*4.2. Microbial Agents Alter NET Formation*

Lipopolysaccharide is a key component of Gram-negative bacterial cell walls, where it maintains the structural integrity, stability and negative charge of the bacteria. LPS does not have the capacity to directly induce neutrophils to release NETs; however, there is a growing belief that LPS can activate platelets, which subsequently initiate NET release. It has recently been discovered that TLR4 is present on platelets, which is indicative of platelets having the capacity to recognize and respond to LPS from Gram-negative bacteria [76]. Early studies identified *P. gingivalis*, *Agregatibacter actinomycetemcomitans* and *Tannerella forsythia* as causative agents in periodontal disease and found them to be involved in NET-related processes [77]. NET formation is dependent on the activation of protease-activated receptor 2 (PAR2) by *P. gingivalis*-derived proteases. *P. gingivalis* is found in the oral cavity, where it is implicated in periodontal disease. Furthermore, a novel role has also been demonstrated for proteases as bacterial virulence factors antagonizing the antibacterial activity of NETs [55]. Additionally, the suggested generation of NETs in the periodontium leads to increased inflammation and can be considered another virulence strategy used by *P. gingivalis*. The presentation of intracellular self-antigens modified by gingipains may have immunological consequences, as the excessive presentation of cryptic antigens creates a developed part of systemic diseases associated with periodontitis [78]. Hirschfeld et al. indicated that some bacteria (*Propionibacterium acnes*, *Veillonella parvula* and *Streptococcus gordonii*) led to an enhancement of NET-derived DNA production, via NADPH oxidase-independent mechanisms [71]. It was previously mentioned in this review that NET formation depends strongly on the formation of ROS for its release. Periodontal bacteria produce DNases that reduce NET release levels, and pathogen colonization might increase in the periodontal tissue. Most aggressive pathogens release DNase, disseminating NET contents that lead to the liberation of their antimicrobial components in the surrounding tissue, resulting in a harmful effect on periodontal tissue [54].

As neutrophils are the major and first immune cell to reach the infected area, they are involved in the initial steps of the inflammatory response. Therefore, neutrophils are a determinant component of the immune response in periodontal status [15]. It is reasonable to assume that NET production or effectiveness in periodontitis may be reduced, a reduction in the effectiveness of the NET function would allow easier bacterial infiltration of periodontal tissues, leading to more inflammatory response in the infected area and resulting in tissue destruction. The digestion of NETs via DNase leads to the liberation of NET-associated antimicrobial peptides, which in turn leads to more tissue destruction [44].

#### *4.3. Defective Neutrophils and Impaired NET Formation in Periodontitis*

Previous evidence has already shown that neutrophils show hyperactivity to bacterial species found in subgingival plaque and an upregulated ROS release [15,79]. Neutrophils in healthy periodontal tissue are moved towards dental biofilms, in which they are stimulated by oral bacteria and their components to form NETs. The migrated oral neutrophil is a viable cell with a hyperactive phenotype, as evidenced by the increased adhesion and internalization of microbes and 13 times more NET formation capacity than the circulating neutrophils [18]. In 2017, Hirschfeld et al. suggested that the variability in neutrophils, such as deficiencies in the number or abnormal function of neutrophils toward various bacteria, might contribute to the pathogenesis of periodontal disease [71]. Periodontitis patients presented with over four times higher oral neutrophil counts compared to healthy periodontal tissue, which was a predictor for protease activity. More oral neutrophils were apoptotic in periodontitis patients than in healthy ones [80,81]. The neutrophil-mediated antimicrobial action fails to stop the bacteria in cases of periodontitis, leading to tissue damage and destruction of both bacterial and immune origin. NET formation is also considered a potential factor changing the influence of the individual course of periodontitis [82]. Periodontitis in Papillon–Lefèvre (PLS) syndrome arises from the failure to eliminate periodontal pathogens because of cathepsin C deficiency [83]. PLS neutrophils reduced the capacity for NET production, characterized by the absence of the NET-related proteins such as chorionic gonadotropin, MPO and NE. ROS formation was higher in PLS [72]. The failure of activities of neutrophil antimicrobial proteins might maintain the stimulus for the wrongful recruiting of highly responsive neutrophils in periodontal tissues, providing a reasonable explanation for the acute inflammation and bone loss that characterize PLS periodontitis patients [84,85]. Interestingly, individuals with PLS do not suffer any systemic infections—rarely are there any skin abscesses. Therefore, the defects of neutrophils appear to be localized in areas of the human body more susceptible to a direct and chronic bacterial challenge, such as the oral cavity [86].

