*Review* **Rheumatoid Arthritis-Associated Mechanisms of** *Porphyromonas gingivalis* **and** *Aggregatibacter actinomycetemcomitans*

#### **Eduardo Gómez-Bañuelos , Amarshi Mukherjee, Erika Darrah and Felipe Andrade \***

Division of Rheumatology, The Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA **\*** Correspondence: andrade@jhmi.edu; Tel.: +1-410-550-8665; Fax: +1-410-550-2072

Received: 30 July 2019; Accepted: 21 August 2019; Published: 26 August 2019

**Abstract:** Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology characterized by immune-mediated damage of synovial joints and antibodies to citrullinated antigens. Periodontal disease, a bacterial-induced inflammatory disease of the periodontium, is commonly observed in RA and has implicated periodontal pathogens as potential triggers of the disease. In particular, *Porphyromonas gingivalis* and *Aggregatibacter actinomycetemcomitans* have gained interest as microbial candidates involved in RA pathogenesis by inducing the production of citrullinated antigens. Here, we will discuss the clinical and mechanistic evidence surrounding the role of these periodontal bacteria in RA pathogenesis, which highlights a key area for the treatment and preventive interventions in RA.

**Keywords:** Rheumatoid arthritis; *Porphyromonas gingivalis*; *Aggregatibacter actinomycetemcomitans*; periodontitis; periodontal disease; citrullination; peptidylarginine deiminase; ACPA; anti-CCP

#### **1. Introduction**

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology characterized by synovial inflammation, joint destruction, and high titer autoantibodies [1]. The disease affects 0.5–1% of the adult population worldwide and is associated with increased mortality rates compared with the general population [2]. A microbial origin in RA has been hypothesized for more than a century [3–6], yet, different to chronic diseases that have an infectious origin, a causal agent for RA has not been identified. Instead, numerous studies support the idea that the etiology of RA is multifactorial with a complex interplay between genetic and environmental factors [7]. Multiple risk factors have been associated with RA, including specific HLA (human leukocyte antigen) alleles, female sex, smoking, obesity, infections, and menopause, among others [8]. Nevertheless, these factors are also common in the general population, which makes it difficult to understand why only few individuals develop RA and not others with similar risk. Even in monozygotic twins, the disease concordance is rather low, varying between 8.8 and 21% [9–12]. Although stochastic events may explain the discordance in disease development among individuals at risk, the current model of RA relies on the contribution of an environmental factor, most likely a microbial agent, which may be responsible for triggering the disease in susceptible individuals.

Assuming that a microorganism is involved in RA pathogenesis, at least two hypotheses may explain why this causal agent has not yet been found. First, RA may be triggered by a rare pathogen that is exclusive to patients with RA, which has not yet been discovered because of the lack of proper technologies. Second, different to other chronic infectious diseases that are linked to a single pathogen, RA may result from the individual or interacting effects of different microbial agents likely unified by triggering common arthritogenic pathways. Moreover, like other risk factors in RA, microbial species with arthritogenic potential may also be common in the general population, but only drive RA in the perfect setting of other predisposing elements.

While it cannot be excluded that a single unknown pathogen may cause RA, current studies suggest that dysbiotic microbiomes may play a role in the pathogenesis of the disease [13–15]. In this regard, several bacterial candidates have been mechanistically linked to RA either by creating a pro-inflammatory environment or by inducing the production of autoantibodies [13–15]. Here, we will discuss the evidence surrounding the potential role of two of these microbial agents, *Porphyromonas gingivalis* (*P. gingivalis*) and *Aggregatibacter actinomycetemcomitans* (*Aa*), in the mechanistic model of RA. Importantly, these pathogens are not exclusive to RA. Indeed, they are causal agents of periodontitis, a common disease in the adult population initially associated with RA in the late 1800s [16].

#### **2. The Focal Infection Theory, Oral Sepsis, and RA**

In order to discuss the potential role of periodontal pathogens in the etiology of RA, it is important to first review the historical background leading to the link between the mouth and RA. In contrast to other arthritides known for several centuries, the first description of RA (initially termed "primary asthenic gout") was not made until 1800 by Augustin Jacob Landré-Beauvais [17]. Later, Alfred Garrod in 1859 proposed the name "rheumatoid arthritis" for diseases previously known as chronic rheumatic arthritis and rheumatic gout, and provided a detailed clinical description to distinguish RA from gout [18]. Once RA was established as an independent entity, numerous investigators tried to identify the etiology of this disease. Due to the inflammatory nature of RA, it was thought that the etiology was infectious, leading to the search for microbial organisms in the joints of patients with RA.

Between 1887 and the early 1900s, several investigators claimed to identify different bacteria in RA synovial fluid and tissue [3–6]. Although the results were inconsistent and not reproducible [19–22], these initial studies reinforced the idea that RA had an infectious origin, either due to bacteria within the joint itself or to a toxin produced by microorganisms in some other part of the body [23]. During the late 1800s, it was widely accepted that an infection in one part of the body may have effects on a different anatomical site (e.g., the syphilitic chancre is followed by systemic infection [24]). Thus, this notion fueled speculation that similar to other chronic diseases [25], RA was the result of microbial dissemination from distant sites of chronic focus of infection, including nasal sinuses, gut, lungs, genitourinary tract and the mouth [26–30]. This idea, known as the focal infection theory, defined the cause and treatment of a broad range of diseases, including RA, for several decades in the early 1900s [25,31,32].

In 1891, Willoughy D. Miller first conjectured that oral pathogens play a significant role in the production of local and systemic diseases [33,34]. This notion was further promoted by William Hunter in 1900, who was responsible for highlighting that the mouth was a major focus of infection driving diseases in other organs of the body. In particular, he reached the incorrect conclusion that pernicious anemia was the result of infective gastritis caused by oral sepsis [35–38]. Following these observations, oral sepsis (including caries, gingivitis, stomatitis, periodontitis, and tonsillitis) was considered a major focus of infection causing systemic diseases in which the etiology was unknown, such as RA [25,30,39,40]. This notion was further supported by anecdotal reports of patients with "rheumatism" cured after the removal of carious teeth [41], leading to the use of tonsillectomies and tooth extractions as the standard of treatment [29,31]. In the 1930s, this theory was refuted by the demonstration that neither tooth extraction nor tonsillectomy provided clinical benefit to patients with RA [42–44].

#### **3. Periodontitis and RA**

Among the different causes of oral sepsis that were linked to RA, an association with "pyorrhea alveolaris" was noticed in 1895 [16]. Pyorrhea alveolaris, also known as Riggs' disease and currently termed periodontal disease (PD) or periodontitis, was described by John W. Riggs in 1875 [45]. Periodontitis is a bacterial-induced chronic inflammatory disease affecting the tissues that support the teeth, including the alveolar bone. The disease results from dysbiosis of the oral microbiota and is associated with destruction of periodontal tissue, potentially leading to tooth loss [46]. Since the

implantation site of the teeth is also an articulation, it was initially thought that the destruction of the periodontal membrane and alveolar bone in patients with RA and pyorrhea alveolaris was part of same RA-induced damage affecting the dentoalveolar joint [16]. Nevertheless, the establishment of the focal infection theory tilted the paradigm to underscore periodontitis as a cause (not as a consequence) of RA in the early 1900s [26–28].

However, after the downfall of the focal infection theory as the cause of RA in the 1930s, the study of periodontitis in the pathogenesis of RA was left behind. Later, at least two major advances renewed interest in periodontitis as a component of the mechanistic model of RA. The first occurred between the 1960s–1990s, when significant advances were made in the immunopathogenesis of periodontitis, which suggested important mechanistic similarities with autoimmune diseases [47], in particular RA [48]. These include common mechanisms of immune-mediated tissue damage, patterns of pro-inflammatory cytokines (e.g., interleukin (IL)-1, IL-6 and tumor necrosis factor α), immune complex deposition and complement activation [48–50]. Rapidly progressive periodontitis is also associated with *HLA-DRB1* alleles linked to RA [51,52]. Moreover, B cells and plasma cells predominate in the affected sites in chronic periodontitis [53–55], autoantibodies such as rheumatoid factor (RF) and anti-collagen antibodies are found in the periodontal lesion [56–59], and RF can be detected in dental periapical lesions from patients with RA [60]. The second advance was in 1999, when it was discovered that an important periodontal pathogen (*P. gingivalis*) secretes a peptidylarginine deiminase (PAD)-like enzyme [61], which was incorrectly thought to be equivalent to human PADs at the time [62]. As PAD enzymes are responsible for generating citrullinated proteins, major targets of autoantibodies in RA [63], it was hypothesized that periodontitis drives RA via the production of citrullinated antigens by *P. gingivalis* [62]. Together, these findings have provided the basis for the renewed theory that periodontitis and RA may be mechanistically related and potentially linked by a common etiologic factor.

