The pathogenesis of S. pneumoniae is an intricate and dynamic process. The pathogen expresses multiple virulence factors, which can combine to induce invasive pneumococcal disease [42]. The flexibility of S. pneumoniae facilitates responses to evolutionary pressures that provide a significant advantage during infection, such as evasion of host immune defenses or developing antibiotic resistance [43]. In addition, EndA in S. pneumoniae plays a dual role in DNA uptake and contributes to the genetic flexibility that characterizes the pathogen [44].

It is also crucial to understand the neutrophils’ general structure and mechanism since the nucleolytic activity of EndA could facilitate the degradation of NETosis (NETs). Neutrophils are recognized as essential effector cells of innate immunity and critical regulators of both natural and adaptive immune responses [45]. Neutrophils have a short half-life of less than 24 hours [46] and will differentiate and exit the bone marrow before dying in the bloodstream in the absence of infection. When the host becomes infected, tissue-resident macrophages and other sentinel cells emit inflammatory cytokines and chemo-attractants to recruit and stimulate neutrophils. In response to the production of these chemicals, neutrophils exit the bloodstream and penetrate the infected tissues via a selection and integrin-mediated process known as extravasation [46]. Neutrophils are the first effector cells to reach the infection site, thus making them essential in pathogen clearance, recruitment, activation of other immune cells, and tissue repair [46].

Mitochondrial DNA was revealed as a significant structural component of NETs in an early finding reported by Brinkmann et al. [47]. Physically, by using electron microscopy, the NET structure was found to be made up of fibrous DNA stretches with widths ranging from 15 nm to 17 nm and globular protein domains around 25 nm that may aggregate to more extensive threads with diameters exceeding 50 nm [47]. Von Köckritz-Blickwede et al. [48] also confirmed the absence of membrane proteins and cytoplasmic fragments within the NET structures, as well as highlighting the presence of histones as intricate components [48]. Furthermore, these structures are primarily composed of nuclear or mitochondrial DNA, serving as the foundation for antimicrobial peptides, enzymes, and histones, whereas some researchers argue that the contribution of DNA from mitochondria to NETs is minimal [49]. These large extracellular structures provide a physical barrier by increasing the local concentration of antimicrobial effectors and could prevent microbial dissemination [50, 51]; hence, understanding their structure and physiology could provide us with knowledge to avoid the further spread of pathogens upon infection. Microbiological stimuli from bacteria, fungi, viruses, and protozoa are examples of NETosis inducers. Besides, additional chemical factors and reactive oxygen species (ROS) may contribute to NETosis, as listed in Table 1.

Neutrophils are the first immune cells that reach the site of infection and support the clearance of harmful bacteria through numerous processes, including developing NETs, as illustrated in Figure 2. Initially, phagocytosis and degranulation were considered the main mechanisms carried out by neutrophils to eliminate pathogens. As demonstrated in Figure 2, the first sign of NET formation via cell death or suicidal NETosis mechanism is changing the morphology of the nuclease, which loses its characteristic lobulated form. Neutrophil elastase (NE), generally stored in neutrophil granules, translocates into the nucleus during NETosis and cleaves histones by promoting chromatin decondensation [70]. Nuclear membranes and chromatin decondensed into the cytoplasm while the plasma membrane remains intact. Ultimately, the plasma membrane ruptures, leading to the release of NETs. The receptors responsible for initiating NET release and the molecular mechanisms through which ROS drives this process are poorly understood.

However, certain pathogenic microorganisms possess the capacity to express virulence factors that enable them to successfully establish an infection and evade the host immune system. For example, S. pneumoniae induces NET formation but has evolved virulence mechanisms to avoid NETs. The invasive pneumococcus type TIGR4 expresses EndA, which enables escape from NETs by degrading the extracellular traps, thereby increasing their virulence in vivo [71] (Figure 2a). The evasion of NETs facilitates the dissemination of bacteria from the upper respiratory tract to the pulmonary region and from the lungs to the bloodstream [52].

