Periodontitis

Periodontitis (PD) is a common chronic inflammatory disease that is caused by bacterial infection in the subgingival microbiome and affects the periodontal tooth-supporting tissues of select teeth or rarely the entire oral structure (gingiva, periodontal ligaments, and alveolar bone). Due to the persistence of periodontal pathogens and an imbalance of the immune response that they encode, PD is characterized by periodontal attachment loss, bone resorption, and can finally lead to tooth loss [82]. Besides tooth loss, PD can influence systemic health, when oral microorganisms enter the bloodstream by crossing damaged oral mucosa [72]. Consequently, PD could affect systemic diseases, i.e., cardiovascular disease [83], rheumatoid arthritis [84], type 2 diabetes [85], and cancer [86]. The main function of the human immune system is to differentiate between commensal bacteria, related to a commensurately oral microbiome, and pathogenic bacteria. Thus, in a healthy balance and established homeostasis, common microorganisms interact with the immune system without provoking a proinflammatory response [87].

The most likely cause for PD to occur is due to an accumulation of pathogenic bacteria on the tooth surfaces and in the gingiva, followed by inflammation [30] caused by activation of signaling pathways in PRRs [20], and thereby, generation of proinflammatory cytokines. In recent years, studies have shed light on the processing steps of these cytokines. Inter alia, this process is dependent upon an intracellular innate immune sensor, the NLRP3 inflammasome that also might influence the PD activity, in response to various bacterial, physical, and chemical agents [30,88,89].

The IL-1 family of cytokines, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), are proinflammatory cytokines, which are involved in the pathogenesis of several bone-affecting inflammatory diseases. Furthermore, they mediate bone loss when produced unbalanced. An unbalanced production is due to higher recruitment and differentiation of osteoclasts in the tissues via activation of the receptor activator of nuclear factor kappa-B ligand (RANKL) in osteoblasts [21,90]. Above all, as an osteoclastogenic factor, RANKL regulates the formation and activity of osteoclasts and upregulates alveolar bone loss [91]. It has been demonstrated that NLRP3 deficiency can significantly decrease RANKL, indicating the relevance of the NLRP3 inflammasome in supporting osteoclast-genesis in PD [20]. Studies demonstrated IL-1β to be a potential marker of this disease, due to its higher level in serum, saliva, and gingival tissue of PD patients [92,93,94]. In the gingival tissue and crevicular fluid of patients with PD, increased expression of IL-1β and IL-18 has been positively correlated with increased expression of NLRP3 mRNA [30]. Moreover, an increased expression of NLRP3 mRNA in the oral epithelium [95] and in the saliva [96] of patients has been found with the simultaneous downregulation of NLRP3 inhibitors [95,97]. Many studies demonstrated that the NLRP3 inflammasome is involved in the development of gingival inflammation and subsequent bone loss, due to an exaggerated immune response [20,30,88,95,96,98]. In a murine model with bacterial plaque-retentive ligatures placed around the teeth, Marchesan et al. [99] further support the NLRP3 upregulation in experimental periodontitis.

In response to several bacterial ligands acting as PAMPs, e.g., lipopolysaccharide (LPS) [14,100], peptidoglycan [19], bacterial and viral RNA [9,101], and flagellin [4], NLRP3 regulates the maturation and secretion of proinflammatory cytokines, like IL-1β [102] via proCASP1. Furthermore, an upregulation of the NLRP3 inflammasome complex leads to an increase in the genesis of IL-1β [30,97] and IL-18 [88,103,104].

IL-1β is mainly produced in monocytes, which are declared to express NLRP3 mRNA [105,106]. Sutterwala et al. [14] found that this process was highly induced by bacterial LPS. On the other hand, there was no production of IL-1β in NLRP3-deficient macrophages, even though bacterial stimulations occurred [19,23,101,107].

Treatment with an IL-1 receptor antagonist of patients with rheumatoid arthritis cancelled clinical symptoms, suggesting a cause–effect relationship between IL-1β production and the development of the disease [108]. Based on an inhibition of the NLRP3 inflammasome, some studies also presented therapeutic pathways in the treatment of experimental PD [109,110].

