The pathogenic mechanism of oral bacteria and treatment with inhibitors

Abstract Objectives The objective of this study was to introduce the evidence obtained through extensive research that periodontitis increases risk of many systemic diseases. Method Analysis of some oral bacteria (P. gingivalis, T. denticola, T. forsythia, A. actinomycetemcomitans, and F. nucleatum) and its related treatments and mediators by the specific methods (western blot, ELISA, etc). Results This article reviews in detail the evidence obtained through extensive research that periodontitis increases risk of many systemic diseases, including cardiovascular disease, rheumatoid arthritis, and Alzheimer's disease. These diseases are known to be associated with some certain specific gram‐negative bacteria as periodontal pathogens, which induce inflammation and related diseases through TLR receptors, kinases, transcriptional factors and other cytokines. We also reviewed the latest research for inhibitors against inflammation and related diseases that have potential to be further applied clinically. In addition, based on a large amount of research evidence, we draw two tables about the mechanism of disease caused by periodontal bacteria, so that readers can easily search and analyze these research results. Discussion This review details how the periodontal bacteria and their virulence factors can trigger host immune defense and induce many systemic diseases via inflammation and invasion. This Review also addressed the latest research around inhibitors against inflammation.


| INTRODUCTION
The link between dental diseases and systemic diseases has long been a controversial subject, which can be traced back to ancient Greece when Hippocrates reported a cure of arthritis after teeth extraction.
During the early to mid-20th century, the "focal infection theory" was widely accepted, which claimed that localized infections cause diseases elsewhere in the body, with oral infection being the prime focus. Many therapeutic edentulations were performed during this period even though that there was no convincing evidence pinpointing teeth to be the "foci" of infection. The focal infection theory era ended around 1950 after researches showed that teeth Thuraya Elgreu, Sean Lee, and Sabrina Wen contributed equally to this work. This research did not receive any specific grants from funding agencies in the public, commercial, or not-profit sectors. extraction did not consistently cure diseases, such as rheumatoid arthritis. However, the revival of the focal infection theory started to gain momentum in the 1990s after researches linked periodontal disease to atherosclerotic vascular disease. Since then, emerging evidence has shown that periodontitis potentially increases the risk of many systemic diseases, including cardiovascular disease, rheumatoid arthritis, and Alzheimer's disease (Holmer et al., 2018).
Periodontitis is an inflammatory disease caused by a host reaction to a mixed bacterial infection. Out of 700 bacterial species, some specific bacteria, such as Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum are regarded as the principal periodontal pathogens. They play an important role in the occurrence and development of periodontal diseases and other diseases (Kim & Amar, 2006).
The causes behind periodontal pathogenic inflammation and other related systemic diseases have become increasingly intriguing and complicated. A large number of studies have found that periodontal pathogens transmit their signals into animal cells or tissues through cell receptors, including Toll-Like receptors 2/4 (TLR2/4), induce phosphorylation of related kinases, express transcriptional factors, regulate cytokines (Jia et al., 2019), and finally induce various diseases (Kim & Amar, 2006). However, a few questions remain: How do these major periodontal pathogens transmit signals through cell receptors such as TLR2/4? Why do the signals of these pathogens induce different factors, cytokines, and diseases, even though they go through the same receptors? To answer these questions, it is necessary to understand this through the following review on how periodontal bacteria induce diseases.
With a deeper understanding of the mechanisms of how pathogens infect animals and cause inflammation and disease (Hasturk & Kantarci, 2015), there will be more opportunities for us to develop effective inhibitors or drugs. So far, a large number of inhibitors or drugs specific to suppression of periodontal pathogens have been developed (Maksylewicz et al., 2019). Since the information about these inhibitors has been in disarray, we provide the following review on inhibitors used for periodontal bacteriainduced Inflammation.

| CELLS AND METHODS
The oral bacteria, mammalian cells, and the experimental methods cited below should be approved by the related school's Institutional Animal Care and Use Committee if they are involved in human subjects or vertebrate animals (reference below for examples of the different cells and methods that have been individually used).
Examples of oral bacteria and mammalian cells include: P. gingivalis, T. denticola, T. forsythia, T. denticola, A. actinomycetemcomitans cells, neutrophils, macrophages, primary bone marrow macrophage, endothelial cells, brain cells, or tumor cells. Examples of methods include: A treatment of cells with lipopolysaccharides (LPS), protein array, enzyme-linked immunosorbent assay, and western blot analysis, and so forth.

