**Antonio Magán-Fernández 1, Sarmad Muayad Rasheed Al-Bakri 1, Francisco O'Valle 2,3, Cristina Benavides-Reyes 4,\*, Francisco Abadía-Molina 5,6,**† **and Francisco Mesa 1,**†


Received: 3 January 2020; Accepted: 17 June 2020; Published: 19 June 2020

**Abstract:** Neutrophils are key cells of the immune system and have a decisive role in fighting foreign pathogens in infectious diseases. Neutrophil extracellular traps (NETs) consist of a mesh of DNA enclosing antimicrobial peptides and histones that are released into extracellular space following neutrophil response to a wide range of stimuli, such as pathogens, host-derived mediators and drugs. Neutrophils can remain functional after NET formation and are important for periodontal homeostasis. Periodontitis is an inflammatory multifactorial disease caused by a dysbiosis state between the gingival microbiome and the immune response of the host. The pathogenesis of periodontitis includes an immune-inflammatory component in which impaired NET formation and/or elimination can be involved, contributing to an exacerbated inflammatory reaction and to the destruction of gingival tissue. In this review, we summarize the current knowledge about the role of NETs in the pathogenesis of periodontitis.

**Keywords:** innate immunity; periodontitis; neutrophil functions; neutrophil extracellular traps

#### **1. Periodontitis**

Periodontitis is a chronic inflammatory disease that affects the tooth-supporting tissues and exhibits a wide range of clinical, microbiological and immunological manifestations. It is associated with, and caused by, a multifaceted dynamic interaction among specific infectious agents, host immune responses, hazardous environmental exposure and genetic propensity [1]. The process of developing the disease starts with the accumulation of a complex bacterial biofilm. The composition of this biofilm has been estimated in approximately 700 species [2]. This biofilm creates a coat for the dental root and its structure is capable of protecting against antimicrobial substances. In healthy subjects, there is a homeostasis between the periodontium and the host response. However, when the plaque biofilm persists in a susceptible host it generates an inflammatory reaction that causes a dysbiosis, where periodontal pathogens thrive [3]. This leads to a chronic inflammatory state, which consequentially causes the destruction of the connective tissue. The process can progress to destroying surrounding support tissues—gingiva, cementum, periodontal ligament and alveolar bone—and eventually end in the loss of the affected teeth [4].

As a result of gingivitis, the bacteria penetrate the sulcus between the gum and the tooth, and then attack the gum attachment to progress deeper along the root. During this migration, toxins produced by bacteria and consequent inflammatory reactions will irreversibly destroy the attachment and the tooth-supporting tissues. This leads to the formation of periodontal pockets, which are located between the deep periodontal tissues and the tooth and are considered to be the main clinical feature of periodontitis [5].

The frequency and severity of periodontitis increases with age, with incidence peaking around the age of 60 [6]. Periodontitis is considered the main cause of tooth loss in people older than 40, having a higher prevalence than caries [7]. A high prevalence of periodontitis has been reported, with more than 47% of adults (more than 60 million) in the USA affected, and the prevalence continues to grow every year [8,9].

Periodontal inflammation is characterized by a chronic inflammatory infiltrate of varying intensity. This infiltrate is mainly composed of lymphocytes, plasma cells and macrophages distributed in patches on the lamina propria, frequently surrounding vascular structures [10]. Neutrophils are abundant in the periodontal inflammatory-immune response infiltrate and are considered a first-line cell defense mechanism against bacterial invasion [11]. However, in a susceptible host in which neutrophils do not properly contribute to the restraint of the invading bacteria, the homeostasis between the biofilm and the host response is altered, leading to an increase in tissue destruction [12,13]. Due to this immune-inflammatory component, periodontitis has been related to several systemic diseases, including rheumatoid arthritis (RA) [14]. Previous studies have indicated that neutrophils derived from patients with periodontitis are hyperactive and have an increased activity and production of reactive oxygen species (ROS) in response to a microbial invasion [15].