Moreover, Nrf2 has been demonstrated to directly suppress transcription of NLRP3-associated genes, including pro-IL-1β, and pro-IL-1α [111,112], suggesting Nrf2 to be a potential therapeutic inhibitor of PD.

Delineating the likely role of several oral microbiota associated with the development of PD is rather complex. Among the thousands of bacterial species in the oral cavity, few Gram-negative anaerobic bacteria were related to PD genesis. Periodontopathogen species dispose of several virulence factors that allow them to survive in the host environment by selectively adapting the host’s immune-inflammatory response. The “red cluster” of periodontopathogenic bacteria, consisting of P. gingivalis, Treponema denticola, Prevotella intermedia, A. actinomycetemcomitans, and T. forsythia, contribute to the initiation and progression of severe PD [113,114].

Therefore, we want to present and discuss representative periodontal pathogens, which play a crucial role in activating inflammation in PD, with special attention to the roles of NLRP3 and Nrf2.

4. Periapical Periodontitis

Besides PD in the traditional sense of term, i.e., gingival PD, periapical PD is one of the most common inflammatory diseases in adults. In response to caries, tooth fracture, or trauma, oral microorganisms can enter the initial sterile tooth pulp and trigger inflammation, which may result in pulp necrosis [174,175]. Symptoms are varied, implicating sensitivity to pressure or cold, pain, periapical radiolucency, and edema [176]. There is a distinction between acute and chronic periapical PD showing different symptoms [175]. Most of endodontic bacteria are located in the root canal [177]; thus, the therapy of choice is a root canal treatment, aiming to remove the inflamed dental pulp [178,179]. Surgical apicoectomy is required when endodontics is insufficient and the inflamed part of the bone includes the tooth apex [180]. Etiology of this odontogenic infection is due to bacterial species and their virulence, as well as the interaction with immunological host responses [175]. It was shown that apical PD is responsible for generating cytokines by recruiting inflammatory cells, i.e., host immune response to inflammatory processes [181].

The most common pathogen in periapical PD was demonstrated to be Enterococcus faecalis (E. faecalis), a Gram-positive coccus [182,183,184]. It was already shown that E. faecalis is able to promote CASP1 activation and pro-IL-1β expression, which subsequently increases IL-1β levels [185]. Moreover, increasing IL-1β production during periapical PD [186] might be associated with an interplay between this inflammatory disease and the NLRP3 inflammasome.

5. Oral Squamous Cell Carcinoma

With an incidence of 90%, oral squamous cell carcinoma (OSCC) is the most common oral cancer [203] with a low 5-year survival rate of only 30% [204]. Despite the frequent inspection of the oral cavity by dentists assuming patients’ responsibility for oral health, most OSCC lesions were missed at an early stage, which may explain the high mortality rate. OSCC is known to occur three times more frequently in men than in women [205]. Besides smoking and alcohol consumption [206], risk factors of OSCC also include chronic inflammation [207]. Several studies have shed light on inflammation as a possible cancer basis, as Hussain et al. [208] already determined in 2003 that inflammation is the cause of one in four cancers. Furthermore, oral bacterial species are reported to be responsible for these inflammatory disorders via influencing key processes, which may be contributing to oral carcinogenesis [209,210].

As already described before, P. gingivalis and F. nucleatum induce the development of proinflammatory cytokines, at least partially, through the NLRP3 pathway. Therefore, it was proved that these periodontopathogenic bacteria are potential etiological agents for oral cancer [211]. Yang et al. [212] provided evidence that Fusobacteria can be associated with cancer staging of OSCC. Interestingly, Tezal et al. [213] evaluated patients whom had never used tobacco and alcohol, but suffered from PD. These patients showed a higher probability of 32.8% for poorly differentiated OSCC than patients of good oral health at 11.5%. A very recent study by Yao et al. [214] from 2021 developed a new mechanism connecting periodontopathogenic bacteria (P. gingivalis and F. nucleatum) and OSCC, by showing that these bacterial species upregulate NLRP3 expression and drive inflammasome activation. It was examined that this pathway can level up tumor growth and tumor proliferation. P. gingivalis has been reported to be carcinogenic. Studies demonstrated a positive correlation between P. gingivalis infection and size or invasiveness [143,144], as well as a late tumor–node metastasis (TNM) stage, and low differentiation [145] of head and neck squamous cell cancer (HNSCC). In addition, Sinha et al. [215] first demonstrated that P. gingivalis is enriched in feces from patients with colorectal cancer. Wang et al. [216] later confirmed that P. gingivalis can be held responsible for enhancing colorectal cancer, due to the activation of the NLRP3 inflammasome.

