains because they release OMVs that inhibit NET adherence (159).Furthermore, Neisseria meningitidis releases OMVs as potent NET inducers.NETs are unable to kill NET-bound meningococci, but they slow down their proliferation rate.The bacteriostatic effect of NETs is counteracted by spontaneously released OMVs from N. meningitidis, which reduces their adherence to NETs (75).

Charge surface alteration
NET-releasing proteins attach to negatively charged phospholipids in pathogen membranes with electrostatic affinity, which promotes death (175).Nonetheless, certain bacteria modify their cell surfaces, which reduces their affinity for attachment to NETreleasing peptides.For example, the surface-associated protein M1 protein contributes to GAS virulence by interfering with NETmediated killing, which sequester and neutralize cathelicidin LL-37 through the N-terminal hypervariable (HV) region and A repeat region that encode the type-specific immunologic epitopes of the M1 serotype (15,153).In addition, the N-terminal portion of the M1 protein binds and inactivates histones before they reach their cell wall target of action and mediate resistance against released extracellular histones in NETs, allowing the pathogen to tolerate high concentrations of histones and promote survival in NETs (14).Streptococcal collagen-like protein 1 (Scl-1) in GAS serotype M1T1 confers resistance to NET-mediated killing, which may be in large part due to the antimicrobial peptides present within the NETs; Scl-1 has an additional role in suppressing the release of MPO, which ultimately limits the production of NETs (156).Haptoglobin is an abundant acute-phase protein produced upon infection, which binds to human neutrophils and monocytes and inhibits their functions (176,177).The T4 antigen, the pilus backbone protein of GAS M4, sequester the host haptoglobin.Coating M4 GAS with haptoglobin causes a reduction in susceptibility to cathelicidin LL-37 and murine cathelicidin-related antimicrobial peptide (CRAMP) and promotes resistance to NET-mediated killing (154).
Modification of lipid A of meningococcal LPS with phosphoethanolamine shields N. meningitidis from the action of NET-bound cathepsin G (75).In a process known as nutritional immunity, host organisms restrict the availability of trace nutrients in the blood or secretions to prevent the growth of invading microorganisms (178).The outer membrane receptor ZnuD of N. meningitidis is crucial for Zn 2+ uptake at very low concentrations (179).N. meningitidis utilizes high affinity absorption systems for critical ions on mucosal surfaces, producing ZnuD, which absorbs Zn 2+ ; contribute to survival within NETs (75).

Antioxidant enzymes secretion
Finally, the initiation of the oxidative burst is important for the induction of NETs, and oxidants contribute to microbicidal activity within the NETs (9,180).Noticeable H. influenzae expresses the bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX) and catalase (encoded by hktE), which confer resistance of NTHI to oxidative killing and thus promote the survival of NTHI within NET structures and persistence in vivo in the lung and middle ear.In addition, exogenous catalase partially rescued NTHI from NETmediated killing in vitro.The expression of both peroxiredoxinglutaredoxin and catalase is a mechanism by which NTHI combats the effects of NETs (158).

Concluding remarks
Neutrophils are type of white blood cell that play a crucial role in the immune system's defense against infections.There are different subsets of neutrophils that have different functions and responses to various stimuli.NET release is a form of neutrophil immune response that, depending on the context in which it is examined, can have both pathogenic and physiological effects.They can entangle and immobilize bacteria, preventing their spread, and facilitating their destruction by other immune cells.Additionally, dysregulation of NET release has several implications for the immune system and overall health.On the other hand, impaired NET formation may lead to reduced ability to fight off infections.Therefore, maintaining a balanced and controlled NET release is essential for the proper functioning of the defense system and overall health.In recent years, there has been a significant increase in research focused on the role NETs in response to pathogens and the mechanisms involved in the modulation of their release.Bacteria have developed various mechanisms to evade or resist NET release, allowing them to escape the immune system's defenses and several key findings and advancements have emerged from these studies.In this review, we have discussed the strategies used by various bacteria to counteract NET-mediated antimicrobial effects as such inhibiting NET release, deactivating their components, degrading their net-like framework, or blocking their contact, resulting in infection dissemination and immune system inactivation.Bacteria possess a vast array of mechanism for modulating NET activity, and ongoing investigations are expected to uncover new molecules and pathways involved in controlling NET release.Current understanding of different inflammatory mediators in modulate NET release is quite limited and need be further elucidated.Overall, the recent focus on NETs and their modulation has provided a deeper understanding of the complex interplay between neutrophils, pathogens, and the immune system.This knowledge has the potential to identify pharmacological targets and drive the development of therapeutic and diagnostic approaches to counteract bacterial evasion strategies and combat the role of NETs in inflammatory and autoimmune diseases.It is crucial to understand that various factors affect whether NETs are advantageous or harmful, with the dose and the timing of NET release and clearance being critical factors.A better understanding of the roles of NETs and their effects on hosts will make it possible to inhibit the adverse attributes without affecting the beneficial ones, which will ultimately enable strategies related to NETs to be used in disease treatment.

FIGURE 1
FIGURE 1 Bacterial factors associated with the degradation of NET components.(1) DNAses cleave NET-associated DNA, and Eap bind to the termini of linearized DNA and expresses an intrinsic DNA aggregation activity.(2) Adenosine synthase act together with the nuclease for generating deoxyadenosine fragments that are apoptotic for macrophages.(3) Phosphatase remove phosphate group from DNA, showing a cation chelating effect.(4) Apds cleave N-terminal amino acid from cathelicidin LL-37 and lose its helical structure.(5) ComK increase glucose and DNA uptake and downregulate ROS production.(6) FnBPB, in preference to FBG, H3 binds to the tunnel between the N2 and N3 of FnBPB, and then causes a conformational shift that allow the complex to stabilize by insertion of the terminal residues of the N3 extension (red arrow).Next, PLG binds to a N3 site, activated by t-PA to PLM, and cleaves FnBPB-bound H3. (7) PAD4 change histone arginines into histone citrullines, and losing its interactions with bacterial membranes.(8) Gingipains, cysteine protease, cleave protease-activated receptor-2 on the neutrophil surface.dAdo, deoxyadenosine; ROS, reactive oxygen species; HGT, horizontal gene transfer; PAD4, protein arginine deiminases 4; MPO, myeloperoxidase; FnBPB, fibronectin binding protein B; FBG, fibrinogen; H3, histones 3; PLG, plasminogen; t-PA, tissue plasminogen activator; PLM, plasmin; Eap, extracellular adhesion protein; Cit, citrullin.S. aureus, Staphylococcus aureus.Created with BioRender.com.

TABLE 1
Factors associated with the inhibition of NET release in bacterial infections.

TABLE 2
Lists the molecules for degradation of NET release in bacterial infections.