#### **2. Periodontal Neutrophils**

Neutrophils are the most abundant cell type of the granulocyte family (95%) and represent 50% to 70% of the blood leukocytes [11], approximately (1–2) <sup>×</sup> 1011 neutrophils are produced daily and released from the bone marrow into the bloodstream [16]. Peripheral blood neutrophils are eventually recruited from the bloodstream into the site of the infection. Naturally present in the oral cavity, neutrophils attach to the endothelial cells through the interaction with selectin and integrin receptors; by extravasation they abandon the bloodstream and migrate from the periodontal sulcus into the oral cavity. In case of infection, neutrophils are the first of the immune cells to arrive at the site through periodontal tissues and into gingival crevices as part of normal immune control. Although neutrophils are one of the predominant immune cells present in the oral cavity, T cells in periodontal tissue constitute the prevalent immune cell type [17]. Additionally, oral neutrophils have been found to show different chemotactic and antimicrobial functions than circulating neutrophils [18,19].

Mutations in genes affecting neutrophil differentiation and egression from the bone marrow have been related to periodontitis. Severe periodontitis has been described in patients with Severe Congenital Neutropenia due to mutations in the neutrophil elastase (NE) ELA2/ELANE or the HAX1 gene (hematopoietic cell-specific Lyn substrate) 1-associated gene X1 [20]. Patients with warts, hypogammaglobulinemia, immunodeficiency and myelokathexis (WHIM) syndrome have been reported to present with severe periodontitis [21,22]. WHIM is an autosomal dominant immunodeficiency caused by mutations in the CXCR4 chemokine receptor leading to defects in neutrophil exiting from the bone marrow.

Different neutrophil defects have been described affecting all stages of neutrophil recruitment and extravasation to periodontal tissue: tethering, rolling, adhesion and endothelial transmigration [23,24]. Most notably, leukocyte adhesion deficiency-I (LAD-I) immunodeficiency, which alters neutrophil extravasation into tissues, presents with periodontitis [25]. LAD-I results from mutation in the CD18 gene [26] preventing normal integrin dimerization and leukocyte adhesion and extravasation. Endothelial cell-derived developmental endothelial locus-1 (DEL-1) inhibits neutrophil adhesion to the endothelial cells [27] thereby restraining neutrophil transmigration; consequently, both DEL-1 upregulation and deficiency have been related to periodontitis [28].

Periodontitis is associated with reduced neutrophil chemotaxis. Dysfunctional neutrophil chemotaxis may predispose patients with periodontitis to disease by increasing tissue transit times, thereby exacerbating neutrophil-mediated collateral host tissue damage [29]. The absence of tissue neutrophils due to defective recruitment and extravasation [23] can also lead to persistent periodontal inflammation and bone loss [30,31]. Both an excessive presence or absence of neutrophils in the tissue can lead to periodontitis, indicating how important neutrophil balance is in periodontal homeostasis. A comprehensive understanding of defective neutrophil behavior in periodontitis would help in the development of new therapeutic approaches.

#### **3. Neutrophil Extracellular Traps (NETs)**

### *3.1. NET Formation*

Neutrophils contribute to host defense at sites of tissue injury by patroling through the circulatory system [32]. The function of eliminating invading pathogens in periodontal tissues is mediated through ROS production, phagocytosis, extracellular and intracellular degranulation [11] and most recently neutrophil extracellular trap (NET) production. Brinkmann first described NETs as bactericidal traps, disarming and promoting the elimination of extracellular bacteria [33]. The formation of NETs involves the extrusion of nuclear chromatin into the extracellular space through the rupture of the nuclear and plasma membranes, and this extruded chromatin is embedded with cytoplasmic granule-derived proteins [34]. The term NETosis has been used in the past years to describe the combination of NET formation and neutrophil death [35]. However, concerns have been raised and the use of this term has been discouraged in some reports, and other terms such as "NET formation" or "NETotic cell death" have been proposed [36,37]. NETs are web-like structures of decondensed nuclear chromatin fibers combined with various antimicrobial compounds, including histones and antimicrobial peptides (AMPs) from azurophilic granules, specific granules and tertiary granules (gelatinase) released out of the neutrophil after activation. These AMPs were found to be effective not only against bacterial species but also against viruses, fungi and other microorganisms [38,39].