In the last 10 years, numerous epidemiological studies, extensively reviewed elsewhere [64–66], have reported a positive association of RA with PD when compared to healthy (non-RA) controls. Overall, a recent meta-analysis found that patients with RA had a 13% greater risk of periodontitis compared to healthy controls, ranging from 4 to 23% (RR: 1.13; 95% CI: 1.04, 1.23; *p* = 0.006) [65]. In addition, a case-control study from the Medical Biobank of Northern Sweden found that periodontitis, characterized as marginal jawbone loss, precedes the clinical onset of RA [67], supporting a potential role for PD in RA pathogenesis. Not every study, however, has confirmed this association either by comparing RA with healthy controls [68,69] or with patients with osteoarthritis (OA) [65]. Although these studies have methodological differences that may explain their discrepancies, a causal relation between RA and periodontitis may be difficult to sustain based purely on association studies.

A major caveat in the epidemiological association between RA and periodontitis is that PD is likely the most frequent chronic infectious disease in humans worldwide. The overall rate of PD in the adult US population is 47%, with 38% over age 30 and 64% over age 65 having either severe or moderate periodontitis [70]. Moreover, severe forms of periodontitis affect 11.2% of the global adult population [71]. Considering that almost half of the adult population has some form of PD, it may be hard to demonstrate a causal relationship with RA, since its prevalence is only 0.5–1% of the adult population [2]. Indeed, the relative risk of periodontitis in patients with RA is only 1.13 when compared to healthy controls, and of 1.10 compared to OA [65]. Despite these potential shortcomings, additional studies have been centered on addressing whether periodontitis, and in particular periodontal pathogens, may have a mechanistic role in RA through the production of citrullinated antigens.

#### **4. Citrullination and RA**

The discovery that the majority of patients with RA have antibodies to citrullinated proteins (known as ACPAs) [63,72,73] marked an important advance in understanding potential pathogenic mechanisms in RA [1]. Citrullination is an enzymatic process mediated by the peptidylarginine deiminases (PADs) in which arginine residues are deiminated to generate citrulline residues [74]. Five PADs have been identified in humans (PAD1–4 and 6) [1], but only PAD1–4 have citrullinating activity [75]. PAD2 and PAD4 have gained prominence as potential candidates that drive citrullination of self-antigens in RA due to their increased expression in rheumatoid synovial tissue and fluid [76–78]. PADs are calcium dependent enzymes. Four, five, and six calcium-binding sites were identified in the structure of PAD1, PAD4, and PAD2, respectively, with calcium binding inducing conformational changes required to generate the active site cleft [79–81]. PADs are highly specific for peptidylarginine residues, requiring at least one additional amino acid residue N-terminal to the site of modification [74,82]. Thus, these enzymes can only citrullinate arginine residues within polypeptide chains but not at their termini (i.e., they are endodeiminases). Different from arginine deiminases (ADI), which catalyze the deimination of free L-arginine, PADs cannot generate citrulline from free L-arginine [74]. PADs 2, 3, and 4 form homodimers, whereas PAD1 is monomeric in solution [79–81]. Each PAD monomer contains a C-terminal catalytic domain and an N-terminal domain involved in substrate binding and protein–protein interactions [79–81]. The PADs are highly conserved and share 50%–55% sequence identity [79], but exhibit distinct substrate preferences and tissue expression [83,84].

Citrullination is a normal process across multiple tissues in humans [85]. More than 200 proteins are citrullinated in different healthy human tissues, with the highest levels found in the brain and lungs [85]. Together, this set of proteins is referred to as the citrullinome. Large amounts of citrullinated proteins are found in RA synovial fluid, including more than 100 proteins that are normally citrullinated among different normal tissues [85–91]. This unique pattern of citrullination that includes proteins spanning the range of molecular weights is termed hypercitrullination [87]. Similar to the RA joint, it is noteworthy that periodontitis is also characterized by the accumulation of large amounts of citrullinated proteins with similar patterns of hypercitrullination found in RA [92,93], supporting the notion that PD is a potential source of citrullinated autoantigens. Understanding which components in periodontitis are responsible for driving hypercitrullination may, therefore, provide important mechanistic insights into RA pathogenesis.

### **5.** *P. gingivalis* **in RA Pathogenesis**

*P. gingivalis* was linked to periodontal disease in the early 1960s, initially named *Bacteroides melaninogenicus*(*B. melaninogenicus*) [94], which was later found to include two species, *B. melaninogenicus* (later *Prevotela melaninogenica*) and *B. asaccharolyticus* [95]. The name *B. gingivalis* was later proposed to distinguish oral from non-oral *B. asaccharolyticus* [96,97], and in the late 1980s, *B. gingivalis* was further reclassified in a new genus, the *Porphyromonas* [98]. *P. gingivalis* has been implicated in the pathogenesis of periodontitis by subverting host immune defenses, leading to overgrowth of oral commensal bacteria, which causes inflammatory tissue destruction [99,100].

Different approaches have been used to address a potential association between *P. gingivalis* and RA, many of which have shown inconsistent or inconclusive results. These include: (1) bacterial detection in gingival tissue, subgingival plaque and/or gingival crevicular fluid (GCF) either by staining using anti-*P. gingivalis* antibodies, bacterial culture, and/or DNA amplification (PCR) [101–107]; (2) metagenomic sequencing in saliva and subgingival plaque, which surveys complex microbial communities [103,106,108–112]; and (3) measuring antibodies to *P. gingivalis* in serum [101,113–125], which in contrast to the other assays that detect the existing bacteria in the mouth, it is an indirect test that determines past or present exposure to *P. gingivalis*.

Due to its convenience, the detection of antibodies to *P. gingivalis* (mainly IgG) has been the most widely used assay to study the relationship between this pathogen and RA. However, it is important to highlight that there is not a standard assay to measure such antibodies. All studies used in house ELISAs with different types of *P. gingivalis* antigens. These include bacterial cells either as intact bacteria (not fixed or fixed with formalin) or bacterial extracts generated by sonication [101,113–119], purified recombinant proteins (e.g., bacterial PAD, arginine gingipain (RgpB), or hemin binding protein 35 (HBP35)] [120–124,126], HtpG peptides (HSP90 homologue) [111], *P. gingivalis*-specific

lipopolysaccharide or outer membrane antigens [125,127]. In regard to the bacterial strains, studies have included either laboratory strains or clinical isolates, which may have some antigenic differences [128]. Considering conflicting conclusions from these studies [101,113–125] and others not cited in this review, an association between anti-*P. gingivalis* antibodies and RA is difficult to sustain. However, based on a selected number of publications, two meta-analysis have reported a significant association between higher antibody titers to *P. gingivalis* and RA when compared to healthy controls with unknown PD status [129], and to healthy controls with and without PD [130]. Anti-*P. gingivalis* antibody levels also positively correlated with ACPAs in one study [129]. Nevertheless, a significant association was not found between antibodies to *P. gingivalis* and RA when compared to non-RA controls (e.g., population-based case-controls) [129]. This finding may be explained by the presence of individuals with chronic diseases and high prevalence of severe PD in the non-RA population, supporting the idea that the relative abundance of *P. gingivalis* is associated with PD severity regardless of RA [111]. Moreover, one meta-analysis showed that only antibodies to whole *P. gingivalis*, but not anti-RgpB antibodies, were positively associated with RA [129], highlighting that the anti-*P. gingivalis* antibody assays are not comparable. Interestingly, one study reported that anti-RgpB antibody concentrations increase before the onset of RA symptoms [123], suggesting a temporal relationship between *P. gingivalis* infection and the clinical onset of RA.

Unlike the detection of antibodies to *P. gingivalis,* which has shown some positive association with RA, a number of studies detecting *P. gingivalis* by PCR and metagenomic sequencing in saliva, subgingival plaque and/or GCF failed to confirm such an association [103–111]. In one of these studies, however, *P. gingivalis* was detected more frequently in a small subgroup of patients with recently diagnosed, never-treated RA, in comparison with patients with established RA or healthy controls; although this finding could be explained by a higher prevalence of severe periodontitis in those patients [111]. More recently, an increase in the relative abundance of *P. gingivalis* was found at healthy periodontal sites in at risk anti-CCP antibody positive individuals compared with anti-CCP negative healthy controls, though the significance of this finding is unclear [112].

From these studies, it is difficult to reach a definitive conclusion as to the relationship between RA and *P. ginigvalis*. A complicating factor is that every assay has different implications with regard to the status of *P. gingivalis* in the patient (e.g., carrier, active infection, or past and present exposure), and their significance is likely influenced by the source of antigen (in the case of antibodies), periodontal sample collection, and the control group used for comparison (e.g., OA, non-RA, healthy controls with or without PD). Overall, it is likely that positive associations with *P. gingivalis* may reflect PD severity rather than RA status, which importantly, neither supports nor excludes a pathogenic role for *P. gingivalis* in RA.