Taken together, oral health and, subsequently, the oral microbiome and its interplay with the host immune response may play a critical role in the development of OSCC. The importance of an efficient and healthy oral microbiome has been underlined by developing a novel strategy of detecting OSCC due to the utilization of saliva, which makes the oral microbiome a noninvasive diagnostic tool [149,212].

Standard therapy of OSCC is a treatment using 5-Fluorouracil (5-FU), which inhibits pyrimidine metabolism and DNA synthesis. It was determined that treatment with 5-FU leads to increased intracellular ROS and is ascertained to upregulate NLRP3 and IL-1β expression in human OSCC cell lines. This subsequently mediates a chemoresistance of OSCC to 5-FU lying on multiple aspects, suggesting tumor-associated macrophages to be responsible. Moreover, survival rates of patients decreased with higher NLRP3 expression, and NLRP3 deficiency enhanced the antitumor effect of 5-FU [217]. This might confirm that NLRP3 is responsible for not only the progression of OSCC but also its limited treatment effectiveness.

As NLRP3 is likely to be critically involved in OSCC occurrence, progression, and proliferation, few studies have shed light on possible strategies for oral cancer treatment, regarding the NLRP3-inflammasome. MicroRNAs are known to act as regulators of carcinogenesis in general. Feng et al. [218] implicated microRNA-22 to inhibit OSCC proliferation due to interference of the NLRP3 pathway.

Yang et al. [221] showed that also bitter melon-derived extracellular vesicles (BMEVs) may downregulate the NLRP3 activation and, moreover, reduce the drug resistance of 5-FU. Additionally, BMEVs could inhibit OSCC proliferation due to the development of reactive oxygen species.

In summary, OSCC treatment targeting NLRP3 is highly promising, and thus, necessitates further research.

It is known that oxidative stress plays an important role in the development of cancer. Nrf2, as an oxidative stress marker, was found to be associated with carcinogenesis and progression of OSCC [224] when hyperactive, while it inhibited carcinogenesis of normal cells [225]. This might suggest a prognostic value of Nrf2 and its potential role as therapeutic target.

6. Dental Implants

Today, the use of dental implants is indispensable in the treatment of edentulism. For the success and persistence of an implant, a connection between implant and living bone tissue is needed. Unlike a natural tooth, which is bound to the surrounding bone indirectly by the periodontal ligament, implants are directly engaged to the bone [226]. Implant stability can be divided into an early stage due to mechanical alliance to the bone, and secondly, into a stage of stability depending on regeneration and remodeling of the bone and tissue close to the inserted implant [227], named osseointegration [228]. Overall, the interaction between bone, tissues, implant surface, and the host immune response has to be compensated for, revealing true osseointegration [229]. Trindade et al. [230] confirmed that titanium implants activate the immune system and lead to inflammation, indicating a two-step osseointegration: first, recognition of the implant as a foreign body; second, development of a bone-forming environment to shield the foreign material from host tissues.

Once again, this shows the importance of a healthy and balanced interplay between the oral microbiome and the immune response, as criteria for implant success and in avoidance of uncontrolled inflammation leading to bone loss and subsequent loss of the implant. Despite advanced technology, failure of implantation (around 1.9–3.6% of dental-implant subjects) and subsequent loss of the implant cannot be ruled out [231]. Besides triggering factors such as medication [232], increasing prevalence of bad systemic health with higher age (≥75 years) [233], or smoking [234], the fundamental reason for implant failure is known to be an overreaction of the immune system, leading to bone loss [235]. Pathogen invasion from the implant surface structure [236], or bad oral hygiene [237] constitute a potential trigger for inflammation, and further, genesis of periimplantitis.

