Molecular Signatures of Senescence in Periodontitis: Clinical Insights.

Most of the elderly population is afflicted by periodontal diseases, creating a health burden worldwide. Cellular senescence is one of the hallmarks of aging and associated with several chronic comorbidities. Senescent cells produce a variety of deleterious secretions, collectively termed the senescence-associated secretory phenotype (SASP). This disrupts neighboring cells, leading to further senescence propagation and inciting chronic inflammation, known as "inflammaging." Detrimental repercussions within the tissue microenvironment can trigger senescence at a younger age, accelerate biological aging, and drive the initiation or progression of diseases. Here, we investigated the biological signatures of senescence in healthy and diseased gingival tissues by assessing the levels of key senescence markers (p16, lipofuscin, and ß-galactosidase) and inflammatory mediators (interleukin [IL]-1ß, IL-6, IL-8, matrix metalloproteinase [MMP]-1, MMP-3, and tumor necrosis factor-α). Our results showed significantly increased senescence features including p16, lipofuscin, and ß-galactosidase in both epithelial and connective tissues of periodontitis patients compared with healthy sites in all age groups, indicating that an inflammatory microenvironment can trigger senescence-like alterations in younger diseased gingival tissues as well. Subsequent analyses using double staining with specific cell markers noted the enrichment of ß-galactosidase in fibroblasts and macrophages. Concurrently, inflammatory mediators consistent with SASP were increased in the gingival biopsies obtained from periodontitis lesions. Together, our findings provide the first clinical report revealing susceptibility to elevated senescence and inflammatory milieu consistent with senescence secretome in gingival tissues, thus introducing senescence as one of the drivers of pathological events in the oral mucosa and a novel strategy for targeted interventions.

TLR9 Mediates Periodontal Aging by Fostering Senescence and Inflammaging.

TLR9 is a critical nucleic acid sensing receptor in mediating periodontitis and periodontitis-associated comorbidities. Emerging evidence implicates TLR9 as a key sensor during aging, although its participation in periodontal aging is unexplored. Here, we investigated whether TLR9-mediated host responses can promote key hallmarks of aging, inflammaging, and senescence, in the course of periodontitis using a multipronged approach comprising clinical and preclinical studies. In a case-control model, we found increased TLR9 gene expression in gingival tissues of older (≥55 y) subjects with periodontitis compared to older healthy subjects as well as those who are younger (<55 y old) with and without the disease. Mechanistically, this finding was supported by an in vivo model in which wild-type (WT) and TLR9-/- mice were followed for 8 to 10 wk (young) and 18 to 22 mo (aged). In this longitudinal model, aged WT mice developed severe alveolar bone resorption when compared to their younger counterpart, whereas aged TLR9-/- animals presented insignificant bone loss when compared to the younger groups. In parallel, a boosted inflammaging milieu exhibiting higher expression of inflammatory/osteoclast mediators (Il-6, Rankl, Cxcl8) and danger signals (S100A8, S100A9) was noted in gingival tissues of aged WT mice compared to the those of aged TLR9-/- mice. Consistently, WT aged mice displayed an increase in prosenescence balance as measured by p16INK4a/p19ARF ratio compared to the younger groups and aged TLR9-/- animals. Ex vivo experiments with bone marrow-derived macrophages primed by TLR9 ligand (ODN 1668) further corroborated in vivo and clinical data and showed enhanced inflammatory-senescence circuit followed by increased osteoclast differentiation. Together, these findings reveal first systematic evidence implicating TLR9 as one of the drivers of periodontitis during aging and functioning by boosting a deleterious inflammaging/senescence environment. This finding calls for further investigations to determine whether targeting TLR9 will improve periodontal health in an aging population.

Human Bone Marrow Stromal Cell Exosomes Ameliorate Periodontitis.

Human bone marrow stromal cell (hBMSC)-derived exosomes are promising therapeutics for inflammatory diseases due to their unique microRNA (miRNA) and protein cargos. Periodontal diseases often present with chronicity and corresponding exuberant inflammation, which leads to loss of tooth support. In this study, we explored whether hBMSC exosomes can affect periodontitis progression. hBMSC exosomes were isolated from cell culture medium through sequential ultracentrifugation. miRNAs and proteins that were enriched in hBMSC exosomes were characterized by RNA sequencing and protein array, respectively. hBMSC exosomes significantly suppressed periodontal keystone pathogen Porphyromonas gingivalis-triggered inflammatory response in macrophages in vitro. Transcriptomic analysis suggested that exosomes exerted their effects through regulating cell metabolism, differentiation, and inflammation resolution. In vivo, weekly exosome injection into the gingival tissues reduced the tissue destruction and immune cell infiltration in rat ligature-induced periodontitis model. Collectively, these findings suggest that hBMSC-derived exosomes can potentially be used as a host modulation agent in the management of periodontitis.

The Ubiquitin System and A20: Implications in Health and Disease.