#### *5.1. P. gingivalis in Experimental Models of Arthritis*

In addition to epidemiological data, experimental studies using different animal models of immune-mediated arthritis (e.g., collagen-induced arthritis (CIA), methylated bovine serum albumin-induced arthritis, and the SKG mouse model) and different routes of bacterial inoculation (including oral, intraperitoneal and subcutaneous chambers) reached the conclusion that *P. gingivalis* can increase the incidence and/or exacerbate the disease [131–141].

However, many of these studies have important caveats. Several studies have reported that DBA/1 mice (a susceptible strain to induce CIA) are resistant to oral colonization by *P. gingivalis* and therefore, have recommended the use of non-oral routes of inoculation or different strains of mice to study the effect of *P. gingivalis* in experimental arthritis [131,136,142]. These findings contrast with others that have successfully induced PD with *P. gingivalis* in DBA/1 mice [132,134,135]. Among DBA/1 mice that developed periodontitis, however, there are also differences observed, with some studies showing that PD exacerbates CIA [132,134], but not others [135]. Using the SKG model of spontaneous arthritis, there are also discrepancies in the induction of PD by *P. gingivalis*, and whether this process aggravates arthritis [141,142]. Although these conflicting data may be explained by differences in

mouse strains or environmental conditions that may affect the immune system in mice, the data certainly highlights the lack of reproducible mouse models to study the effect of periodontitis and *P. gingivalis* in autoimmune arthritis.

Using models of arthritis in rats, periodontitis induced by *P. gingivalis* had no effect in the development or severity of pristane-induced arthritis in Dark Agouti rats [143]. More recently, however, one study showed that *P. gingivalis*-associated periodontitis was sufficient to induce erosive arthritis in Lewis rats, providing the first direct evidence that *P. gingivalis* may have the capacity to induce arthritis [144].

Several mechanisms have been proposed by which *P. gingivalis* may promote autoimmune arthritis in humans and in experimental animals. These include the induction of a systemic Th17 cell response [132–134], induction of autoantibodies [131,138,143,144], cross-reactivity between bacterial and host antigens [145], by promoting C5a generation [141], through direct dissemination of *P. gingivalis* into the joints [136], and by swallowing the bacteria, which may alter the gut microbiota and gut immune system [137]. Nevertheless, the production of citrullinated autoantigens by a PAD enzyme released from *P. gingivalis* is considered the most important mechanistic evidence to support a role of *P. gingivalis* in RA pathogenesis [14,146].

#### *5.2. P. gingivalis PAD (PPAD)*

The existence of an arginine deiminase-like enzyme in *P. gingivalis* was suggested in the early 1990's [147], and the enzyme was purified and characterized in 1999 [61]. PPAD is not evolutionarily related to mammalian PADs. Structurally, it is a close relative of agmatine deiminases, which are found across bacteria [148]. Different to mammalian PADs, PPAD does not require calcium for catalysis, it is only composed of a ~40 kDa catalytic domain, and it can convert free L-arginine to free L-citrulline [61,148,149]. In addition, PPAD only modifies C-terminal arginine residues (i.e., an exodeiminase), as those generated after peptide cleavage by arginine gingipain (Rgp) [149], which is also secreted by *P. gingivalis*. Several PPAD variants have been identified through the analysis of *P. gingivalis* genomes and clinical isolates [150]. One of these variants (termed PPAD-T2) has two-fold higher catalytic activity compared with PPAD from the reference strain (PPAD-T1) [150]. Whether PPAD variants are relevant for *P. gingivalis* pathogenesis is unknown.

PPAD is detected in the outer membrane (OM) fractions of *P. gingivalis* and as a secreted enzyme, which is found in a soluble form and in association with outer membrane vesicles (OMVs) [120,151,152]. In most clinical isolates (termed type I isolates), extracellular PPAD is mainly found in secreted OMVs and to a minor extent in a soluble form. In contrast, a small subset of clinical isolates (termed type II isolates) showed minimal levels of PPAD in OMVs and most of the enzyme in the soluble form [152]. Type II isolates are associated with a lysine residue at position 373 in PPAD [152]. The clinical significance of *P. gingivalis* type I and type II subsets in the pathogenesis of PD and RA is unknown. Secreted PPAD is believed to be a major virulence factor of *P. gingivalis* due to its capacity to generate ammonia during deimination of arginine to citrulline. Ammonia may protect *P. gingivalis* during acidic cleansing in the mouth [153], and promote periodontal infection by inhibiting neutrophil function [154,155].

#### *5.3. PPAD-Mediated Citrullination of Bacterial and Host Proteins*

A potential association between PPAD and citrullination in RA was initially suggested as a hypothesis in 2004 [62]. The first experimental evidence, published in 2010, provided two major findings [156]. The first was that clinical isolates of *P. gingivalis* were highly enriched in citrullinated proteins, suggesting that Rgp and PPAD are actively cleaving and citrullinating a large number of substrates in *P. gingivalis*. The second was that after cleavage by Rgp, PPAD citrullinates C-terminal arginine residues in fibrinogen and α-enolase, two important targets of ACPAs in RA. Together, these data provided initial evidence to suggest that bacterial and host proteins citrullinated by PPAD might initiate the loss of tolerance to citrullinated autoantigens in RA [156].

Subsequent studies, however, did not confirm the abundant citrullination initially found in *P. gingivalis* [120,128]. Indeed, although PPAD may citrullinate some proteins in *P. gingivalis*, this feature appears to be exclusive to a few bacterial strains (it was only detectable in reference strain W83 and the clinical isolate MDS45), and potential citrullination seems to be limited to only six bacterial proteins [128]. Whether C-terminal citrullinated peptides derived from these bacterial proteins are specific targets of ACPAs is unknown. Interestingly, PPAD is stable at low pH, resistant to limited proteolysis, and retains significant activity after boiling [61]. Therefore, depending on the processing of *P. gingivalis* for protein analysis, it is possible that citrullination of bacterial proteins may occur in vitro following cell lysis [120]. As an artifact, this may explain the reports of abundant citrullination (including endocitrullination) in lysates from *P. gingivalis* [121,157], as well as the finding that some monoclonal ACPAs cross-react with citrullinated outer membrane antigens (OMAs) from *P. gingivalis* lysates [157].

Regarding autoantigen citrullination by *P. gingivalis* [156], it is important to highlight that neither Rgp nor PPAD have substrate specificity. The finding that fibrinogen and α-enolase are cleaved and citrullinated by these enzymes is, therefore, not surprising, as any other protein would be predicted to undergo the same processing when exposed to these enzymes. Since cleavage and citrullination by Rgp and PPAD is not specific for RA autoantigens, additional evidence is required to support a role of PPAD in the lack of tolerance to citrullinated autoantigens. For example, this could be achieved by demonstrating that C-terminal citrullination mediated by Rgp and PPAD enhances immunoreactivity to autoantigens in RA.

#### *5.4. Self-Endocitrullination of Pro-PPAD*

PPAD is expressed as a pro-enzyme (thereafter pro-PPAD) that contains four domains: an N-terminal pro-peptide, a catalytic domain, an immunoglobulin-superfamily (IgSF) domain and a C-terminal domain (CTD) [148,149]. The mature enzyme only contains the catalytic and IgSF domains [148]. N-terminal processing appears to maintain enzyme activity and stability [120,158], while C-terminal cleavage is required for cell surface translocation of PPAD [159,160]. During the production and immunogenic analysis of recombinant pro-PPAD, it was noticed that the pro-enzyme undergoes self-endocitrullination when expressed in *E. coli,* and that this modified protein was preferentially recognized by RA sera when compared with the native pro-enzyme [121]. Using 13 cyclic PPAD peptides (termed CPP1-CPP13, which should not be confused with cyclic citrullinated peptides or CCPs) encompassing 18 potential citrullination sites in pro-PPAD, the study further identified immunodominant endocitrullinated peptides recognized by RA antibodies [121]. Although these results were surprising, because PPAD has no endocitrullination activity, it was assumed that these findings were analogous to PPAD from *P. gingivalis*. Thus, the data were interpreted to suggest that loss of tolerance to citrullinated proteins in RA may originate from an antimicrobial immune response directed against citrullinated PPAD [121].

Nevertheless, further studies demonstrated that self-endocitrullination of pro-PPAD was an artifact that resulted from the lack of N-terminal processing of the enzyme expressed in *E. coli* [120]. The absence of endocitrullination in PPAD has been additionally confirmed in structural studies using processed PPAD expressed in *E. coli* or generated in *P. gingivalis* [148,149]. In accordance with these findings, it was also demonstrated that although patients with RA have antibodies to PPAD, these antibodies have no preferential reactivity to the citrullinated pro-enzyme made in *E. coli* [120]. Together, these data demonstrated that self-endocitrullination of pro-PPAD in *E. coli* and the serum reactivity to this modified protein are unlikely to reflect the function and immunogenicity of PPAD from *P. gingivalis* [14,120,161].