| MAJOR PERIODONTAL BACTERIA
In 1998, Socransky et al. 1998 classified microbials in subgingival plaque into multiple complexes based on cluster and ordination analysis. The red complex consisted of P. gingivalis, T. denticola, and T. forsythia are gram-negative late colonizers that relate closely to clinical measurements of periodontal disease. The name, P. gingivalis, originates from the Greek word porphyromonas (which means purple) due to its hemin forming property when growing on blood agar plates. Their fimbriae promote aggregation and induces pro-inflammatory cytokine expression. The outer membrane sheds evaginations (outer membrane vesicles) into the environment. These vesicles contain some of the most important virulence factors of P. gingivalis: Gingipains, and LPS.
Gingipains are cysteine proteases that cleave many host proteins to allow P. gingivalis to escape the immune system and weaken the inflammatory response. In addition to directly degrading cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), some of its other well-known functions are to inhibit the phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) pathway, reduce cluster of differentiation 14 (CD14) expression, and cleave complement component 5 (C5) into C5a and C5b, which further leads to a complement-TLR crosstalk that suppresses the immune response. On the other hand, LPS elicits the immune response through binding with TLR-2 and TLR-4. Moreover, there have been reports of crosstalk between TLRs-2, 4, and 9. TLRs have a TIR domain, which is responsible for signal transduction. Myeloid differentiation primary response 88 (MyD88), TIR-domain-containing adapter-inducing interferon-β (TRIF), and TIRAP are toll/interleukin-1 receptor (TIR) domaincontaining adaptor proteins downstream of TLRs, which further activates signaling pathways such as nuclear factor kappa B (NF-κB), Activator protein 1 (AP-1), mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinases (JNK), PI3K, or extracellular signal-regulated kinases (ERK) (Jia et al., 2019). P. gingivalis also produces peptidylarginine deiminase (PADs) and heat-shock proteins (GroEL), which have both been linked to non-oral diseases (Lin et al., 2015).
T. denticola was originally described as "spirochaete denticola" and later proposed as a distinct species "T. denticola" (Chan et al., 1993) after three strains were isolated from deep periodontal pockets. Studies have indicated that T. denticola triggers the immune response through TLR2 and its periplasmic flagella is crucial for this stimulation (Ruby et al., 2018). The outer membrane, or more commonly known as the outer sheath, hosts major sheath proteins (MSP), and lipooligosaccharide (LOS). They both exhibit the ability to induce pro-inflammatory cytokines. A trademark virulence factor of T. denticola is its prolyl phenylalanine-specific peptidase (dentilisin), which can degrade host cell proteins and activate polymorphonuclear neutrophils (PMNs) via the complement C3 pathway (Yamazaki et al., 2006).
T. forsythia was originally referred to as Bacteroides forsythus and later renamed due to it being discovered by Dr. Anne Tanner of the Forsyth Institute. A unique surface layer formed of glycosylated proteins covers the outer membrane of T. forsythia and is responsible for adhesion to and invasion of cells and coaggregation with other species. It also attenuates pro-inflammatory cytokine expression by evading immune recognition. BspA, a cell surface-associated and secreted protein can induce chemokine expression through binding with TLR2. Its LPS has also been associated with inflammation.
Another significant periodontal pathogen is A. actinomycetecomitans, which is considered to be an early colonizer and has been found to be highly associated with localized aggressive periodontitis, as well as chronic periodontitis. Its virulence factors LPS, leukotoxins, and cytolethal distending toxins (Cdts) can all induce inflammation (Belibasakis et al., 2019). Leukotoxins plays a crucial role in the development of aggressive periodontitis through its specificity to macro- F. Nucleatum is also an important periodontal pathogen due to its ability to coaggregate with other species, therefore, playing a key role in the formation of dental plaque. It has received attention from the scientific community due to its association with multiple diseases such as adverse pregnancy outcomes and gastrointestinal disorders. It produces virulence factor FadA, which is considered an adhesin/invasin has been hypothesized to be responsible for both a direct invasion of host cells and increased endothelial permeability for other bacteria invasion (Fardini et al., 2011). A recent systematic review has reported consistent detection of periodontal bacteria DNA in coronary atherosclerotic plaque, with P.