Inflammation is triggered by stimulation of innate sensors that recognize pathogens, chemical and physical irritants, and damaged cells subsequently initiating a well-orchestrated adaptive immune response. Immune cell activation is a strictly regulated and self-resolving process supported by an array of negative feedback mechanisms to sustain tissue homeostasis. The disruption of these regulatory pathways forms the basis of chronic inflammatory diseases, including periodontitis. Ubiquitination, a covalent posttranslational modification of target proteins with ubiquitin, has a profound effect on the stability and activity of its substrates, thereby regulating the immune system at molecular and cellular levels. Through the cooperative actions of E3 ubiquitin ligases and deubiquitinases, ubiquitin modifications are implicated in several biological processes, including proteasomal degradation, transcriptional regulation, regulation of protein-protein interactions, endocytosis, autophagy, DNA repair, and cell cycle regulation. A20 (tumor necrosis factor α-induced protein 3 or TNFAIP3) is a ubiquitin-editing enzyme that mainly functions as an endogenous regulator of inflammation through termination of nuclear factor (NF)-κB activation as part of a negative feedback loop. A20 interacts with substrates that reside downstream of immune sensors, including Toll-like receptors, nucleotide-binding oligomerization domain-containing receptors, lymphocyte receptors, and cytokine receptors. Due to its pleiotropic functions as a ubiquitin binding protein, deubiquitinase and ubiquitin ligase, and its versatile role in various signaling pathways, aberrant A20 levels are associated with numerous conditions such as rheumatoid arthritis, diabetes, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, Sjögren syndrome, coronary artery disease, multiple sclerosis, cystic fibrosis, asthma, cancer, neurological disorders, and aging-related sequelae. Similarly, A20 has recently been implicated as an essential regulator of inflammation in the oral cavity. This review presents information on the ubiquitin system and regulation of NF-κB by ubiquitination using A20 as a representative molecule and highlights how the dysregulation of this system can lead to several immune pathologies, including oral cavity-related disorders mainly focusing on periodontitis.

Increasing popularity of waterpipe tobacco smoking and electronic cigarette use: Implications for oral healthcare.

Cigarette smoking increases the risk of developing several systemic conditions including cancer, cardiovascular and pulmonary diseases. Cigarette smoking is also detrimental to oral health as it increases the incidence and severity of oral cancer, periodontal diseases and peri-implantitis, as well as impacting negatively on the dental patients' response to therapy. Therefore, consideration of smoking behavior and recommendation of smoking cessation are important parts of dental treatment planning. However, cigarettes are no longer the most popular form of tobacco use among adolescents in the United States and globally. In recent years, tobacco smoking using a waterpipe ("hookah," "shisha") and use of electronic cigarettes (ECIGs) has increased significantly. Thus, dental clinicians likely will treat more patients who are waterpipe and/or ECIG users. Yet, the literature on the health effects of waterpipe and ECIGs use is sparse. Both waterpipe and ECIGs deliver the dependence-producing drug nicotine. Waterpipe tobacco smoking has been associated with periodontitis, dry socket, premalignant lesions, and oral and esophageal cancer. The health effects of long-term ECIG use are unknown. The purpose of this review is to inform healthcare professionals about waterpipes and ECIGs, highlight emerging evidence on the biological effects of these increasingly popular tobacco products, and introduce perspectives for dental patient management and future research.

Microbial Nucleic Acid Sensing in Oral and Systemic Diseases.

One challenge in studying chronic infectious and inflammatory disorders is understanding how host pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), sense and respond to pathogen- or damage-associated molecular patterns, their communication with each other and different components of the immune system, and their role in propagating inflammatory stages of disease. The discovery of innate immune activation through nucleic acid recognition by intracellular PRRs such as endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) and cytoplasmic proteins (absent in melanoma 2 and DNA-dependent activator of interferon regulatory factor) opened a new paradigm: Nucleic acid sensing is now implicated in multiple immune and inflammatory conditions (e.g., atherosclerosis, cancer), viral (e.g., human papillomavirus, herpes virus) and bacterial (e.g., Helicobacter pylori, pneumonia) diseases, and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis). Clinical investigations reveal the overexpression of specific nucleic acid sensors in diseased tissues. In vivo animal models show enhanced disease progression associated with receptor activation. The involvement of nucleic acid sensors in various systemic conditions is further supported by studies reporting receptor knockout mice being either protected from or prone to disease. TLR9-mediated inflammation is also implicated in periodontal diseases. Considering that persistent inflammation in the oral cavity is associated with systemic diseases and that oral microbial DNA is isolated at distal sites, nucleic acid sensing may potentially be a link between oral and systemic diseases. In this review, we discuss recent advances in how intracellular PRRs respond to microbial nucleic acids and emerging views on the role of nucleic acid sensors in various systemic diseases. We also highlight new information on the role of intracellular PRRs in the pathogenesis of oral diseases including periodontitis and oral cavity cancer, which might offer future possibilities for disease prevention and therapy.