Although some RA sera appear to react with cyclic citrullinated peptides derived from pro-PPAD (i.e., CPPs) [121,162], it is unclear whether these peptides are recognized by antibodies to native PPAD or if similar to commercial CCPs, CPPs may function as non-specific targets to detect ACPAs [161]. In this regard, although it was justified that CPPs were generated without knowledge that endocitrullination of pro-PPAD in *E. coli* was an artifact, there is no scientific support to continue their use in the study of RA. Nevertheless, antibodies to CPPs are still being used as biomarkers to link PPAD self-endocitrullination and RA pathogenesis. In particular, serum reactivity against the peptide termed CPP3 has been used to define clinical associations between *P. gingivalis* and RA [122,123,163,164], and to demonstrate that *P. gingivalis* can induce antibodies to citrullinated PPAD in rats [143]. Since the significance of CPPs is unclear, the findings generated with these peptides should be taken with caution. More importantly, serum reactivity to CPPs should not be considered as a maker of citrullination induced by PPAD.

#### *5.5. PPAD in Experimental Models of Arthritis*

To establish a direct role of PPAD in the production of ACPAs in RA, several experimental models of arthritis have been used to demonstrate that *P. gingivalis* induces or exacerbates arthritis *via* PPAD-mediated autoantigen citrullination and ACPA production. However, while several studies appear to confirm this hypothesis [131,138,144], these studies also highlight important misunderstandings in the study of protein citrullination and ACPAs in RA. For example, in a model of *P. gingivalis* and CIA, citrullinated proteins were mistakenly quantified by a colorimetric method, which measures both free citrulline and peptidylcitrulline [131]. In addition, unconventional methods have been used to detect or define ACPAs. This include the quantification of IgG levels against ACPA (i.e., anti-ACPA antibodies) rather than antibodies to the citrullinated substrates themselves [138]. More recently, one study showed that *P. gingivalis* drives ACPAs (detected by the anti-CCP2 assay) and erosive arthritis in Lewis rats, providing the first evidence that this microbial agent may be arthritogenic [144]. However, it also demonstrated that the antibody response was not specific to the citrullinated forms of antigens [144].

The failure to demonstrate a role of PPAD in autoantigen citrullination and ACPA production in mice and rats is not surprising. Although these animal models express synovial citrullinated proteins and recapitulate several features of the human disease [140,165], they do not produce antibodies specific for citrullinated proteins as observed in RA [165–169]. Moreover, they have a high amount of false-positive reactivity toward citrullinated peptides [166], which is frequently misinterpreted as ACPA positivity when non-citrullinated peptide controls are not included to confirm specificity [131,139,140]. Considering these important shortcomings, even if PPAD plays a critical role in the induction of ACPAs in RA, this hypothesis will be hard to demonstrate using current models of immune-mediated arthritis. Indeed, the data from some animal models of arthritis strongly suggest that *P. gingivalis* may exacerbate or induce disease by mechanisms independent of ACPA production, which may involve the activity of PPAD as a virulence factor targeting either free L-citrulline or protein substrates [131]. Thus, although epidemiological and experimental data may suggest a potential role of *P. gingivalis* in RA pathogenesis, there is no experimental evidence to support that this process is mediated by the induction of ACPAs against host or bacterial proteins citrullinated by PPAD.

### **6.** *Aa* **in RA Pathogenesis**

#### *6.1. Infection Due to Aa*

*Aa*, initially named *Bacterium actinomycetem comitans*, is a Gram-negative oral pathobiont first described in 1912 as a co-isolate from actinomycosis lesions [170]. It was reclassified as *Actinobacillus actinomycetemcomitans* and *Haemophilus actinomycetemcomitans* [171], and finally transferred in 2006 to a new genus, the *Aggregatibacter* [172]. For many years, it was thought that *Aa* was unable to cause infection, except in conjunction with *Actinomyces* [173,174]. This idea was initially supported by the finding that injection of a pure culture of *Aa* in the skin of a normal individual produced no infection [174]. In the 1960's, however, several cases of *Aa* infection not associated with actinomycosis were reported, which were mainly associated with endocarditis [175–177]. *Aa* is now considered part of the HACEK (*Haemophilus*, *Aggregatibacter* (previously *Actinobacillus*), *Cardiobacterium*, *Eikenella*, *Kingella*) group of bacteria that are an unusual cause of infective endocarditis [178]. In addition, it is

recognized as a cause of serious infections that can affect almost any organ, although these are very uncommon [179,180].

Although *Aa* is a rare cause of infection, this bacterium has attracted special attention since the 1980s because of its strong association with severe forms of periodontitis [181–185], both localized aggressive periodontitis (LAP) and chronic periodontitis [181,182,186–195]. *Aa* expresses several virulence factors [196]. Among these, the production of leukotoxin A (LtxA) is considered the major pathogenic component in the progression of aggressive periodontitis [195]. LtxA is a member of the repeats-in-toxin (RTX) family of pore-forming proteins [197]. Lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and αXβ<sup>2</sup> (CD11c/CD18) act as receptors for LtxA (CD18 harbors the major binding site for LtxA), which accounts for the selective killing of human leukocytes [198]. By secreting LtxA, *Aa* induces cytolysis of target cells, thereby disabling host immune defenses and permitting escape from immune surveillance. *Aa* isolates exhibit variable virulence potential [195]. Strains of the serotype b JP2 genotype, characterized by a 530-bp deletion in the promoter region of the leukotoxin operon, has been shown to be highly virulent [199,200]. This genotype, which expresses 10- to 20-fold-higher levels of LtxA due to deletion of a transcriptional terminator [201], is associated with LAP primarily in subjects of African descent [202]. A more detailed analysis of the pathogenic mechanisms associated with the induction of periodontitis by *Aa* have been reviewed recently. [203]

#### *6.2. Aa-Induced Hypercitrullination and the Production Citrullinated RA Autoantigens*

A role of *Aa* in the pathogenesis of RA was suggested in 2016, during the search for periodontal pathogens that may explain the presence of hypercitrullination in periodontitis [92]. The study of gingival tissue from patients with PD using anti-citrulline antibodies provided initial evidence that protein citrullination is increased in PD [204,205], supporting the idea that periodontitis is a potential source of citrullinated autoantigens. In addition, the data suggested the possibility that this process was linked to the activity of PPAD [205]. However, although further analysis by mass spectrometry (MS) of GCF confirmed that protein citrullination is increased in PD, this approach also provided two novel findings [92]. First, GCF from patients with PD is highly enriched in citrullinated proteins, mimicking patterns of cellular hypercitrullination found in the rheumatoid joint [92]. Second, peptide spectra of citrullinated proteins in periodontitis were consistent with endocitrullination [92], suggesting that an abnormal activation of host PADs, rather than PPAD, may be responsible for this process. Importantly, the presence of endocitrullination in periodontitis has been further confirmed by MS in GCF and periodontal tissue from patients with PD, both with and without RA [93].

Among different microbial species associated with severe periodontitis, in vitro studies identified *Aa* as the only pathogen that could reproduce the citrullinome found in periodontitis and RA, including the production of well-known targets of ACPAs [92]. In contrast to *P. gingivalis*, however, *Aa* does not encode a PAD-like enzyme. Instead, *Aa* drives citrullination by hyperactivating host PADs through the activity of LtxA. This toxin targets neutrophils, which are enriched in PAD enzymes and constitute the major immune cells in PD and the RA joint [206–208]. Since PADs are calcium dependent, the prominent calcium influx generated by the lytic effect of LtxA hyperactivates these enzymes, driving global hypercitrullination of a wide range of cellular proteins [92]. During this process, cell membrane integrity is eventually lost and membrane rupture occurs with release of citrullinated contents into the extracellular space [92]. Interestingly, this process is similar to the induction of hypercitrullination by host immune-effector pathways (i.e., perforin and complement) in the RA joint [87], suggesting that membranolytic damage by pore-forming proteins may be a unifying mechanism in the production of citrullinated autoantigens [209]. The form of cell death induced by these pore-forming mechanisms has recently been named leukotoxic hypercitrullination (LTH) [209], to distinguish it from other forms of neutrophil death that do not induce hypercitrullination.

#### *6.3. Aa Exposure and RA Pathogenesis*

Similar to *P. gingivalis*, epidemiological studies to identify an association between *Aa* and RA have been done by detecting antibodies in serum. In particular, IgG antibodies to LtxA (used as markers of *Aa* infection) have been measured in two large cohorts of patients with RA [92,210]. In one study that included patients with established RA, anti-LtxA antibodies were significantly associated with RA when compared to healthy individuals without PD [92]. Similarly, anti-LtxA antibodies were associated with chronic periodontitis in patients without RA, and the strongest association was observed in individuals with severe periodontitis [92]. Antibodies to LtxA may, therefore, identify a subgroup of RA patients with moderate to severe PD. Interestingly, this study also found that the association between *HLA-DRB1* susceptibility alleles (known as shared epitope alleles) and RA autoantibodies was restricted to patients who had evidence of *Aa* exposure. This suggested that in susceptible individuals, LtxA-induced hypercitrullination may play a role in ACPA production [92]. In support of this hypothesis, ACPA production and RA-like symptoms were recently reported in a patient with *Aa* endocarditis who had strong a genetic susceptibility to RA conferred by three HLA alleles linked to ACPA-positive RA [211].