This hyper-reactivity may come from the excessive NET formation in response to periodontal pathogens and/or local mediators [66]. The implication of the neutrophils and their enzymes is supported by the fact that high levels of NETs remain in the tissue for an extended period. In addition, this supports the hypothesis that NET formation is dependent on ROS formation, which has been shown to be higher in periodontitis [15]. The neutrophil function in periodontitis may be a key determinant of the patient's periodontal health status.

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

In addition, increased neutrophil ROS formation is associated with elevated IFNα levels in periodontitis, indicating that this class of signaling proteins is also important in NET formation [75,87]. High levels of NETs within periodontal tissue could stimulate an autoimmune response, resulting in augmented neutrophil levels and causing more tissue destruction [73]. This hypothesis of NETs' hyperactivity in periodontitis is supported by Vitkov et al. They investigated NETs in exudate samples from the gingiva of periodontitis patients and compared the results with previous examinations of abscesses. In addition, they found that the samples collected had high levels of NETs and that in seven samples 22 trapped bacteria were associated with the NETs. In addition, based on the use of electron microscopy and analysis of gingival biopsies, patients with chronic periodontitis showed the presence of NETs [69]. In a recent study from the same authors, they hypothesize that there is a dissemination phenomenon of bacterial species, LPS and antigens citrullinated by NETs from the infected periodontal tissue. This dissemination could contribute to exacerbated autoimmune diseases such as RA via the activation of TLR receptors [78]. Therefore, both mechanisms of NET formation may be responsible for tissue destruction [88]. The impaired degradation of NETs and the escape of pathogens from the effect of NETs by virulence factors leads to a response from neutrophils, upregulating the release of NETs, resulting in the immobilization and localization of neutrophils instead of trapping bacteria, which leads to tissue destruction [34] (Figure 3).

**Figure 3.** NET release. NET formation may be one of the main neutrophil functions in periodontal tissue. NET production starts with chromatin decondensation, which is then embedded with cytoplasmic antimicrobial peptide granules. NETs are then released into the extracellular space after cell membrane rupture to exert their antimicrobial effect and later removed from the tissue. If NET removal fails, persistent high levels of NETs could cause damage to periodontal tissues.

#### **5. Role of NETs in Systemic Diseases**

The formation of NETs could promote thrombosis via histones [61]. NETs in combination with platelets may damage the blood vessels during sepsis, destroying endothelial cells and causing vascular occlusions [76,89]. On the other hand, it has been reported that NETs might promote the implantation of metastases through the uptake of circulating malignant cells [90]. Garley et al. indicated in 2018 that the neutrophils of patients with oral inflammation with stage I/II cancer produce increased formation of NETs compared to the neutrophils of healthy humans. However, the amount of NETs in stage III/IV cancer patients was lower than the amount of NETs in inflammation and early-stage cancer patients [91].