Periimplantitis is an irreversible disease characterized by inflammation of the supporting bone and connective tissues surrounding a dental implant, resulting in unsuccessful osseointegration and subsequent implant failure [238]. A systematic review from Rakic and colleagues [239] in 2018 showed a prevalence of periimplantitis in 12.8% of all implants used. Another study from 2019 revealed that 1/3 of all patients and 1/5 of all implants underwent periimplantitis [240]. Moreover, it has been shown that the incidence of periimplantitis increases with implant age [241].

Studies showed that proinflammatory cytokines are expressed at higher concentrations in the crevicular fluid of healthy implants than around teeth [242]. Furthermore, levels of proinflammatory cytokines in the peri-implant crevicular fluid are again higher around implants with periimplantitis than around healthy implants [243]. Many studies associated IL-1β to with playing a critical role in the occurrence of periimplantitis [244] and peri-implant bone loss [245], which is similar to PD, suggesting that the NLRP3 inflammasome plays, at least, a partial role.

Titanium implants release Ti ions into surrounding tissues [246], which further leads to the secretion of IL-1β, TNF-α, and RANKL in Jurkat T-cells [247], and might aggravate inflammation. Li et al. [248] confirmed these facts, and further, showed that Ti ions activate the NLRP3 inflammasome, increasing the release of ROS.

Candida species were found to be associated with periimplantitis [249] and triggered the NLRP3 inflammasome-mediated pyroptosis in macrophages [250]. As mentioned before, LPS from P. gingivalis—another bacterial species related to periimplantitis— also activates NLRP3 [191].

Taken together, inflammasomes, and mainly the NLRP3 inflammasome, may play a critical role in the development of periimplantitis. Considering the absence of therapeutic agents for the treatment of periimplantitis, further studies are needed to follow up on targeting inflammasome pathways as a future therapeutic option.

We suggest further molecular biologic studies be undertaken on interactions between dental implants and Nrf2.

7. The Alveolar Bone

It is already confirmed that the NLRP3 inflammasome attenuates osteogenesis by mediating inflammation and inducing osteoblast pyroptosis due to the processing of GSDMD and CASP1, and the following secretion of proinflammatory cytokines [251]. As NLRP3 affects bone homeostasis through the regulation of osteoclasts, osteoblasts, and other cell types, one might suggest that NLRP3 plays a vital role in the metabolism of the alveolar bone. However, due to unbalanced NLRP3 activation, alveolar bone homeostasis is disrupted, leading to local dysregulations, or acting as a foundation for systematic bone diseases [182,251]. Qu et al. [252] demonstrated hyperactive NLRP3 expression in osteoclasts, encouraging osteolysis in the absence of systemic inflammation. Furthermore, uncontrolled activation of NLRP3 is associated with osteopenia, a preliminary stage of osteoporosis [253,254].

Activation of the NLRP3 inflammasome in macrophages due to P. gingivalis infection or related to bisphosphonate therapy may also lead to bone loss due to increased IL-1β production [128,129]. Aging, estrogen deficiency, or hyperparathyroidism provide a chronic inflammatory microenvironment and enhance NLRP3 activation, which can further lead to bone resorption and genesis of osteoporosis [254,255,256,257].

On the one hand, Zang et al. [258] already stated that the inflammasome mediates age-related alveolar bone loss, on the basis of a correlation between alveolar bone loss in aged mice and elevated levels of IL-1β. On the other hand, NLRP3-deficient mice demonstrated improved bone mass qualities, presented by an increased bone density [253,258]. Treatment with an inhibitor of NLRP3, i.e., MCC950, significantly suppressed alveolar bone loss [258]. Another study outcome revealed an improvement of alveolar bone healing in diabetic rats [259], indicating that interfering with NLRP3 activation might be a potential therapy regarding alveolar bone loss.

Osteoarthritis (OA) is an age-related inflammatory process of the joints and can affect jaw joints, too [260]. It is characterized by the proliferation of the subchondral bone and the degeneration of articular cartilage [261]. As inflammation is the basis of OA, NLRP3 [262], proinflammatory cytokines such as IL-1β or IL-18, and ROS [263] are related to the development and progression of OA. Chen et al. [260] demonstrated that inhibition of Nrf2 expression and, further, its antipyroptosis effects, upregulate NLRP3 activation in vitro, suggesting that OA therapies targeting the Nrf2/HO-1 signal pathway might be a promising strategy.