Many stimuli have been revealed to induce NET formation, such as viruses, fungi, parasites and host-derived components, such as cytokines and activated platelets [40]. Three main forms of NET formation have been identified. The classical form of NET formation is defined as a programmed cell death, different from necrosis and apoptosis, characterized by disruption of the nuclear membrane that lasts from two to four hours, which gives neutrophils the ability to fight pathogens beyond their lifespan. NET formation starts with the recognition of several stimuli (e.g., bacteria, fungi, viruses) through neutrophil receptors (such as toll-like receptors (TLRs), IgG-Fc receptors and cytokine receptors) [41]. Then, the mobilization of stored calcium ions from the endoplasmic reticulum would also be crucial for the process, the calcium being necessary for the citrullination of the histones and for the activation of protein-arginine deiminase 4 (PAD4) and the release of ROS [42]. The histone deamination by PAD4 is known as a major event in the decondensation of chromatin and the release of NET. ROS play an essential part in promoting the breakdown of the nuclear membrane. NE and deferoxamine are involved in the further decondensation of the nuclear chromatin phenomenon [43]. In addition, NE and myeloperoxidase are dismissed from azurophilic granules and then translocate into the nucleus. Then the nuclear chromatin is extruded into the extracellular space; suicidal NETosis can be recognized microscopically by the presence of disrupted neutrophils in the tissue (Figure 1). NET extrusion from cell death would cause damage of periodontal tissues through an autoimmune phenomenon [44]. However, in 2012, Pilsczek et al. offered another mechanism and stated that the neutrophils formed NETs during highly developed infection with *Staphylococcus aureus* (*S. aureus*), but the neutrophils are still viable, and have the normal function of vital neutrophils in terms of phagocytosis and other purposes. NET formation involves the use of vesicles that carry the chromatin without extracellular release of DNA [45]. This phenomenon is very rapid; it takes place between 5 and 60 min after stimulation and does not involve NADPH oxidase. In this second form, called vital NET

formation, neutrophils create NETs but there is no breakdown of the plasma or nuclear membranes [46]. More recently, NET formation from mitochondrial DNA in viable neutrophils has been described [47]; mitochondrial DNA is released instead of nuclear DNA. Mitochondrial NET formation is not related to cell death but is dependent on ROS formation [47]. Mitochondrial NETs are identified in neutrophils within 15 min when stimulated with C5a or lipopolysaccharide (LPS). These findings are not in line with those of Brinkmann et al., who stated that NET formation leads inexorably to the death of the neutrophil [48]; moreover, it is not clear whether the mitochondrial DNA content would be enough for the amount of DNA detected in the traps [40]. The mechanisms that result in the formation of NETs through the release of mitochondrial DNA or through viable cells are still unknown. Interestingly, mitochondrial NETs may be a faster antimicrobial mechanism, which allows cells to remain viable and to prevent the extrusion of phagocytosed bacteria [49], an event that to our knowledge has not yet been studied in relation with periodontitis.

**Figure 1.** Transmission electron microscopy micrograph from a gingival tissue sample with periodontitis. An emptied disrupted neutrophil alongside an intact one are shown. Scale bar, 2¦Ìm.

#### *3.2. Microbicidal E*ff*ects of NETs*

Since 2004, many studies have highlighted the ability of NETs to participate in destroying infectious agents, such as bacteria, parasites, fungi and more recently viruses. Bacteria are powerful stimuli that activate the release of NETs [50]. NETs can trap microorganisms and slow their spread from the initial site of infection, probably through the electrostatic interactions between cationic components of NETs and the anionic surface of the pathogen [48]. NETs can also inactivate the virulence factors of pathogenic microorganisms; whose function is to modify and destroy the host cells. This had already been confirmed in the first evidence on NETs, where extracellular NE as a component of NETs actively targeted bacterial virulence factors of *Shigella* spp., such as the adhesin IcsA protein and the invasion plasmid antigen B. The antimicrobial activity of NETs depends on the structure of the NETs, as it provides a high local concentration of proteins with anti-infectious activity in the direct proximity of

the trapped pathogen [33]. These proteins' proteases include enzymes such as antimicrobial peptides and lysozyme. Histones, the most abundant proteins of NETs, also possess a strong ability to kill microorganisms. NETs are involved in the elimination of Gram-positive and Gram-negative bacteria. Among Gram-positive bacteria, *S. aureus* can be destroyed by a mechanism dependent on peroxidase activity of the NET's MPO [51]. NETs can also kill Gram-negative bacteria, including *Shigella flexneri*, *Escherichia coli* and *Salmonella typhimurium* [34].