In contrast, the first mechanism discovered is vital NET formation [72] (Figure 2b) which occurs via slow lytic cell death. A few NETs are produced when neutrophils quickly release their nuclear content by vesicular secretion. Their nuclear content expulsion via vesicles to intact cytoplasts that continue crawling and digesting microbes using phagocytosis or chemotaxis [73]. The significance of these structures in the immune response is highlighted by the description of pathogen compounds that can impede, destroy, and elude the antimicrobial action of NETs (Figure 2) [74].

NETs have been shown to play a physiological protective role in infection sites and inhibit the spread of pathogens. In response to their phagocytic function, NETs provide several benefits to reduce disease spreading by concentrating host antimicrobial agents at infection sites. For example, it has been shown that NETs in staphylococcal skin infections inhibit the penetration of pathogens into the bloodstream [75]. Investigations on S. pneumoniae-infected humans and animals have shown increased NETs within alveolar gaps [76]. Moreover, an in vitro investigation has shown that NETs have vigorous antibacterial activity against S. pneumoniae [53]. These results demonstrated the importance of NETs, particularly in pneumococcal pneumonia.

Despite the variable efficiency of the antimicrobial properties of NETs, some microbes have evolved mechanisms and strategies to reduce the effectiveness of NETs. The pathogenic systems have three functions: deactivate the parts of NETs that function to trap and eliminate pathogens, inhibit the growth of NETs, and create defense mechanisms against the antimicrobial components of NETs [77]. Previous studies have demonstrated the various mechanisms utilized by pathogens to escape from NETs by utilizing the bacterial polysaccharide capsule, formation of biofilms, altering the electric charge of the cell surface, inhibiting ROS formation or inhibition of NET production by peptidase and DNase generation [78].

As part of their defensive strategies, bacteria produce nucleases to protect themselves by breaking down the NETs. This is because the DNA backbone of NETs contains bactericidal molecules such as histones or cathelicidin that can kill entrapped bacteria. Membrane-bound bacterial REases, which are released into the surrounding environment in response to NETs might thus be considered as a protective virulence factor of the bacteria [79]. Numerous S. pneumoniae strains have been shown to produce an endonuclease that renders them entirely immune to NET-induced death and has been shown to compromise the functional integrity of NETs [72]. In his early study, Beiter et al. [73] reported that EndA represents a major nuclease that allows pneumococci to degrade the DNA scaffold of NETs and then escape from NETs efficiently. By using the mouse intranasal model, Beiter et al. [73] showed that during pneumonia, pneumococci travelled more easily from the upper airways to the lungs and into the bloodstream when they managed to escape out of the NETs, contributing to bacterial resistance to innate immunity. Therefore, understanding the endonuclease mechanism in escaping NETs could give a clear perspective on manipulating nuclease secretion, making S. pneumoniae less invasive to humans.

Similarly, Peterson et al. [42] reported that the surface EndA of S. pneumoniae performs two crucial roles in pneumococcal illness. Initially, EndA was responsible for the transport and destruction of DNA during transformation, which adds to the genomic variety of pneumococci. Secondly, as illustrated in Figure 3a, it allows the extracellular breakdown of the DNA component of NETs, which promotes pneumococcal spread and raises the risk of invasive infection [42]. EndA deletion in the study reduced transformation efficiency, potentially impeding the genetic diversity responsible for pneumococcal pathogenicity. Furthermore, in experiments on mice, pneumococci lacking EndA exhibit decreased invasive infection and cannot escape from NETs [72]. Specifically, the previous findings on mutant knock-out for EndA could mitigate the invasion of S. pneumoniae due to the inability to escape through NETs, leading to unsuccessful colonization of S. pneumoniae in the host. Therefore, these would give promising attenuating pneumococcal pathogenesis to increase immunogenicity protection and offer a novel target for controlling pneumococcal disease (see Figure 3b).

However, excessive NETs have been reported to be associated with reduced clinical stability hazards and increased pneumonia mortality [80]. Higher concentrations of NETs released by activated neutrophils promoted tissue damage, including lung injury [81] and sepsis [82]. The component mechanisms that are responsible for NET-induced tissue damage are NE components and other proteases that induce cell death in multiple cell types [83–87]. Instead of pneumonia, NETosis contributes to developing complications for the infected lung, including bronchial asthma, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD). Before the occurrence of the COVID-19 pandemic, a clinical experiment found a link between the severity of community-acquired pneumonia and the content of NETs [80]. Two decades after the discovery of NETs, a significant correlation emerges between the release of NETs and the immune system and various diseases [88].

