Meta-Analysis of Two Human RNA-seq Datasets to Determine Periodontitis Diagnostic Biomarkers and Drug Target Candidates

Periodontitis is a chronic inflammatory oral disease that affects approximately 42% of adults 30 years of age or older in the United States. In response to microbial dysbiosis within the periodontal pockets surrounding teeth, the host immune system generates an inflammatory environment in which soft tissue and alveolar bone destruction occur. The objective of this study was to identify diagnostic biomarkers and the mechanistic drivers of inflammation in periodontitis to identify drugs that may be repurposed to treat chronic inflammation. A meta-analysis comprised of two independent RNA-seq datasets was performed. RNA-seq analysis, signal pathway impact analysis, protein-protein interaction analysis, and drug target analysis were performed to identify the critical pathways and key players that initiate inflammation in periodontitis as well as to predict potential drug targets. Seventy-eight differentially expressed genes, 10 significantly impacted signaling pathways, and 10 hub proteins in periodontal gingival tissue were identified. The top 10 drugs that may be repurposed for treating periodontitis were then predicted from the gene expression and pathway data. The efficacy of these drugs in treating periodontitis has yet to be investigated. However, this analysis indicates that these drugs may serve as potential therapeutics to treat inflammation in gingival tissue affected by periodontitis.     

The Role of Matrix Metalloproteinases (MMP-8, MMP-9, MMP-13) in Periodontal and Peri-Implant Pathological Processes

Severe periodontitis, a destructive inflammatory disease of the supporting tissues of the teeth, ranks sixth in terms of global spread, affecting about 11% of the population. Metalloproteinases (MMPs) are extracellular matrix (ECM) macromolecules that are important in cellular development and morphogenesis, and they are capable of activating growth factors in their proximity, cell surface receptors, and adhesion molecules. MMPs are part of a major family of zinc-dependent endopeptidases, and their activity is modulated and regulated by certain inhibitors known as tissue metalloproteinase inhibitors (TIMPs). Because type I collagen is the major component of the periodontal extracellular matrix, special attention has been paid to the role of collagenases, especially MMP-8 and MMP-13 and gelatinases, MMP-2 and MMP-9, in periodontal diseases. In fact, MMP-8 (or collagenase 2) is currently one of the most promising biomarkers for periodontitis in oral fluids. Among them, salivary MMP-9 has been shown to be a more sensitive marker for periodontal inflammation during orthodontic treatment, which opens new perspectives in reducing periodontal hazards during such treatments. Both MMP-8 and MMP-9 are extremely valuable diagnostic tools in treating periodontitis, and future studies and healthcare policies should focus on implementing more accessible methods of chairside testing in order to reduce the prevalence of this disease.     

Periodontitis and Gestational Diabetes Mellitus: A Potential Inflammatory Vicious Cycle

Periodontitis is a chronic inflammatory immune disease associated with a dysbiotic state, influenced by keystone bacterial species responsible for disrupting the periodontal tissue homeostasis. Furthermore, the severity of periodontitis is determined by the interaction between the immune cell response in front of periodontitis-associated species, which leads to the destruction of supporting periodontal tissues and tooth loss in a susceptible host. The persistent bacterial challenge induces modifications in the permeability and ulceration of the sulcular epithelium, which facilitates the systemic translocation of periodontitis-associated bacteria into distant tissues and organs. This stimulates the secretion of pro-inflammatory molecules and a chronic activation of immune cells, contributing to a systemic pro-inflammatory status that has been linked with a higher risk of several systemic diseases, such as type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). Although periodontitis and GDM share the common feature of systemic inflammation, the molecular mechanistic link of this association has not been completely clarified. This review aims to examine the potential biological mechanisms involved in the association between periodontitis and GDM, highlighting the contribution of both diseases to systemic inflammation and the role of new molecular participants, such as extracellular vesicles and non-coding RNAs, which could act as novel molecular intercellular linkers between periodontal and placental tissues.     