Novel inflammatory pathways in periodontitis.

New insights into the biological mechanisms involved in modulating periodontal inflammation and alveolar bone loss are paving the way for novel therapeutic strategies for periodontitis. The neutrophil adhesion cascade for transmigration in response to infection or inflammation is a key paradigm in immunity. Developmental endothelial locus-1 (Del-1) is one of several newly identified endogenous inhibitors of the leukocyte adhesion cascade. Del-1 competes with intercellular adhesion molecule-1 (ICAM-1) on endothelial cells for binding to the LFA-1 integrin on neutrophils, thereby regulating neutrophil recruitment and local inflammation. In animal periodontitis models, Del-1 deficiency resulted in severe inflammation and alveolar bone loss, but local treatment with recombinant Del-1 prevented neutrophil infiltration and bone loss. The expression of Del-1 is inhibited by the pro-inflammatory cytokine IL-17. Nucleic-acid-receptor-mediated inflammatory responses may be important in periodontal disease pathogenesis. Bacterial nucleic acids released during inflammation are detected by host microbial DNA sensors, e.g., Toll-like receptor-9 (TLR-9), leading to the activation of pro- and/or anti-inflammatory signaling pathways. DNA from periodontitis-associated bacteria induced pro-inflammatory cytokine production in human macrophage-like cells through the TLR-9 and NF-κB signaling pathways, but had less effect on human osteoblasts. Inhibition of TLR-9 signaling in human macrophages reduced cytokine production in response to P. gingivalis DNA. Differential expression of a polymorphic site in the TLR-9 gene promoter region and increased TLR-9 gene and protein expression were reported in chronic periodontitis. Further research to confirm that periodontal bacterial DNA contributes to destructive inflammation in vivo could provide alternative therapeutic targets to control periodontitis.

Single nucleotide polymorphisms of pattern recognition receptors and chronic periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial disease influenced partly by genetics. Activation of pattern recognition receptors (PRRs) can lead to the up-regulation of inflammatory pathways, resulting in periodontal tissue destruction. Hence, functional polymorphisms located in PRRs can explain differences in host susceptibility to periodontitis. This study investigated single nucleotide polymorphisms of PRRs including toll-like receptor (TLR)2 (G2408A), TLR4 (A896G), TLR9 (T1486C), TLR9 (T1237C) and CD14 (C260T) in patients with chronic periodontitis and in periodontally healthy subjects. METHODS: One-hundred and fourteen patients with chronic periodontitis and 77 periodontally healthy subjects were genotyped using TaqMan® allelic discrimination assays. Fisher's exact test and chi-square analyses were performed to compare genotype and allele frequencies. RESULTS: The frequency of subjects with the CC genotype of CD14 (C260T) (24.6% in the chronic periodontitis group vs. 13% in the periodontally healthy group) and those expressing the T allele of CD14 (C260T) (CT and TT) (75.4% in the chronic periodontitis group vs. 87% in the periodontally healthy group) was statistically different among groups (p = 0.04). Homozygocity for the C allele of the CD14 (C260T) polymorphism (CC) was associated with a two--fold increased susceptibility to periodontitis (p = 0.04; odds ratio, 2.49; 95% confidence interval, 1.06-6.26). Individuals with the CC genotype of TLR9 (T1486C) (14.9% in the chronic periodontitis group vs. 28.6% in the periodontally healthy group) and those expressing the T allele of TLR9 (T1486C) (CT and TT) (85.1% in the chronic periodontitis group vs. 71.4% in the periodontally healthy group) were also significantly differently distributed between groups without adjustment (p = 0.03). Further analysis of nonsmokers revealed a significant difference in the distribution of genotypes between groups for TLR9 (T1486C; p = 0.017) and CD14 (C260T; p = 0.03), polymorphisms again without adjustment. CONCLUSION: The CC genotype of CD14 (C260T) is related to susceptibility to chronic periodontitis in Caucasians. In addition, differences observed in the distribution of TLR9 (T1486C) genotypes between groups warrant further investigation.

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines.

Toll-like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP-1). THP-1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA-initiated cytokine production was determined either by blocking TLR9 signaling in THP-1 cells with chloroquine or by measuring IL-8 production and nuclear factor-kappaB (NF-kappaB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL-1beta, IL-6, and TNF-alpha) was increased significantly in bDNA-stimulated cells compared with controls. Chloroquine treatment of THP-1 cells decreased cytokine production, suggesting that TLR9-mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9-transfected cells demonstrated significantly increased IL-8 production (P < 0.001) and NF-kappaB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF-kappaB genes in response to periodontal bDNA in THP-1 cells, suggesting that cytokine induction is through NF-kappaB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.

Fibrinogen-neutrophil interactions in response to fMLP and Porphyromonas gingivalis fimbrial peptides.

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61-80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61-80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171-185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.