gingivalis and A. actinomycetemcomitans being most frequently detected (Joshi et al., 2019). These bacteria and their virulence factors can induce cardiovascular endothelial dysfunction through invasion and inflammation, which further favors atheroma formation (Sanz et al., 2020). Experiments on apolipoprotein E knockout mice have shown that P. gingivalis LPS-induced inflammation is highly associated with atherosclerotic lesion progression. The bacterial heat shock protein GroEL of P. gingivalis also contributes to atherosclerosis through the up-regulation of TLR4 (Huang et al., 2016). Furthermore, immune cross-reaction to GroEL with the human heat shock protein hHSP60 expressed by arterial endothelial cells could also result in endothelial dysfunction. Periodontal pathogens and their virulence factors have also been found in the brain tissue of patients with Alzheimer's disease. Indeed, a connection between chronic periodontitis and cognitive impairment and Alzheimer's disease has been drawn after studies show a continuous pattern (Holmer et al., 2018). Inflammation induced by periodontal pathogens potentially alters the blood-brain barrier transport and accumulation of amyloid-ß protein, a characteristic of Alzheimer's disease. Both LPS and gingipain of P. gingivalis induced inflammation in brain cells through upregulation of cytokines such as TNF-α, IL-1β, and IL-6 and resulted in neurodegeneration or learning disability (Ilievski et al., 2018). A. actinomycetemcomitans cells also upregulate pro-inflammatory cytokines and induce accumulation of amyloid-ß peptides in brain cells through its LPS. Furthermore, they induce apoptosis and establish a pro-inflammatory milieu in blood- Another disease that has been linked with periodontitis is type 2 diabetes mellitus, in which the bi-directional relationship has been well documented. P. gingivalis LPS has been shown to induce secretion of pro-inflammatory adipokines leptin and resistin, IL-6, and monocyte chemoattractant protein 1 (MCP-1). It also promotes oxidative stress, which together may participate in obesity and insulin resistance (Le Sage et al., 2017). P. gingivalis inoculation led to more proinflammatory cytokine production in diabetic mice compared to normal mice (Nishihara et al., 2009).
The role that periodontal bacteria may play in Rheumatoid Arthritis has also been investigated. P. gingivalis can citrullinate proteins through its virulence factors gingipain and PADs. The antibodies that target these citrullinated proteins may cross-react with physiological citrullinated proteins, an autoimmune characterization of rheumatoid arthritis (G omez-Bañuelos, 2019).
Periodontal pathogens also play a role in adverse pregnancy outcomes, as P. gingivalis invasion of the amniotic cavity was found in women with threatened premature labor (Le on et al., 2007). F. nucleatum has been detected in chorion tissue from high-risk pregnant women. Furthermore, LPS from F. nucleatum induces IL-6 and corticotrophin-releasing hormones in chorion-derived cells, indicating that the pro-inflammatory environment and dysregulated parturition condition induced by periodontal pathogens may lead to pregnancy complications (Tateishi et al., 2012).
Some of the other diseases that have been linked to periodontal disease but its relationship less studied include chronic obstructive pulmonary disease (COPD), pneumonia, and cancer. COPD is mainly caused by smoking but may be exacerbated by infection and inflammation of the lungs. A recent study demonstrated that periodontal pathogens, especially F. nucleatum, induced IL-6 and IL-8 in bronchial epithelial cells and IL-8 in alveolar epithelial cells (Hayata et al., 2019). Another lung-related disease, Pneumonia, is caused by infection of the lungs by a variety of microorganisms, including bacteria. A mice study showed that intratracheal inoculation of P. gingivalis led to lung damage accompanied by elevated levels of TNF-α, IL-6, IL-17, and C-reactive protein (CRP). These effects were dependent on the virulence factor gingipain, as inoculation with gingipain-null P. gingivalis did not cause increased levels of IL-6 and TNF-α (Benedyk et al., 2016). Lastly, periodontitis has been associated with multiple types of cancer. Positive correlation between cyclooxygenase-2 (COX-2) products, which can be activated by periodontal bacteria-induced cytokines, and the development of tumor and metastatic sites have been reported (Malinowski, 2019).
Furthermore, other mechanisms linking the two diseases have been suggested. A study demonstrated that P. gingivalis GroEL enhances tumor growth by promoting the neovasculogenesis of tumor cells (Lin et al., 2015). Another study showed that P. gingivalis and F. nucleatum coinfection stimulated oral squamous cell carcinoma tumorigenesis via direct interaction with oral epithelial cells through TLRs (Gallimidi et al., 2015). It has also been suggested that tumor suppressor gene p53 mutations, commonly detected in patients with pancreatic cancer, may be caused by the enzymes of periodontal bacteria (Ö grendik, 2016).