Since NET formation and elimination were unregulated and led to severe effects, the focus of NET research has changed from innate immune defense to NET involvement in severe consequences [88]. Recent in vitro and in vivo experiments using animal models demonstrated the crucial role of NETs in pathogenesis and increasing morbidity and mortality [88]. Multiple organ failure and poor prognoses have been linked to high levels of circulating NETs in sepsis patients [89–91]. In addition, NETs have been linked to pathologic changes in autoimmune and autoinflammatory illnesses [92, 93] due to the circulating free DNA (cfDNA) exacerbating inflammation by inducing TNF-α mRNA [94]. At the same time, it has been demonstrated that these inflammatory substances increase NETosis and pathology in these patients without causing infection [95]. For example, the deposition of monosodium urate crystals in the joints is an autoinflammatory disease called gout that stimulates an immune response by attracting leukocytes to induce NETs, hence promoting inflammation [95]. Undoubtedly, excessive NET formation and poor elimination mechanisms of NETs can cause extreme tissue damage and pathology that lead to inflammatory and autoimmune pathologies [96].

There are still many unsolved essential concerns about the exact functional roles that NETs play in the etiology of various illnesses. The release of enzymes and other proteins during NETosis causes tissue injury, and EndA produced by many strains of S. pneumoniae, enables them to escape NETs by degrading their functional integrity. Buchanan et al. [52] found that increasing bacterial migration from the upper airways to the lungs was linked to nuclease digestion of the DNA scaffold of NETs, possibly contributing to a 20–30% increase in cases of invasion into the bloodstream. However, reducing this virulence factor of pathogens does not address all issues, as it is crucial to consider all possible mechanisms and their consequences. Therefore, understanding the role of bacterial nucleases in degrading NETs is important, as they can balance NET secretion and potentially serve a protective role in managing infections.

Above all, the exploration of nucleases becomes attractive. Researchers have come out with various findings to understand the mechanism of nucleases as one of the NETs’ therapeutics [97, 98]. In addition, EndA is a desirable target for antimicrobial therapies due to its function as a virulence component in pneumococcal infection. A previous study by Peterson et al. [42], using a high throughput screening assay, uncovered six small molecules that were potent EndA inhibitors. These small-molecule inhibitors were shown to be a promising starting point for the development of pharmacologic EndA probes. By doing this, the virulence of pneumococcal bacteria might be addressed, and it could be identified as a target for novel compounds to fight pneumococcal infection [42].

DNase I and anti-histone antibodies have also become exciting targets for researchers instead of drugs aimed at destroying NETs. In the beginning, the recombinant DNase I significantly facilitates the course of ARDS [98] and COPD [99] in animal model studies. Since then, it has been effectively applied to practically all models of NETosis-related disorders. For instance, Pulmozyme (Dornase alfa), a recently discovered highly pure solution of recombinant human deoxyribonuclease I (rhDNase), was commercialized by Genentech [100]. This enzyme’s selectively cleaving DNA mechanism becomes a key factor of success due to hydrolyzing the DNA in the sputum/mucus of cystic fibrosis patients, reducing lung viscosity and promoting clearance of secretions [101].

Meanwhile, the impact of rhDNase on NETs has been assessed in various investigations. According to some studies, a decrease in NETosis will decrease neutrophil infiltration and the inflammatory response [102, 103]. Findings from an alternative approach show that in septic circumstances, early concurrent treatment with DNase I and antibiotics improved survival and decreased bacteremia and organ dysfunction [104]. Kaplan et al. [105] suggested that possible combination therapy will assist in controlling NETosis. These enzymes may significantly impact human medicine in the future by providing novel therapeutic options for conditions where multi-drug resistance had otherwise caused treatment failure. However, further studies are needed to validate whether these effects are due primarily to NET inhibition.