Osteoimmunology in Periodontitis: Local Proteins and Compounds to Alleviate Periodontitis

Periodontitis is one of the most common oral diseases resulting in gingival inflammation and tooth loss. Growing evidence indicates that it results from dysbiosis of the oral microbiome, which interferes with the host immune system, leading to bone destruction. Immune cells activate periodontal ligament cells to express the receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) and promote osteoclast activity. Osteocytes have active roles in periodontitis progression in the bone matrix. Local proteins are involved in bone regeneration through functional immunological plasticity. Here, we discuss the current knowledge of cellular and molecular mechanisms in periodontitis, the roles of local proteins, and promising synthetic compounds generating a periodontal regeneration effect. It is anticipated that this may lead to a better perception of periodontitis pathophysiology.     

Relationship between NAFLD and Periodontal Disease from the View of Clinical and Basic Research,and Immunological Response

Periodontal disease is an inflammatory disease caused by pathogenic oral microorganisms that leads to the destruction of alveolar bone and connective tissues around the teeth. Although many studies have shown that periodontal disease is a risk factor for systemic diseases, such as type 2 diabetes and cardiovascular diseases, the relationship between nonalcoholic fatty liver disease (NAFLD) and periodontal disease has not yet been clarified. Thus, the purpose of this review was to reveal the relationship between NAFLD and periodontal disease based on epidemiological studies, basic research, and immunology. Many cross-sectional and prospective epidemiological studies have indicated that periodontal disease is a risk factor for NAFLD. An in vivo animal model revealed that infection with periodontopathic bacteria accelerates the progression of NAFLD accompanied by enhanced steatosis. Moreover, the detection of periodontopathic bacteria in the liver may demonstrate that the bacteria have a direct impact on NAFLD. Furthermore, Porphyromonas gingivalis lipopolysaccharide induces inflammation and accumulation of intracellular lipids in hepatocytes. Th17 may be a key molecule for explaining the relationship between periodontal disease and NAFLD. In this review, we attempted to establish that oral health is essential for systemic health, especially in patients with NAFLD.     

The Emerging Regulatory Role of Circular RNAs in Periodontal Tissues and Cells

Periodontitis is a chronic complex inflammatory disease associated with a destructive host immune response to microbial dysbiosis, leading to irreversible loss of tooth-supporting tissues. Regeneration of functional periodontal soft (periodontal ligament and gingiva) and hard tissue components (cementum and alveolar bone) to replace lost tissues is the ultimate goal of periodontal treatment, but clinically predictable treatments are lacking. Similarly, the identification of biomarkers that can be used to accurately diagnose periodontitis activity is lacking. A relatively novel category of molecules found in oral tissue, circular RNAs (circRNAs) are single-stranded endogenous, long, non-coding RNA molecules, with covalently circular-closed structures without a 5’ cap and a 3’ tail via non-classic backsplicing. Emerging research indicates that circRNAs are tissue and disease-specific expressed and have crucial regulatory functions in various diseases. CircRNAs can function as microRNA or RNA binding sites or can regulate mRNA. In this review, we explore the biogenesis and function of circRNAs in the context of the emerging role of circRNAs in periodontitis pathogenesis and the differentiation of periodontal cells. CircMAP3K11, circCDK8, circCDR1as, circ_0062491, and circ_0095812 are associated with pathological periodontitis tissues. Furthermore, circRNAs are expressed in periodontal cells in a cell-specific manner. They can function as microRNA sponges and can form circRNA–miRNA–mRNA networks during osteogenic differentiation for periodontal-tissue (or dental pulp)-derived progenitor cells.     

Targeting S1PRs as a Therapeutic Strategy for Inflammatory Bone Loss Diseases—Beyond Regulating S1P Signaling