To make it easier to understand the mechanism of pathogeninduced inflammation/diseases, we have organized the following into Many types of inhibitors have been proposed to be used in adjunct to periodontal treatment, but the only agent that has been approved by the United States Food and Drug Administration (US FDA) and used clinically so far is a submicrobial-dose doxycycline, which functions by downregulating matrix metalloproteinases (MMPs) and mediators such as IL-6, TNF-α, and prostaglandin E2 (PGE2). This approach, termed as "host modulation therapy", was initially discovered by Golub and colleagues in the 80s, when experiments showed that orally administered minocycline in diabetic rats resulted in inhibition of pathologically excessive collagenase activity in their gingival tissues (Golub et al., 1985). Nevertheless, many other anti-inflammatory agents have and are still being examined for their effects against periodontal inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) initially showed promise in periodontal treatment (Salvi & Lang, 2005).
They downregulate inflammation by blocking the cyclooxygenase (COX enzymes), therefore regulating arachidonic acid metabolite prostaglandins. Their effects on the treatment of periodontal disease were widely researched starting in the early 80s (Das, 2005). Although nonselective Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, flurbiprofen, ibuprofen, and COX-2 selective NSAIDs such as celecoxib showed promise in both animal and human studies, as they helped stabilize periodontal conditions by reducing the rate of alveolar bone resorption (Salvi & Lang, 2005), they can also lead to gastrointestinal problems, decreased platelet aggregation, renal and hepatic impairment, and cardiovascular issues. Furthermore, it was later discovered that bone loss accelerates when NSAIDs are stopped abruptly due to the rebound effect. Therefore, the routine adjunct use of NSAIDs has been precluded from periodontal treatment. However, alternative methods of NSAID use in periodontal treatment are still being examined up until this day, as a recent study demonstrated that the cyclic regimen of diclofenac potassium (50 mg BID, use for 2 months, stop for 2 months, then use again for 2 months) after initial non-surgical periodontal treatment suppressed the increase of gingival crevicular fluid (GCF) IL-1β levels at 6 months compared to the control group that only took placebos. Significantly lower levels of Prostaglandin E2 (PGE2) were also found compared to the control group at 6 months. No adverse effects were detected during this time. However, some rebound effects of PGE2 levels were noticed during the 2 month cessation period (Oduncuoglu et al., 2018). Another strategy utilizing NSAID derivatives claims to achieve greater results with reduced side effects. This rat study demonstrated that a hydrogen sulfide-releasing derivative of the NSAID drug ketoprofen reduced expressions of LPS-induced IL-1β, TNF-α, COX-2, and inducible nitric oxide synthase (iNOS) and also showed less gastric damage compared to ketoprofen. However, the rebound effect was not addressed (Gugliandolo et al., 2018). Further studies will be needed to clarify the practicality of these new methods.
Another type of promising anti-inflammatory therapy that has yet to find its way into routine periodontal treatment is anti-cytokine agents, despite widespread use in treating cancer and various autoimmune diseases such as rheumatoid arthritis and psoriasis. Cytokines are cell-signaling molecules that can either be pro-inflammatory or anti-inflammatory. Agents that aim to down-regulate proinflammatory cytokines or up-regulate anti-inflammatory cytokines have the potential to significantly reduce inflammation. They achieve down-regulation of pro-inflammatory cytokines through utilizing antibodies against specific cytokines, blocking cytokine receptors with cytokine antagonists, using soluble cytokine receptors to block its action, or disrupting the upstream signaling pathways of the cytokines (Lai & Dong, 2016). The drug infliximab, a neutralizing antibody to TNF-α used in the treatment of rheumatoid arthritis among other autoimmune diseases, has been evaluated for its effect against periodontal disease in both animals and humans. Infliximab showed significant anti-inflammatory and bone-protective effects in a Wistar rat model with experimentally induced periodontitis, as it reduced gingival IL-1β, TNF-α, and myeloperoxidase levels, and also diminished MMP-1/-8, receptor activator of nuclear factor κB (RANK), and RANK-Ligand (RANKL) bone immunolabeling compared with the control group (Gonçalves et al., 2014). Studies also evaluated the effect of Infliximab on rheumatoid arthritis patients with co-existing periodontitis and noticed that they exhibited decreased periodontal attachment loss, and had lower periodontal indices and TNF-α gingival crevicular fluid levels (Mayer et al., 2009). Soluble cytokine receptors have also been studied for its effect against periodontal disease.