In a different study using a large cohort of patients with early RA, anti-LtxA antibodies were also significantly enriched in RA compared to healthy controls and patients with other inflammatory arthritides (in both groups PD status was unknown) [210], supporting the high prevalence of *Aa* exposure in RA, both in early and established disease. However, this study found that the interaction between *HLA-DRB1*-SE and anti-CCP positivity was not exclusive to anti-LtxA-positive patients with RA [210]. Major differences among these cohorts may explain this discrepancy [212].

Analogous to *P. gingivalis*, metagenomic sequencing and PCR analysis in subgingival plaque have not found a significant association between the presence of *Aa* and RA [104,105,108,110]. In one study, however, analysis of GCF using commercial DNA probes (micro-Ident) identified *Aa* as the only periodontal pathogen showing significant differences between RA and non-RA controls (both with and without PD) [107]. Thus, similar to *P. gingivalis*, positive associations with *Aa* likely indicate PD severity irrespective of RA status.

Few studies have explored the potential role of *Aa* in experimental arthritis [142,213]. However, defining the causal effect of *Aa* in animal models of arthritis has the same limitations as those described for the study of *P. gingivalis*. Moreover, LtxA is known to have activity against leukocytes in primates, with no toxicity on rodent cells [214]. Therefore, different to the human model, any effect that *Aa* may have in the induction of experimental arthritis in rodents is unlikely to be driven by LtxA-induced hypercitrullination and the production of ACPAs.

#### **7.** *P. gingivalis* **and** *Aa* **in the Mechanistic Model of RA: Causal Agents, Risk Factors, Disease Modulators, or Research Distractors**

Assuming that periodontitis has a mechanistic role in RA, *P. gingivalis* and *Aa* may influence disease pathogenesis at different phases of RA development. The production of autoantibodies is an early and asymptomatic event that precedes the clinical onset of RA by several years [215,216]. The presence of a chronic and amplifying immune response against bacterial-induced RA autoantigens, in particular citrullinated antigens is, therefore, one of the most attractive hypothesis to explain disease initiation and potentially a causal association. In this context, this model explains why there is so much interest in establishing a causal relationship between *P. gingivalis* and the production of ACPAs in either experimental arthritis or RA. Although the current evidence neither demonstrate nor exclude the possibility that PPAD generates citrullinated autoantigens, other possibilities may explain a potential role of *P. gingivalis* in RA pathogenesis. Interestingly, protein citrullination is a process that is increased during inflammation [217]. However, while transient inflammation may not be sufficient to initiate an immune response to citrullinated proteins, periodontitis is a chronic non-fatal illnesses that can start during adolescence and progress over decades [218]. During this time, it is possible that some individuals at risk may develop autoantibodies to citrullinated proteins. *P. gingivalis,* a keystone

microorganism in periodontitis, is certainly a strong candidate for promoting chronic inflammation [100], which may indirectly drive the production of autoantibodies in susceptible individuals.

In contrast to *P. gingivalis*, a potential role of *Aa* in the lack of tolerance to RA-associated autoantigens involves the induction of lytic hypercitrullination [92]. In this regard, different factors such as *Aa* virulence (i.e., LtxA production) and bacterial load may define the risk of developing autoantibodies in the context of *Aa* infection in susceptible individuals [92,211]. Importantly, as other bacterial species also produce pore-forming toxins [219], it is possible that other pathogens may induce hypercitrullination and ACPAs at other mucosal sites, including the lung and gut [1,209]. LTH as a mechanism of autoantigen production by different pathogens may explain why every patient with seropositive RA does not have exposure to *Aa* and/or PD.

Interestingly, patients with RA show abnormalities in central and peripheral B cell tolerance checkpoints [220–222], likely due to an intrinsic genetic predisposition [223,224]. This results in the accumulation of autoreactive and polyreactive B cells that can recognize immunoglobulins and citrullinated peptides [220,221]. Depending on genetic and environmental risk factors, autoreactive or polyreactive naïve B cells may have a selective advantage to develop into high affinity autoreactive memory B cells through somatic mutations and affinity maturation. In this scenario, autoantigens generated by arthritogenic bacteria may promote the expansion and maturation of already existing autoreactive cells, rather than initiating the loss of tolerance to host antigens. A prerequisite of having autoreactive/polyreactive B cells to induce pathogenic autoantibodies by *P. gingivalis* or *Aa* may explain why only a limited number of individuals with PD may develop RA.

Although the hypothesis that periodontal pathogens (e.g., *P. gingivalis* or *Aa*) are causal agents of RA is an attractive idea, it is also possible that these bacteria may only be relevant as risk factors for the development of ACPA-positive RA. Alternatively, once RA has been established, persistent low-grade inflammation associated with PD may modulate RA progression and severity. Lastly, despite the enormous enthusiasm of demonstrating that periodontitis is relevant for RA pathogenesis, it cannot yet be excluded that patients with RA may have a higher risk of developing PD (even during the pre-clinical phase of RA), but neither periodontitis, *P. gingivalis*, nor *Aa* may play a pathogenic role in the disease.

#### **8. Therapeutic Implications**

The possibility of identifying a factor that triggers a multifactorial disease, such as RA, has critical implications for treatment and preventive interventions. The success of any therapy, however, may depend on targeting such a factor at the correct time during the evolution of the disease [225]. In this regard, although a systematic review and meta-analysis suggested that periodontal treatment may decrease some markers of disease activity in RA [226], these findings remain controversial [227]. The lack of an efficient response to PD treatment on RA disease activity may suggest that periodontitis has no pathogenic role in RA, or that it is most important for disease initiation, during the pre-clinical phase, and that treatment during established disease is no longer effective. If a periodontal pathogen is relevant for the initial breech of tolerance to arthritogenic autoantigens, aiming to treat asymptomatic autoantibody positive (e.g., anti-CCP, RF, or any other autoantibody) individuals may still be too late to stop the progression to symptomatic RA. In this scenario, preventive therapies (e.g., oral hygiene and potentially vaccines against periodontal pathogens) should be initiated in at-risk individuals prior to the development of these serologies. In contrast, if periodontitis is important for the amplification of the autoimmune response in preclinical RA, there is a window of opportunity to target seropositive asymptomatic individuals. Indeed, to date, this is the most feasible hypothesis that could be therapeutically addressed. However, if periodontal pathogens only play a role in the induction of specific subtypes of autoantibodies (e.g., ACPAs), eradication of these agents during the pre-clinical phase of the disease may only decrease the risk of developing certain autoantibodies (and potentially further RA severity), but may not change the course of the disease, including the production of antibodies to other autoantigens. Interestingly, recent evidence suggest that periodontitis is a potential

source of proteins modified by malondialdehyde-acetaldehyde adducts and carbamylation [228], which are also common targets of autoantibodies in RA. [229] Thus, independent of the periodontal pathogen, preventing, or targeting periodontitis may be the best exploratory option to prevent RA.

#### **9. Conclusions**

Although some epidemiological and mechanistic data supports a role for *P. gingivalis* and *Aa* in the pathogenic model of RA, it is important to underscore that these oral pathobionts do not fulfill the Henle-Koch's postulates of causation [230–232]. These microbial agents neither occur in every case of the disease, nor are specific for patients with RA. In the context of inducing ACPA production in vivo to satisfy the third postulate, it will require the use of animal models that can truly reproduce the autoantibody response observed in RA. Considering these shortcomings, demonstrating or discarding a role of *P. gingivalis* and *Aa* in the pathogenesis of RA may require a different view of how to define disease causality by these microbial agents [212].

In the case of *P. gingivalis*, it is imperative to recognize that its potential role in RA involves elucidation of at least three different questions. The first is whether *P. gingivalis* is relevant for RA pathogenesis, either by initiating or exacerbating the disease. The second is whether *P. gingivalis* is important for RA because of the production of PPAD or because it plays a critical role in the induction of periodontitis by inciting inflammation. Indeed, it appears that ligation-induced periodontitis (without additional infection with *P. gingivalis*) is sufficient to exacerbate CIA in Wistar rats [233]. The third is whether PPAD drives RA due to the production of citrullinated antigens and the induction of ACPAs in the host or because this enzyme is a virulence factor that facilitates bacterial growth and the establishment of PD. Although these questions have been somewhat addressed, definitive conclusions cannot be reached from the available data. Defining the role of PPAD in the production of citrullinated antigens and whether C-terminal deimination offers an advantage for peptide immunogenicity remains a high priority. Importantly, other potential arthritogenic mechanisms induced by *P. gingivalis*, besides autoantigen citrullination, should be kept in consideration.