#### *3.3. Microorganisms' Strategies to Escape the Action of NETs*

Identifying strategies to escape NETs in various microorganisms highlights the long coexistence of neutrophils and infectious agents in evolution, as well as the importance of this mechanism for combating infections [50]. Among these strategies, some bacteria produce DNases and other extracellular nucleases in order to destroy the DNA backbone of NETs and therefore evade this mechanism. This has been demonstrated with *S. aureus* [52] and *Streptococcus pneumonia* (*S. Pneumonia*) [53]. DNase production has been reported by a wide range of periodontal bacterial species and this expression appeared to be a trait in species that have been classically considered as periodontal pathogens, such as species from red (*Porphyromonas gingivalis* (*P. gingivalis*) and *Tannerella forsythia* (*T. forsythia*)), orange (*Fusobacterium nucleatum* (*F. nucleatum*), *Prevotella intermedia* (*P. intermedia*) and *Prevotella nigrescens* (*P. nigrescens*)) and yellow (*Streptococcus gordonii* (*S. gordonii*)) microbial complexes. As *P. gingivalis* is one of the most important periodontal pathogens, the DNase expression of six different strains was analyzed, showing all of them had different degrees of DNase activity [54]. *P. gingivalis* is a potent inducer of NET formation that is mediated by gingipains, but its proteolytical activity has shown to inactivate the bactericidal components of NETs through the activation of protease-activated receptor-2 [55]. Several mutant and wild-type strains of *P. gingivalis* have been analyzed and their results showed that mutant strains induced a characteristic NET formation [56]. *P. intermedia* has also shown a strong nuclease activity when compared with other periodontal bacterial species, suggesting that this species could have a major role in the biofilm ability to evade the action of NETs. In the same study, another major periodontal pathogen such as *Aggregatibacter actinomycetemcomitans* (*A. actinomycetemcomitans*) showed no DNase activity [57].

### *3.4. Removal of NETs*

Many investigations about the removal of NETs have been published recently. While the investigations appreciated that the removal of NETs is essential for tissue homeostasis, the processes involved and time required in removing NETs are not well understood. In 2010, it was reported that NETs produced in vitro were stable for over 90 h. DNase 1 is one of the mechanisms responsible for NET degradation, and the presence of DNase 1 inhibitors or anti-NET antibodies that also blocked the access of the enzyme would be responsible for the removal of impaired NETs in cases of autoimmune diseases such as systemic lupus erythematosus [58]. NETs are degraded by macrophages through lysosomic action. However, the whole specific nuclease pathway involved in this process remains difficult to find. A key to this process is that the mechanism of NET removal is similar to that of apoptosis, whereby macrophages do not release pro-inflammatory cytokines [59]. Recently it has been reported that NET degradation is increased in treated periodontitis patients, what indicates that NET degradation contributes to a decreased pro-inflammatory state [60,61].

#### **4. NETs and Periodontitis**

#### *4.1. NETs in Periodontitis Studies*

In Table 1, we summarize the articles to date that have studied the role of NETs in periodontitis.


*Cells* **2020**, *9*, 1494

**Table 1.**

Summary table of the studies assessing the role of neutrophil

extracellular

 traps (NETs) in

periodontitis

 and the induction of NET formation by





With regard to previous results published by our group, we were able to characterize NETs in tissue samples with periodontitis and gingivitis using immunofluorescence, immunohistochemistry and electron microscopy analysis (Figure 2). The comparison of periodontitis and gingivitis showed that NET composition changed, and the general expression of citrullinated histone H3 was found to be higher in gingivitis. These findings suggested that the potential role of NETs in periodontitis may be associated with early and more acute phases of the inflammatory process [63].

**Figure 2.** Micrographs from gingivitis (**A**,**C**) and periodontitis (**B**,**D**) gingival tissue samples. Immunostaining of citrullinated histone H3 (**A**,**B**) and MPO (**C**,**D**) are shown. Although citrullinated histone H3 expression did not differ between gingivitis and periodontitis (**A**,**B**), a higher MPO expression in gingivitis compared to periodontitis was found. This suggested that NET formation might be more associated with gingivitis. Scale bar, 50 μm.

Previous investigations (see Table 1) showed that periodontitis led to an increased formation of ROS and NETs. In addition, interferon alpha (IFN-α) was found in significant amounts in periodontitis patients. This mediator is very important for stimulating NET formation and the periodontal pocket provides ideal O2 levels and pH for ROS formation [75]. Thus, for all the previous reasons this provides a friendly environment for ROS formation. Investigations suggest that the loss of bone and progression of disease depend on the nature of the inflammatory response of the patient and the type of pathogen.