NETs have been described as a source of auto-antigens in various autoimmune diseases, such as vasculitis, lupus, psoriasis and RA [92]. NETs exhibit proteins normally restricted to the interior of the granules, nucleus or cytoplasm. This exposure would result in immunization against self-antigens and create autoimmune disorders. For example, in anti-neutrophil cytoplasmic antibody-associated vasculitis, proteinase 3 and MPO are self-antigens targeted by auto-antibodies, and these two enzymes are associated with NETs [93]. NETs have also been shown to have adverse effects in pre-eclampsia, where placenta-derived cytokine-activated neutrophils activated NET extrusion. NETs were found in the intervillous space of placental tissue samples [94]. In atherosclerosis, dendritic cell activation by NETs is similar to that which occurs in lupus, and these dendritic cells are one of the cell populations found in atheromatous plaques [95].

#### *The Relationship between Rheumatoid Arthritis, Periodontitis and NETs*

Periodontitis and RA are considered to be two chronic inflammatory diseases with a common pathogenesis. RA is an autoimmune inflammatory disease defined by the destruction and inflammation of joints and internal organs in which citrullination is a central feature leading to the generation of auto-antibodies to citrullinated protein antigens. In periodontitis, citrullination either by NET formation or *P. gingivalis*-derived peptydil arginine deiminase activity has been suggested [96], although citrullination independent of oral bacteria has also been reported [97]. Evidence has suggested that citrullinated antigens in RA are mostly derived from NETs [98]. Patients with periodontitis may have RA and vice versa [99]. A recent publication has suggested that periodontal indices such as gingival index, probing pocket depth (PPD) and bleeding on probing (BOP) have positive relationships with RA. Anti-*P. gingivalis* antibody levels were associated with BOP, PPD and GI and the severity of periodontitis; thus, increasing the values of periodontitis indices could be a sign of advanced disease development in RA patients [100]. In addition, a high level of anti-*P. gingivalis* antibody could be regarded as a warning sign in RA patients suffering from periodontitis [101]. Non-surgical periodontal treatment has shown to improve symptoms in both diseases [102–104]. Previous studies demonstrated that NETs were increased in the synovial fluid, rheumatoid nodules, peripheral blood and skin of RA patients [92]. Increased NET formation in the oral cavity of periodontitis patients perhaps plays a part in the initiation of RA [105]. *P. gingivalis* is the most important pathogen responsible for periodontitis. Further, it was shown that *P. gingivalis* could induce NET generation [55]. Interestingly, a study has demonstrated that patients with periodontitis and RA showed significantly higher serum levels of NETs than the control group. Furthermore, a periodontal cure remarkably decreased the serum levels of NETs in patients with RA and periodontitis [65]. However, more studies are required with a greater number of cases and a longer evolution time in order to understand the relation between the two diseases.

#### **6. Conclusions and Future Research Lines**

NETs trap and/or kill a wide variety of microorganisms, bacteria, fungi and parasites through their antimicrobial agents, such as MPO, NE and proteinase. NET formation has been associated with different diseases, such as inflammatory diseases including periodontitis and autoimmune diseases such as RA. Excess formation of NETs can be harmful to periodontal tissue if they are not correctly removed; consequently, increased NET degradation has been reported following periodontal treatment. As stated by a recently published consensus document, several areas regarding the study of NETs are still controversial. Specifically, the origin of the DNA found in NETs should be identified in order to find a clear way to distinguish NET formation from other forms of programmed cell death, and to identify all the pathways that regulate NET formation, since it is very unlikely that it is mediated by a single pathway. There is also a great need for standardization of the methodologies used for the identification of NETs [37]. Finally, NETs are currently considered potential therapeutic targets. Treatment with Nupharidine, an agent purified from the plant *Nuphar lutea*, has been shown to increase NET extrusions by neutrophil-like cells by 106%. However, the authors claim that whether the increase in NET extrusion by this compound has a detrimental or protective effect on the periodontal tissues requires further in vivo research [64]. Therefore, NETs can be considered as potential therapeutic targets for periodontitis as well as for other diseases of autoimmune origin. Certainly, the role of NETs in periodontitis needs to be further studied to enable a full understanding of their role in the pathogenicity of the disease.

**Funding:** This research has received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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