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Objective Method Results Conclusion Periodontitis is an inflammatory oral disease that occurs as a result of the damaging effects of the immune response against the subgingival microflora. Among the mechanisms involved, the nucleotide‐binding oligomerization domain, leucine‐rich repeat‐containing proteins family member NLRP3 (NLR family pyrin domain‐containing 3), proposed as the key regulator of macrophage‐induced inflammation, is strongly associated with periodontal disease due to the bacterial activators. This paper aimed to present key general concepts of NLRP3 inflammasome activation and regulation in periodontal disease.A narrative review was conducted in order to depict the current knowledge on the relationship between NLRP3 inflammasome activity and periodontal disease. In vitro and in situ studies were retrieved and commented based on their relevance in the field.The NLRP3 inflammasome activity stimulated by periodontal microbiota drive periodontal disease pathogenesis and progression. This occurs through the release of proinflammatory cytokines IL‐1β, IL‐18, and DAMPs (damage‐associated molecular pattern molecules) following inflammasome activation. Moreover, the tissue expression of NLRP3 is dysregulated by oral microbiota, further exacerbating periodontal inflammation.The review provides new insights into the relationship between the NLRP3 inflammasome activity and periodontal disease pathogenesis, highlighting the roles and regulatory mechanism of inflammatory molecules involved in the disease process. [ABSTRACT FROM AUTHOR]

The oral organism Tannerella forsythia is auxotrophic for peptidoglycan amino sugar N‐acetylmuramic acid (MurNAc). It survives in the oral cavity by scavenging MurNAc‐ and MurNAc‐linked peptidoglycan fragments (muropeptides) secreted by co‐habiting bacteria such as Fusobacterium nucleatum with which it forms synergistic biofilms. Muropeptides, MurNAc‐l‐Ala‐d‐isoGln (MDP, muramyl dipeptide) and d‐γ‐glutamyl‐meso‐DAP (iE‐DAP dipeptide), are strong immunostimulatory molecules that activate nucleotide oligomerization domain (NOD)‐like innate immune receptors and induce the expression of inflammatory cytokines and antimicrobial peptides. In this study, we utilized an in vitro T. forsythia–F. nucleatum co‐culture model to determine if T. forsythia can selectively scavenge NOD ligands from the environment and impact NOD‐mediated inflammation. The results showed that NOD‐stimulatory molecules were secreted by F. nucleatum in the spent culture broth, which subsequently induced cytokine and antimicrobial peptide expression in oral epithelial cells. In the spent broth from T. forsythia–F. nucleatum co‐cultures, the NOD‐stimulatory activity was significantly reduced. These data indicated that F. nucleatum releases NOD2‐stimulatory muropeptides in the environment, and T. forsythia can effectively scavenge the muropeptides released by co‐habiting bacteria to dampen NOD‐mediated host responses. This proof‐of‐principle study demonstrated that peptidoglycan scavenging by T. forsythia can impact the innate immunity of oral epithelium by dampening NOD activation. [ABSTRACT FROM AUTHOR]

Tannerella forsythia is strongly implicated in the development of periodontitis, an inflammatory disease that destroys the bone and soft tissues supporting the tooth.  To date, the knowledge of the virulence attributes of T. forsythia species has mainly come from studies with a laboratory adapted strain (ATCC 43037). In this study, we focused on two T. forsythia clinical isolates, UB4 and UB20, in relation to their ability to activate macrophages. We found that these clinical isolates differentially induced proinflammatory cytokine expression in macrophages. Prominently, the expression of the chemokine protein IP-10 (CXCL10) was highly induced by UB20 as compared to UB4 and the laboratory strain ATCC 43037. Our study focused on the lipopolysaccharide component (LPS) of these strains and found that UB20 expressed a smooth-type LPS, unlike UB4 and ATCC 43037 each of which expressed a rough-type LPS. The LPS from UB20, via activation of TLR4, was found to be a highly potent inducer of IP-10 expression via signaling through STAT1 (signal transducer and activator of transcription-1). These data suggest that pathogenicity of T. forsythia species could be strain dependent and the LPS heterogeneity associated with the clinical strains might be responsible for their pathogenic potential and severity of periodontitis. [ABSTRACT FROM AUTHOR]

Periodontitis is a bacterially-induced inflammatory disease that leads to tooth loss. It results from the damaging effects of a dysregulated immune response, mediated largely by neutrophils, macrophages, T cells and B cells, on the tooth-supporting tissues including the alveolar bone. Specifically, infiltrating B cells at inflamed gingival sites with an ability to secrete RANKL and inflammatory cytokines are thought to play roles in alveolar bone resorption. However, the direct contribution of B cells in alveolar bone resorption has not been fully appreciated. In this study we sought to define the contribution of RANKL expressing B cells in periodontitis by employing a mouse model of pathogen-induced periodontitis that used conditional knockout mice with B cell-targeted RANKL deletion. Briefly, alveolar bone loss was assessed in the wild-type, B-cell deficient (Jh), or B-cell-RANKL deleted (RANKLΔB) mice orally infected with the periodontal pathogen Tannerella forsythia. The RANKLΔB mice were obtained by crossing Cd19-Cre knock-in mice with mice homozygous for conditional RANKL-flox allele (RANKLflox/flox). The alveolar bone resorption was determined by morphometric analysis and osteoclastic activity of the jaw bone. In addition, the bone resorptive potential of the activated effector B cells was assessed ex vivo. The data showed that the RANKL producing B cells increased significantly in the T. forsythia -infected wild-type mice compared to the sham-infected mice. Moreover, T. forsythia -infection induced higher alveolar bone loss in the wild-type and RANKLflox/flox mice compared to infection either in the B cell deficient (Jh) or the B-cell specific RANKL deletion (RANKLΔB) mice. These data established that the oral-pathogen activated B cells contribute significantly to alveolar bone resorption via RANKL production. [ABSTRACT FROM AUTHOR]

The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/ Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sjögren's syndrome. [ABSTRACT FROM AUTHOR]

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A4 (LXA4) binds to the G proteincoupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA4 inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA4 thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA4 has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA4 decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA4 blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA4. Furthermore, LXA4 preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule- 1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS). [ABSTRACT FROM AUTHOR]

The objective of this chapter is to provide a detailed protocol for the peptidoglycan (cell wall) labeling of the periodontal pathogen Tannerella forsythia and the development of a laboratory-safe Escherichia coli strain utilizing the N-acetylmuramic acid recycling enzymes AmgK, N-acetylmuramate/N-acetylglucosamine kinase, and MurU, N-acetylmuramate alpha-1-phosphate uridylyltransferase, from T. forsythia. The procedure involves bioorthogonal labeling of bacterial cells with an azido-modified analog of the amino sugar, N-acetylmuramic acid, through "click chemistry" with a fluorescent dye. The protocol is suitable for the generation of fluorescently labeled peptidoglycan molecules for applications in the study of bacterial and peptidoglycan trafficking in the host cells and cell wall recycling in complex microbiomes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The objective of this chapter is to provide a detailed purification protocol for the surface-layer (S-layer) glycoproteins of the periodontal pathogen Tannerella forsythia. The procedure involves detergent based solubilization of the bacterial S-layer followed by cesium chloride gradient centrifugation and gel permeation chromatography. The protocol is suitable for the isolation of S-layer glycoproteins from T. forsythia strains with diverse O-glycan structures, and aid in understanding the biochemical basis and the role of protein O-glycosylation in bacterial pathogenesis.

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).