In the case of *Aa*, this model offers an advantage over other periodontal bacteria because of its capacity to induce neutrophil hypercitrullination and the release of citrullinated autoantigens through osmotic lysis [92]. However, the number of studies linking *Aa* with RA are too limited to truly determine its pathogenic significance for the disease. Similar to *P. gingivalis*, rigorous studies are necessary to support or refute the role of *Aa* in the etiology of RA. Together, these oral pathobionts pose an opportunity to understand whether bacterial-associated citrullination is a mechanism involved in RA pathogenesis. This has important implications for the implementation of preventive interventions in RA.

**Author Contributions:** Conceptualization and writing—original draft preparation: F.A.; writing—review and editing: E.G.-B., A.M., and E.D.

**Funding:** This research was funded by the Jerome L. Greene Foundation, Rheumatology Research Foundation, and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) at the National Institutes of Health (NIH) grant number R01 AR069569. The content of this paper is solely the responsibility of the author and does not represent the official views of the NIAMS or the NIH.

**Conflicts of Interest:** F.A. and E.D. are authors on issued patent no. 8,975,033, entitled "Human autoantibodies specific for PAD3 which are cross-reactive with PAD4 and their use in the diagnosis and treatment of rheumatoid arthritis and related diseases" and on provisional patent no. 62/481,158 entitled "Anti-PAD2 antibody for treating and evaluating rheumatoid arthritis". F.A. serves as consultant for Bristol-Myers Squibb. E.D. received a research grant from Pfizer and from Bristol-Myers Squibb. E.D. previously served on the scientific advisory board for Padlock Therapeutics, Inc. The remaining authors declare that they have no conflicts of interests.

#### **References**

1. Darrah, E.; Andrade, F. Rheumatoid arthritis and citrullination. *Curr. Opin. Rheumatol.* **2018**, *30*, 72–78. [CrossRef]


Antibodies with Elevated Immune Responses to Prevotella intermedia in a Subgroup of Patients With Rheumatoid Arthritis and Periodontitis. *Arthritis Rheumatol.* **2017**, *69*, 2303–2313. [CrossRef]


to Biological Disease-Modifying Antirheumatic Drug in Rheumatoid Arthritis. *PLoS ONE* **2016**, *11*, e0154182. [CrossRef]


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Periodontitis: A Multifaceted Disease of Tooth-Supporting Tissues**

#### **Eija Könönen 1,2,\*, Mervi Gursoy <sup>1</sup> and Ulvi Kahraman Gursoy <sup>1</sup>**


Received: 28 June 2019; Accepted: 29 July 2019; Published: 31 July 2019

**Abstract:** Periodontitis is an infection-driven inflammatory disease in which the composition of biofilms plays a significant role. Dental plaque accumulation at the gingival margin initiates an inflammatory response that, in turn, causes microbial alterations and may lead to drastic consequences in the periodontium of susceptible individuals. Chronic inflammation affects the gingiva and can proceed to periodontitis, which characteristically results in irreversible loss of attachment and alveolar bone. Periodontitis appears typically in adult-aged populations, but young individuals can also experience it and its harmful outcome. Advanced disease is the major cause of tooth loss in adults. In addition, periodontitis is associated with many chronic diseases and conditions affecting general health.

**Keywords:** periodontal disease; alveolar bone loss; gingiva; bacteria; biofilm; immunity; inflammation; smoking

#### **1. Introduction**

Periodontitis is an infection-driven inflammatory disease in tooth-supporting tissues (i.e., the periodontium). Moreover, genetics and environmental and behavioral factors are involved in the development of the disease, the exposure of susceptible individuals to its initiation, and the speed of progression. The structure of the periodontium is diverse; it is composed of the gingiva, the underlying connective tissue, cement on the root surface, alveolar bone, and the periodontal ligament between the cementum and alveolar bone (Figure 1A,B). The junctional epithelium of the gingiva is a unique structure, located at the bottom of the gingival sulcus, which controls the constant presence of bacteria at this site. The most characteristic feature of periodontitis is the activation of osteoclastogenesis and the destruction of alveolar bone as its consequence, which is irreversible and leads to loss of tooth support.

Periodontal disease, especially its mild and moderate forms, is highly prevalent in adult-aged populations all over the world, with prevalence rates around 50% [1], while its severe form increases especially between the third and fourth decades of life, with the global prevalence being around 10% [2]. Certain demographic characteristics, such as age, gender, ethnicity, and socioeconomic status, influence the prevalence of periodontitis. Other strongly contributing factors include smoking, diabetes mellitus, metabolic syndrome, and obesity [3,4]. It is noteworthy that smoking and diabetes can expose individuals to the advanced form of periodontal disease already in adolescence and early adulthood [5–7]. There is also a strong relation of smoking to tooth loss in young individuals [8]. Severe periodontitis, the major cause of tooth loss in adults (https://www.nidcr.nih.gov/research/data-statistics/ periodontal-disease), is typically complicated by the drifting and hypermobility of teeth, eventually resulting in the collapsed bite function of an affected individual [9,10]. Moreover, periodontal disease as well as tooth loss are considered to have an association with a variety of chronic diseases and conditions affecting general health.

**Figure 1.** The anatomical structure of the periodontium in health (**A**) and in periodontitis (**B**). Abbreviations: Alveolar bone (AB), bacterial biofilm (BF), connective tissue (CT), gingiva (G), gingival sulcus (GS), inflammatory cells (IC), junctional epithelium (JE), polymorphonuclear neutrophils (PMN), periodontal ligament (PL), periodontal pocket (PP), root cementum (RC), and tooth (T).

Even in periodontal health, immune cells are constantly present in the gingiva, thus supporting the balance between oral biofilms and the host [11]. This constant communication keeps the immune response active, being a reciprocal, synergistic, and dynamic interaction. In the periodontium, the immune response carries characteristics of that of any other part of the body; the first action against microbes is due to non-specific innate response, while extended pathogenic challenge activates specific adaptive responses.

Excessive dental plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species [12]. The presence of periodontal species with pathogenic potential in the gingival sulcus initiates an inflammatory response in gingival tissue. When allowed to become chronic, this can have drastic consequences in the periodontium of susceptible individuals. Interactions between the components and metabolic activities of the oral microbiota and the host either support the balance (homeostasis) or result in disturbance (dysbiosis) within the microbiota [12]. Periodontal health-associated commensals are important in protecting the balance, for example, by inhibiting the growth of periodontitis-associated pathogens. However, qualitative and quantitative alterations within subgingival biofilms can result in disrupted homeostasis, which consequently can lead to the onset of disease with various degrees of periodontal tissue destruction.

#### **2. Pathogenic Biofilms**

Multispecies biofilm formation and maturation occur on tooth surfaces via intergeneric interactions, where coaggregations occur between different bacterial taxa, and highly diverse bacterial communities are formed at supragingival (above the gumline) and subgingival (below the gumline) sites [13,14]. *Fusobacterium nucleatum*, which belongs to the core anaerobic microbiota of the oral cavity from early years of life onwards [15,16], is seen as an important bridging organism of maturing dental biofilms, allowing late-colonizing species with virulent properties to be colonized [13]. This gradual maturation and shifts in the microbial composition influence the pathogenicity of subgingival biofilms where metabolically highly specialized microorganisms function in physical proximity as interactive microbial communities [12]. Focusing on only its adherence capabilities may lead to the underestimation of other important virulence characteristics of *F. nucleatum*, a relevant bacterium in the initiation and progression of periodontal disease. In a biofilm, this obligate anaerobe can survive and increase its numbers in aerobic environments [17]. Indeed, recent evidence indicates that *F. nucleatum* induces an environmental change through hypoxia [18], which can support the colonization of anaerobic pathogens in dental biofilms. The effects of *F. nucleatum*-induced hypoxia are not limited to the shifts

in the biofilm composition—this hypoxia also directs endothelial cells to an inflammatory state and activates angiogenesis [18].

In subgingival biofilms, anaerobic gram-negative species with their biologically active lipopolysaccharide (LPS)-containing cell wall structure may be essential in awakening the inflammatory reaction in the gingiva and culprits of periodontal destruction to occur in periodontitis-susceptible individuals [11]. Subgingival plaque samples collected from periodontitis patients and periodontitis-free individuals differ from each other. *Porphyromonas gingivalis*, *Tannerella forsythia*, and *Treponema denticola*, the so-called red complex, have shown the strongest association with periodontal disease [19]. In another study by using 454 pyrosequencing of 16S rRNA genes, *P. gingivalis* and *T. denticola* but also *Filifactor alocis*, a gram-positive anaerobe, formed the top three species [20]. Of these, *P. gingivalis* is suggested to be the principal pathogen in the process, causing a disturbed interplay between the subgingival biofilm and the host response [21]. Even as a minor constituent of the subgingival microbiota, it is able to severely affect the ecosystem by influencing the numbers and community organization of commensal bacteria at the site and dysregulate innate immunity pathways. *P. gingivalis*, a highly proteolytic gram-negative anaerobe, is a common recovery from deepened periodontal pockets of adult periodontitis patients. While *P. gingivalis* is rather rare in children and adolescents, its salivary carriage rates increase significantly with aging, and it is detected in the majority of the Finnish population after the age of 55 years [22]. Different from *P. gingivalis*, the carriage of *Aggregatibacter actinomycetemcomitans* was less frequent, without any connection to age. This gram-negative capnophilic coccobacillus and an established periodontal pathogen has been linked to aggressive forms of periodontal disease [23]. Besides these traditional pathogens, open-ended molecular methods have significantly enlarged the list of pathogenic species within periodontitis-associated subgingival biofilms [24].