Besides inflammation and subsequent bone loss due to an unbalanced NLRP3 activation, and further, overexpression, interestingly, NLRP3 expressed at the physiological level may have positive regulatory effects on bone homeostasis at early ages. Detzen et al. [264] showed that NLRP3-depleted mice have a shorter/smaller habitus, due to affected long-bone growth and malfunction in osteoblast metabolism, compared to wild-type mice with unaffected NLRP3 function. Attention should be paid to the fact that this impaired skeletal development was transitory, as bone homeostasis returns to wild-type-level with age. Consequently, activation of inflammasomes might be not only a promotor of inflammation but also an outcome due to inflammatory bone loss, suggesting a positive feedback mechanism of inflammatory bone loss.

8. Calculus

Based on bad oral hygiene, oral bacterial biofilm persists on the teeth, and further, mineralizes when calcium phosphate salts precipitate in the intermicrobial matrix. Thus, dental calculus, i.e., mineralized dental plaque, occurs supra- and subgingivally, with a nonmineralized bacterial biofilm on it [276]. Dental calculus is responsible for irritation and subsequent inflammation of the gingiva [277], as it acts as a plaque-retention factor, suggesting a pathogenic potential.

Previous studies demonstrated a strong relationship between subgingival calculus and periodontal inflammation [278,279,280]. Therefore, scaling and tooth root debridement for removal of calculus is the therapy of choice regarding PD [281], and procedures with ultrasound systems for comfortable patient therapy are more popular [282].

Raudales et al. [283] showed that dental calculus induced IL-1β secretion in human polymorphonuclear leukocytes, human peripheral blood mononuclear cells, and in macrophages from wild-type mice, although, IL-1β production was inhibited in NLRP3-deficient mice. In conclusion, this study determined that, in mice and in humans, dental calculus, and partially, its crystalline structure is responsible for IL-1β formation through the activation of NLRP3.

It is already known that human epithelial cells, as the first line of the host’s defense, express NLRP3 inflammasome components [104]. Furthermore, it was demonstrated that cell death of epithelial cells is mainly induced by the inorganic component of dental calculus, which, in consequence, affects epithelial barrier functions of this cell line. Moreover, an involvement of NLRP3 inflammasome activation was indicated [284].

Cleaning the tooth root surface of periodontopathogenic bacteria and calculus remains the ultimate solution for PD prevention. Qiu et al. [285] suggested differences in the NLRP3 inflammasome activation, due to various treatments of the tooth root surface, i.e., ultrasonic scaling, hand scaling, sandblasting, or a combination. It could be concluded that there is no significant difference in the expression of NLRP3 inflammasome, and further, IL-1β secretion in human gingival fibroblasts among the different mechanical treatments leading to varying tooth root biological interfaces.

Until now, there had been no studies that examined the potential relationship between Nrf2 and dental calculus. Possible connections could be hypothesized, paying attention to the fact that, on the one hand, Nrf2 aggravates atherosclerosis. Cholesterol crystals accumulate in atherosclerotic plaques triggered Nrf2 and NLRP3 inflammasome activation, leading to IL-1 production in mice [34]. As Nrf2 is activated by cholesterol, Nrf2 is shown to be a positive regulator of the NLRP3 inflammasome.

On the other hand, Liu et al. [286] established a link between Nrf2 and intrarenal calcium oxalate crystals, suggesting that an inhibition of further inflammatory injuries to renal tubular epithelial cells is possible via the Nrf2 pathway in vivo and in vitro.

As Nrf2 is at least partly contributing to some inflammatory conditions due to crystalline structures [34,286], one might suggest that this transcription factor is involved in inflammatory responses of the gingiva due to dental calculus. Confirming studies remain missing.

Conclusions

Our review provides insight into the mechanisms and functions of the NLRP3 inflammasome and the Nrf2 transcription factor, related to the field of dental health care. Comprehension of the oral microbiome, first in health, then in disease, is fundamental. It is of great significance to understand the contribution of several proteins, e.g., inflammasomes to diseases, and their crosstalk to signaling pathways, e.g., Nrf2, which may help investigate new therapy strategies, customized for each patient.