As G protein coupled receptors, sphingosine-1-phosphate receptors (S1PRs) have recently gained attention for their role in modulating inflammatory bone loss diseases. Notably, in murine studies inhibiting S1PR2 by its specific inhibitor, JTE013, alleviated osteoporosis induced by RANKL and attenuated periodontal alveolar bone loss induced by oral bacterial inflammation. Treatment with a multiple S1PRs modulator, FTY720, also suppressed ovariectomy-induced osteoporosis, collagen or adjuvant-induced arthritis, and apical periodontitis in mice. However, most previous studies and reviews have focused mainly on how S1PRs manipulate S1P signaling pathways, subsequently affecting various diseases. In this review, we summarize the underlying mechanisms associated with JTE013 and FTY720 in modulating inflammatory cytokine release, cell chemotaxis, and osteoclastogenesis, subsequently influencing inflammatory bone loss diseases. Studies from our group and from other labs indicate that S1PRs not only control S1P signaling, they also regulate signaling pathways induced by other stimuli, including bacteria, lipopolysaccharide (LPS), bile acid, receptor activator of nuclear factor κB ligand (RANKL), IL-6, and vitamin D. JTE013 and FTY720 alleviate inflammatory bone loss by decreasing the production of inflammatory cytokines and chemokines, reducing chemotaxis of inflammatory cells from blood circulation to bone and soft tissues, and suppressing RANKL-induced osteoclast formation.     

Increased Presence of Complement Factors and Mast Cells in Alveolar Bone and Tooth Resorption

Periodontitis is the inflammatory destruction of the tooth-surrounding and -supporting tissue, resulting at worst in tooth loss. Another locally aggressive disease of the oral cavity is tooth resorption (TR). This is associated with the destruction of the dental mineralized tissue. However, the underlying pathomechanisms remain unknown. The complement system, as well as mast cells (MCs), are known to be involved in osteoclastogenesis and bone loss. The complement factors C3 and C5 were previously identified as key players in periodontal disease. Therefore, we hypothesize that complement factors and MCs might play a role in alveolar bone and tooth resorption. To investigate this, we used the cat as a model because of the naturally occurring high prevalence of both these disorders in this species. Teeth, gingiva samples and serum were collected from domestic cats, which had an appointment for dental treatment under anesthesia, as well as from healthy cats. Histological analyses, immunohistochemical staining and the CH-50 and AH-50 assays revealed increased numbers of osteoclasts and MCs, as well as complement activity in cats with TR. Calcifications score in the gingiva was highest in animals that suffer from TR. This indicates that MCs and the complement system are involved in the destruction of the mineralized tissue in this condition.     

Pyroptosis-Mediated Periodontal Disease

Pyroptosis is a caspase-dependent process relevant to the understanding of beneficial host responses and medical conditions for which inflammation is central to the pathophysiology of the disease. Pyroptosis has been recently suggested as one of the pathways of exacerbated inflammation of periodontal tissues. Hence, this focused review aims to discuss pyroptosis as a pathological mechanism in the cause of periodontitis. The included articles presented similarities regarding methods, type of cells applied, and cell stimulation, as the outcomes also point to the same direction considering the cellular events. The collected data indicate that virulence factors present in the diseased periodontal tissues initiate the inflammasome route of tissue destruction with caspase activation, cleavage of gasdermin D, and secretion of interleukins IL-1β and IL-18. Consequently, removing periopathogens’ virulence factors that trigger pyroptosis is a potential strategy to combat periodontal disease and regain tissue homeostasis.     

An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis

To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.     

The Potential Roles of Dec1 and Dec2 in Periodontal Inflammation

Periodontal inflammation is a common inflammatory disease associated with chronic inflammation that can ultimately lead to alveolar attachment loss and bone destruction. Understanding autophagy and pyroptosis has suggested their significant roles in inflammation. In recent years, studies of differentiated embryo-chondrocyte expressed genes 1 and 2 (Dec1 and Dec2) have shown that they play important functions in autophagy and in pyroptosis, which contribute to the onset of periodontal inflammation. In this review, we summarize recent studies on the roles of clock genes, including Dec1 and Dec2, that are related to periodontal inflammation and other diseases.     

Ibuprofen Rescues Abnormalities in Periodontal Tissues in Conditional Presenilin 1 and Presenilin 2 Double Knockout Mice