Many periodontitis-associated species, among those *A. actinomycetemcomitans*, *P. gingivalis*, and *F. nucleatum*, or even polymicrobial aggregates, are capable of invading periodontal tissues [25–27], thus evading many defense mechanisms of the host. This, in turn, has an impact on the persistence of inflammation and progression of periodontal tissue destruction.

For periodontal disease to occur, it is not the presence of a single periodontal pathogen, but the interplay between the composition of the subgingival biofilm and the host response where host factors and specific niches play an important role. In dysbiotic biofilms, there is abundance of immunostimulatory pathobionts and their virulence factors, but also a reduced inhibitory effect of commensal bacteria, thus resulting in an increased inflammatory response [28,29]. In gingival epithelia, cellular responses are especially elicited against polymicrobial biofilms due to their interbacterial metabolic and virulence synergisms [30], leading the way to initial pocket formation and attachment loss. Deepening periodontal pockets with an anaerobic environment, inflammatory conditions, and a large amount of substrates originating from tissue destruction all favor the growth of inflammophilic periodontal pathogens and pathobionts [21]. Notably, daily smoking contributes to further disturbances in the subgingival microbiota, facilitating an abundance of periodontal pathogens and the reduction of beneficial commensals, thus exposing smokers to periodontal disease [31].

There have been intensive research activities targeting periodontitis-associated bacteria and/or the antibodies working against them, with the aim of revealing their involvement in various systemic diseases and conditions. Major periodontal pathogens raise local and systemic antibody responses; it has been shown in multivariate analyses that the main determinant of the systemic antibody response to *P. gingivalis* and *A. actinomycetemcomitans* is the carriage of the pathogen, whereas the presence or degree of periodontal disease has only a modest modifying effect [32]. High serum IgG antibodies to major periodontal pathogens act as risk factors for future cardiovascular events [33–35]. Also, the circulation of LPS of virulent gram-negative pathogens (endotoxemia) accompanied by exaggerated proinflammatory responses is connected to risk for these events [33]. Subgingival bacteria, including *P. gingivalis* and *A. actinomycetemcomitans*, have also been associated with the prevalence of prediabetes in young diabetes-free adults [36]. Interestingly, associations between bacterial measures and prediabetes

are consistently stronger than those between periodontitis and prediabetes. Furthermore, there is accumulating evidence on the role of *P. gingivalis* in rheumatoid arthritis [37,38]. A prospective study on the association between pancreatic cancer and the oral microbiome revealed that the carriage of *P. gingivalis* and *A. actinomycetemcomitans* is a subsequent risk for this highly lethal cancer type [39]. Besides the impact of *F. nucleatum* as the key organism in dental biofilms and its involvement in oral and extra-oral polymicrobial infections [40], recent research has shown its significant carcinogenic potential. Its ability to tolerate oxygen, create hypoxia, and induce an inflammatory environment may explain the role of *F. nucleatum* in the development and progression of colorectal adenocarcinoma [41].

#### **3. Immunologic Players of the Periodontium**

Due to the constant interaction with bacteria, immune cells (neutrophils, macrophages, and lymphocytes) are present in the periodontium to take part in maintaining a healthy equilibrium. Neutrophils continuously transmigrate through the junctional epithelium to gingival sulcus and release antimicrobial peptides (α-defensins) against invading bacteria, while they also stimulate adhesion and the spread of keratinocytes on the tooth surface [42]. Resident cells of the periodontium (keratinocytes, fibroblasts, dendritic cells, and osteoblasts) are not passive barriers against bacterial invasion, but they initiate innate immune response and regulate adaptive immune response [43,44]. An essential component is the complement pathway, which activates, amplifies, and synchronizes innate immune response by opsonizing and killing bacteria as well as activating mast cells, neutrophils, and macrophages of the periodontium [45].

Keratinocytes, which form the majority of the gingival epithelium, are capable of producing and secreting various immune response mediators, among them human β-defensins (hBDs), cathelicidins, proinflammatory cytokines, chemokines, and angiogenetic proteins [46,47]. In the healthy gingiva, innate response is mainly regulated by keratinocytes and neutrophils; keratinocytes secrete hBDs to protect the oral and sulcular epithelium (Figure 2A), whereas neutrophils secrete α-defensins to protect the junctional epithelium (Figure 2B). Gingival keratinocytes recognize pathogen-associated molecular patterns (PAMPs) by their pattern recognition receptors, such as toll-like receptors (TLRs). mRNA expressions of TLR 1–9 are detected in connective tissue and epithelial layers of the gingiva [48]. In addition, bacterial signaling molecules (cyclic dinucleotides and quorum signaling molecules) activate cytokine response in gingival keratinocytes [49,50]. There is also a reciprocal interaction between innate-immune proteins and keratinocytes. For example, proinflammatory interleukins (IL-1α, IL-1β, IL-6) activate the protein expression and secretion of hBDs from keratinocytes [46,51], while keratinocytes can suppress the inflammatory response by secreting monocyte chemotactic protein-induced protein-1 [52].

**Figure 2.** In a healthy gingiva, epithelial defensins (human β-defensins (hBD-2) in red color) are located in the oral (OE) and sulcular (SE) epithelia (**A**), while neutrophilic antimicrobial peptides (α-defensins in brown color) are located in the junctional epithelium (JE) and partly in connective tissue (CT) (**B**). (An original figure by U.K.G.)

Gingival connective tissue, periodontal ligament, and the organic component of the bone are formed of collagen. Fibroblasts are responsible for the synthesis of new collagen bundles and they remove the old collagen by secreting matrix metalloproteinases (MMPs). Overexpression of MMPs by gingival fibroblasts may either induce the release of cytokines and chemokines from the extracellular matrix or cleave cytokines and interrupt immune response signaling cascades [53]. The interplay between neutrophils and gingival fibroblasts is a good example of the bidirectional interactions between resident and immune cells.

Dendritic cells differ from keratinocytes and fibroblasts by acting as phagocytes and antigen-presenting cells. In a healthy environment, dendritic cells are in their immature forms and have high phagocytic capacity against invading microorganisms, but during infection they initiate a maturation process that involves their migration to lymph nodes to activate CD4<sup>+</sup> T cells [54] and promote the polarization of T-helper (Th)1, Th2, Th17, and B cells [55]. Uncontrolled upregulation of Th1 and Th17 cell pathways enhances alveolar bone loss via the induction of osteoclastogenesis [56]. There is also evidence that dendritic cells can differentiate to osteoclasts [57]; however, it is unknown how much of the bone resorption seen in periodontitis is actually induced by dendritic cell-derived osteoclasts.

Neutrophils form the primary defense system in periodontal tissues. Notably, their migration through the junctional epithelium into the gingival sulcus is a continuous process, which may differ from other organs, where transmigration is a hallmark of infection [58]. In the healthy oral cavity, neutrophil populations tend to be parainflammatory, while proinflammatory neutrophil phenotypes are present in periodontal disease [59]. Severe forms of periodontitis can be connected to diseases with neutrophil function defects, such as leukocyte adhesion deficiency 1 (LAD-1). Lack of neutrophil surveillance against bacterial infection is considered the cause of excessive periodontal degradation in neutrophil function deficiencies. However, recent evidence indicates that the absence of neutrophils in LAD-1 leads to the overproduction of IL-17, which eventually enhances the proliferation and differentiation of B cells [60]. Chronic granulomatous disease (CGD) is another genetic disease, which is characterized by defective neutrophilic respiratory burst and bacterial elimination. Although CGD patients are prone to developing bacterial and fungal infections, their periodontitis prevalence does not differ from that of the general population. In the highlights of these two examples, the presence of neutrophils in the periodontal immune response cascade seems to be more important than their ability to kill bacteria, as other phagocytes can take care of bacterial killing [60]. Interestingly, peripheral

blood neutrophils of periodontitis patients release higher levels of proinflammatory cytokines and reactive oxygen species compared to periodontally healthy individuals, and this hyperinflammatory response persists even after successful periodontal treatment [61,62].

Neutrophils have a relatively short lifespan and they are programmed to die via apoptosis. Apoptotic neutrophils are phagocytosed from tissues by macrophages and are eliminated through lymphatics (efferocytosis). Since neutrophils produce and secrete a significant number of inflammatory molecules, their removal is a hallmark of healing. In inflamed periodontal tissues, partly due to pathogenic biofilms, there is an extended recruitment of neutrophils and delayed apoptotic cell death [63]. Instead of enhanced elimination of pathogens, however, neutrophils demonstrate impaired antibacterial function with this uncontrolled and extended immune response activation [64].