Etanercept, a recombinant soluble receptor to TNF-α that is used for treating autoimmune diseases, led to significantly reduced periodontal inflammation, infiltration of neutrophils, and iNOS levels in rats with experimentally induced periodontitis (Di Paola et al., 2007). Other soluble receptors or antagonists to IL-1 or TNF-α were used in monkey models with experimentally induced periodontitis and either showed a significant reduction of inflammatory cell infiltrates, reduction of osteoclast numbers, or reduced attachment and bone loss (Graves et al., 1998). Manipulation of the signaling pathways of LPS-induced cytokines has been explored, as inhibition of p38-α mitogen-activated protein kinase (p38 MAPK) resulted in a significant reduction of TNFα, IL-1, and IL-6 (Kirkwood & Rossa, Jr., 2009). The main concern of anti-cytokine therapy is that the down-regulation of the immune system increases the risk of infection. For instance, an opportunistic infection has been reported when TNF-α was neutralized for rheumatoid arthritis therapy (Keane et al., 2001). Therefore, these safety issues need to be resolved before anti-cytokine therapy can be applied in the field of periodontal treatment.
In addition to anti-cytokine therapy, which either blocks the cytokines from functioning or inhibits the signaling pathways of cytokines,  Another trending anti-inflammatory agent is natural compounds, which are considered relatively safe and inexpensive. They have long been an essential part of traditional medicine in many countries anti-inflammatory and anticonvulsive properties. Several studies demonstrated that they reduced LPS-induced pro-inflammatory cytokines in-vitro and also exhibited significant anti-inflammatory effects in animals . Another example of a natural compound is curcumin, a biologically active polyphenolic compound found in turmeric, an Indian spice derived from the plant Curcuma longa Linn. It exhibits anti-inflammatory, anti-oxidant, and anti-bacterial effects among other properties. Studies have shown that they inhibit proinflammatory cytokines including IL-1β, TNF-α, and IL-6 through blocking signaling pathways of NF-κB and p38 MAPK (Zambrano et al., 2018). Furthermore, animal experiments have demonstrated that curcumin inhibits inflammatory bone resorption, decreases osteoclast counts, and inflammatory infiltrates, and reduces MMPs production (Guimarães et al., 2011). Human studies have shown that the adjunctive use of curcumin with scaling and root planing (SRP) resulted in significant improvement of periodontal parameters compared to SRP alone (Raghava et al., 2019). Furthermore, in order to increase its MMP inhibiting potency, a chemically modified curcumin, CMC2.24 has been shown to be effective in reducing periodontal inflammation and MMP production through inhibition of signaling molecules TLR-2 and p38 MAPK (Deng et al., 2020).
Studies have also examined methods to suppress TLR signaling, including inhibiting TIRAP, inhibiting MyD88, blocking NFkB, or competing with CD14 and LBP (LPS binding protein). Chloroquine, an antimalarial agent that blocks TLR-9 signaling, has been shown to significantly decrease inflammatory cytokines induced by periodontal pathogens (Sahingur et al., 2012). Female sex hormones β-estradiol and progesterone both significantly reduced P. gingivalis LPS-induced TLR2 expression (Jitprasertwong et al., 2016). Lastly, many attempts have been made to inhibit specific periodontal pathogens and their virulence factors. Chlorhexidine, polyphenols, and some protein-derived peptides are among agents that inhibit gingipain (Olsen & Potempa, 2014). The antimicrobial peptide LL-37 is effective against T. denticola and human saliva inhibits dentisilin (Rosen et al., 2012). Some bacteria from subgingival flora can inhibit A.actinomycetemcomitans by degrading its leukotoxin (Johansson et al., 2000).
To make it easier to search and analyze the information about the inhibitors treated for the pathogen-induced diseases, we have organized the following into Tables 2.

| CONCLUSION
Evidence supports a strong association between periodontal disease and various systemic diseases; this link may have direct and indirect effects of periodontal pathogens. Although we investigated how periodontal bacteria and their virulence factors can trigger host immune defense and induce many systemic diseases via inflammation and invasion, further studies will be needed to identify the causative mediators of these association. This review also discusses the latest research regarding inhibitors against inflammation, how the downregulation of the inflammatory mediators induced by periodontal bacteria may reduce the likelihood or result in a resolution of periodontitis and periodontal-related systemic diseases, and the effectiveness of clinical therapy for many patients. Overall, this review will prove to be a helpful reference for researchers that summarizes the past, present, and future beliefs and treatments.

CONFLICT OF INTEREST
Non-conflict of interest in the manuscript.

AUTHOR CONTRIBUTIONS
Xiaoren Tang

DATA AVAILABILITY STATEMENT
Data sharing not applicable -no new data generated, or the article describes entirely theoretical research. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.