We used forebrain-specific conditional presenilin 1 (PS1) and presenilin 2 (PS2) double knockout mice (dKO mice) that exhibit symptoms of neurodegenerative diseases, especially Alzheimer’s disease, to investigate whether ibuprofen can rescue brain and periodontal tissue abnormalities by attenuating the inflammatory response. Mandibles were dissected for alveolar bone-height analysis. Maxillae were fixed and decalcified for histological observation and osteoclast detection. ELISA measurements from the hippocampus, cortex, and gingiva of the mandibular incisor teeth were used to assay inflammatory mediators. We confirmed periodontal tissue abnormalities and inflammatory responses in brain and periodontal tissues in naive nine- and 12-month-old dKO mice. The other two groups of age-matched dKO mice that received 375-ppm ibuprofen treatment for six consecutive months exhibited significantly attenuated damage in periodontal tissues and reduction in several inflammation-related factors in brain and periodontal tissues. Our findings showed that the anti-inflammatory drug ibuprofen significantly decreased inflammation through the cyclooxygenase (COX) pathway in brain and periodontal tissues in dKO mice, and then attenuated abnormalities in periodontal tissues. This suggests that ibuprofen could be an ideal drug for preventing both nervous system and periodontal tissue damage caused by inflammatory responses.     

Locally Secreted Semaphorin 4D Is Engaged in Both Pathogenic Bone Resorption and Retarded Bone Regeneration in a Ligature-Induced Mouse Model of Periodontitis

It is well known that Semaphorin 4D (Sema4D) inhibits IGF-1-mediated osteogenesis by binding with PlexinB1 expressed on osteoblasts. However, its elevated level in the gingival crevice fluid of periodontitis patients and the broader scope of its activities in the context of potential upregulation of osteoclast-mediated periodontal bone-resorption suggest the need for further investigation of this multifaceted molecule. In short, the pathophysiological role of Sema4D in periodontitis requires further study. Accordingly, attachment of the ligature to the maxillary molar of mice for 7 days induced alveolar bone-resorption accompanied by locally elevated, soluble Sema4D (sSema4D), TNF-α and RANKL. Removal of the ligature induced spontaneous bone regeneration during the following 14 days, which was significantly promoted by anti-Sema4D-mAb administration. Anti-Sema4D-mAb was also suppressed in vitro osteoclastogenesis and pit formation by RANKL-stimulated BMMCs. While anti-Sema4D-mAb downmodulated the bone-resorption induced in mouse periodontitis, it neither affected local production of TNF-α and RANKL nor systemic skeletal bone remodeling. RANKL-induced osteoclastogenesis and resorptive activity were also suppressed by blocking of CD72, but not Plexin B2, suggesting that sSema4D released by osteoclasts promotes osteoclastogenesis via ligation to CD72 receptor. Overall, our data indicated that ssSema4D released by osteoclasts may play a dual function by decreasing bone formation, while upregulating bone-resorption.     

PF-3845, a Fatty Acid Amide Hydrolase Inhibitor,Directly Suppresses Osteoclastogenesis through ERK and NF-κB Pathways In Vitro and Alveolar Bone Loss In Vivo

Alveolar bone loss, the major feature of periodontitis, results from the activation of osteoclasts, which can consequently cause teeth to become loose and fall out; the development of drugs capable of suppressing excessive osteoclast differentiation and function is beneficial for periodontal disease patients. Given the difficulties associated with drug discovery, drug repurposing is an efficient approach for identifying alternative uses of commercially available compounds. Here, we examined the effects of PF-3845, a selective fatty acid amide hydrolase (FAAH) inhibitor, on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis, its function, and the therapeutic potential for the treatment of alveolar bone destruction in experimental periodontitis. PF-3845 significantly suppressed osteoclast differentiation and decreased the induction of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific markers. Actin ring formation and osteoclastic bone resorption were also reduced by PF-3845, and the anti-osteoclastogenic and anti-resorptive activities were mediated by the suppression of phosphorylation of rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase, (ERK) and nuclear factor κB (NF-κB) inhibitor (IκBα). Furthermore, the administration of PF-3845 decreased the number of osteoclasts and the amount of alveolar bone destruction caused by ligature placement in experimental periodontitis in vivo. The present study provides evidence that PF-3845 is able to suppress osteoclastogenesis and prevent alveolar bone loss, and may give new insights into its role as a treatment for osteoclast-related diseases.     