Tissue macrophages derive either from circulating monocytes or from embryo-derived precursors [65]. Phenotyping them as inflammatory and resolving macrophages will define their roles in disease and health. Inflammatory macrophages produce and secrete a large group of cytokines (IL-1β, IL-23, IL-6, tumor necrosis factor (TNF)-α) and enzymes (MMPs) that take part in osteoclastogenesis and collagen degradation in periodontitis [66]. A conversion from a destructive inflammatory phenotype to a resolving and bone-forming phenotype requires both signaling molecules and the presence of apoptotic neutrophils [67]. *P. gingivalis* can reverse the conversion of inflammatory macrophages to resolving macrophages by inducing inflammatory cytokines [68]. Impaired elimination of neutrophils by macrophages and defects in the activation of resolving macrophages may in turn lead to the initiation and progression of periodontitis.

Innate immune cells present intra- and extracellular pathogens to lymphocytes. In the gingiva, the most common subset of lymphocytes is CD4<sup>+</sup> T cells, followed by CD8<sup>+</sup> T cells, which are further subgrouped as Th1, Th2, Th17, Th9, regulatory (Treg), and unconventional γδ T cells [69]. Recent evidence indicates the role of Th17 cells as one main regulator of T cell response and bone resorption in the periodontium [70]. In addition, Treg cells can limit the progression of periodontal disease without suppressing the immune response. When chronic gingivitis progresses to periodontitis, there is a shift from T cell dominance to B and plasma cells. Different types of B cells include naive B cells, memory B cells, and antibody-secreting B cells. Antibodies produced against periodontitis-associated pathogens can be found in saliva and in serum as well [32].

Finally, periodontitis is a complex disease with a nonlinear character, and its effects on immune response are rather disproportional [71]. Although knowledge about immune cell functions has considerably increased, it is still difficult to fully understand cellular interactions in periodontal disease pathogenesis due to its multicausal etiology.

#### **4. Inflammatory Process and Periodontal Tissue Destruction**

The junctional epithelium forms a unique seal between the root surface and gingiva, and its main function is to provide protection to the underlying tissues against the constant exposure of oral microbes and their by-products [72]. Various molecular factors involved in adhesion, cell–cell interactions, chemotaxis, proinflammatory cytokines, epithelial growth, MMP activation, and antimicrobial peptide production contribute to the function of the junctional epithelium. If this elegant and well-adapted defense system is overwhelmed by bacterial virulence factors (e.g., *P. gingivalis* gingipains) and prolonged inflammation (clinically seen as gingival bleeding and changes in soft tissue contour and color), the junctional epithelium migrates apically on the root surface and activates collagen destruction, which eventually leads to periodontal pocket formation [72]. It is noteworthy that although gingival inflammation is the precursor of periodontitis and a clinically relevant risk factor for disease progression, not all gingivitis lesions lead to periodontitis [73]. During periodontal pocket formation, new tissue formation by resident cells (keratinocytes, fibroblasts, osteoblasts) is suppressed, whereas tissue degradation by neutrophils, macrophages, and osteoclasts is stimulated; thus, the balance between tissue removal and regeneration is disrupted [74].

Proinflammatory cytokines (IL-1β, IL-6, IL-23, TNF-α), chemokines (IL-8), and antimicrobial peptides produced by keratinocytes, fibroblasts, and dendritic cells are chemoattractant gradients for neutrophils, which migrate into inflamed tissues and stimulate the chemotaxis of nonresident cells (macrophages, lymphocytes, plasma cells, and mast cells) to the site of infection [43,44]. Phagocytic cells mainly aim to eliminate invading pathogens by producing and secreting antimicrobial agents, reactive oxygen species, and enzymes. However, abundant tissue concentrations of collagenolytic MMPs and elastase activate the degradation of type I collagen in the connective tissue and periodontal ligament [75]. During disease, MMP-8 is the major collagenase in periodontal tissues. Irreversible periodontal destruction occurs when the inflammatory cell infiltrate, predominantly containing plasma cells, extends deeper into the connective tissue, leading to tissue damage in periodontal ligament and alveolar bone [76].

Alveolar bone resorption is the principal pathological characteristic of periodontitis. The activation of osteoclasts, multi-nucleated bone-resorbing cells, is regulated by a cascade of inflammatory proteins (cytokines) and enzymes (MMPs). IL-1β, IL-6, and TNF-α are the major proinflammatory cytokines in osteoclastogenesis activation, which is achieved by upregulating the receptor of nuclear factor-kappa ligand (RANKL) expression and inhibiting the differentiation of osteoblasts as well as decreasing osteocalcin production and new bone formation [77]. Due to the upregulated RANKL (stimulator of mature osteoclast formation) and downregulated osteoprotegerin (blocker of RANKL action), degradation of the bone is enabled to progress. MMP-1, -8, and -13 are especially involved in alveolar bone destruction by degrading type I collagen (the main type of collagen in the periodontium), while two gelatinases (MMP-2 and -9) accomplish the degradation of denatured collagen [78]. Furthermore, MMP-9 assists in osteoclast migration and MMP-13 triggers osteoclast activation, which all facilitate type I collagen degradation.

The disease development with fast or slow progress and with stable periods varies among periodontal sites and among individuals. Diagnosis of periodontitis is based on clinical and radiographic information on periodontal attachment and alveolar bone loss. In the current classification system, staging estimates the severity of the disease, while grading aims to estimate the rate of its progression, taking the known risk factors into account [10]. At the early phase of periodontal disease, the clinical signs and symptoms can be lacking or very mild. When periodontal tissue destruction proceeds, deepened pocket depths with alveolar bone loss result in tooth mobility, drifting, flaring, and finally loss of the affected tooth. In advanced cases, where several teeth are affected, these abnormalities lead to the collapse of the bite function.

#### **5. Periodontal Therapy—Impact on Oral and General Health**

The primary goal of periodontal therapy is to reduce the infectious and inflammatory challenge and to halt the progressing tissue destruction. Removal of pathogenic biofilms and suppression of inflammation can discontinue the periodontal tissue degradation; however, only limited regain of lost tissues occurs, depending on the form of tissue defects, systemic health status, and age [79]. In advanced cases, the active anti-infective treatment phase is often combined with surgery to eliminate residual pockets—with the aim of improving the ecology at periodontal sites—or sometimes with adjunctive systemic antimicrobials to reduce pathogen burden. In smokers, however, the treatment outcome is compromised, which makes smoking cessation an essential part of their periodontal therapy [80,81]. The beneficial influence of quitting may partly be due to decreased pathogen numbers and increased abundance of health-associated commensals in subgingival biofilms [82]. Although anti-infective treatment reduces total bacterial counts, proportions of periodontal pathogens, as well as the number of sites colonized with pathogens, many of the species return with time [83]. Therefore, daily oral hygiene of the patient and continuing professional supportive periodontal therapy are necessary to maintain the outcome and strengthen the long-term success of the treatment [84,85]. Moreover, patients with advanced disease and masticatory dysfunction and bite collapse due to severe tooth loss have an obvious need for complex rehabilitation of the bite function as well as esthetic

treatment. After treatment, however, periodontitis patients with prosthodontic reconstructions have still an increased risk for tooth loss, and many patient-related factors such as age, socioeconomic status, non-compliance, and diabetes are associated with abutment tooth loss [86].

Since untreated periodontitis increases systemic low-grade inflammation, another treatment goal is to improve this condition [87]. Although intensive mechanical periodontal treatment of patients with severely damaged periodontal tissues can cause an acute systemic inflammatory response and impair endothelial function, this occurs only transiently and, after six months, a significantly improved endothelial function is reached [88]. Furthermore, periodontal treatment has been shown to reduce atherosclerotic biomarkers (e.g., IL-6, TNF-α) of individuals with cardiovascular disease and/or diabetes [89] as well as to improve the glycemic status (Hba1c levels) of diabetic patients [90,91].

#### **6. Future Considerations**

Periodontal disease is multifactorial and the imbalance between tissue loss and gain can occur due to various reasons, including aggressive infection, uncontrolled chronic inflammation, weakened healing, or all of the above simultaneously. Thus, successful disease management requires an understanding of different elements of the disease at the individual level and the design of personalized treatment modalities, including immunotherapies and modulators of inflammation [92,93]. With the aid of newly developed omics technologies, these novel strategies may become available for clinicians.

**Author Contributions:** Conceptualization: E.K., U.K.G.; Writing: E.K., M.G., U.K.G.; Visualization: M.G., U.K.G. **Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


development of dental caries and periodontal diseases. Consensus report of group 1 of the joint EFP/ORCA workshop on the boundaries between caries and periodontal disease. *J. Clin. Periodontol.* **2017**, *44* (Suppl. 18), 5–11. [CrossRef] [PubMed]


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