The Effects of Omega-3 Supplementation on Serum Oxidative Stress Parameters in Experimental Periodontitis in an Animal Model

Experimental studies suggest that omega-3 may be beneficial at preventing alveolar bone loss (ABL) by modulating host immune response. The aim of this study is to evaluate the effects of omega-3 supplementation on ABL and serum oxidative biomarkers in a ligature-induced periodontitis model. Twenty-four Wistar albino rats are divided into 3 groups: control (C, n = 8); ligature-induced periodontitis (L, n = 8); and omega-3 plus ligature-induced periodontitis (O+L, n = 8). All rats are fed with ad libitum diet, and O+L group is additionally supplemented with omega-3 fish oil by oral gavage for 44 days. Experimental periodontitis is induced by placement of ligatures around the maxillary second molars of rats in L and O+L groups. ABL is measured histomorphometrically and serum 8-hydroxy-2'-deoxyguanosine, total antioxidant capacity, and total oxidant status (TOS) are analyzed. ABL is higher in L and O+L groups than the C group. Omega-3 supplementation is significantly reduced ABL in group O+L, compared to L group. Furthermore, TOS levels are lower in O+L group than L group. It is suggested that omega-3 administration may reduce ABL and serum TOS levels, which supports the antioxidant role of omega-3 on periodontal disease pathogenesis. Practical applications: Adjunctive use of omega-3 supplementation in periodontitis seems beneficial, although biochemical mechanisms underlying the conflicting results have not been completely understood. On the other hand, oxidative stress has been used as an appropriate target for host modulation therapies in periodontal disease. Omega-3 supplementation in ligature-induced periodontitis in an animal model successfully reduces alveolar bone loss by modulating serum total oxidant status, which may provide a novel pathway in periodontal disease pathogenesis.     

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.     

Oral Microbiome in Relation to Periodontitis Severity and Systemic Inflammation

Systemic inflammation induced by periodontitis is suggested to be the link between periodontitis and cardiovascular disease. The aim of this work was to explore the oral microbiome in periodontitis in relation to disease severity and systemic inflammation. The saliva and subgingival microbiome from periodontal pocket samples of patients with severe (n = 12) and mild periodontitis (n = 13) were analyzed using metagenomic shotgun sequencing. The taxa and pathways abundances were quantified. The diversity was assessed and the abundances to phenotype associations were performed using ANCOM and linear regression. A panel of inflammatory markers was measured in blood and was associated with taxa abundance. The microbial diversity and species richness did not differ between severe and mild periodontitis in either saliva or periodontal pockets. However, there were significant differences in the microbial composition between severe and mild periodontitis in the subgingival microbiome (i.e., pocket samples) and, in a lower grade, in saliva, and this is positively associated with systemic inflammatory markers. The “red complex” and “cluster B” abundances in periodontal pockets were strongly associated with inflammatory markers interleukin-6 and the white blood cell count. Our data suggest that systemic inflammation in severe periodontitis may be driven by the oral microbiome and may support the indirect (inflammatory) mechanism for the association between periodontitis and cardiovascular disease.     

The Receptor AT1 Appears to Be Important for the Maintenance of Bone Mass and AT2 Receptor Function in Periodontal Bone Loss Appears to Be Regulated by AT1 Receptor

A large number of experimental studies has demonstrated that angiotensin II (Ang II) is involved in key events of the inflammatory process. This study aimed to evaluate the role of Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors on periodontitis. Methods: Experimental periodontitis was induced by placing a 5.0 nylon thread ligature around the second upper left molar of AT1 mice, no-ligature or ligature (AT1-NL and AT1-L), AT2 (AT2-NL or AT2-L) and wild type (WT-NL or L). Alveolar bone loss was scanned using Micro-CT. Cytokines, peptides and enzymes were analyzed from gingival tissues by Elisa and RT-PCR. Results: The blockade of AT1 receptor resulted in bone loss, even in healthy animals. Ang II receptor blockades did not prevent linear bone loss. Ang II and Ang 1-7 levels were significantly increased in the AT2-L (p < 0.01) group compared to AT2-NL and AT1-L. The genic expression of the Mas receptor was significantly increased in WT-L and AT2-L compared to (WT-NL and AT2-NL, respectively) and in AT1-L. Conclusions: Our data suggest that the receptor AT1 appears to be important for the maintenance of bone mass. AT2 receptor molecular function in periodontitis appears to be regulated by AT1.