Inflammation and tissue remodeling mediator expression during gingivitis: A comparison between experimental, naturally occurring gingivitis, and periodontal health.

BACKGROUND: The aim of this study is to evaluate the immune regulation and tissue remodeling responses during experimental gingivitis (EG) and naturally occurring gingivitis (NG) to provide a comprehensive analysis of host responses. Gingival crevicular fluid (GCF) was obtained from 2 human studies conducted in university settings. METHODS: The EG study enrolling 26 volunteers provided controls for the baseline (Day 0) from healthy disease-free participants, while Day 21 (the end of EG induction of the same group) was used to represent EG. Twenty-six NG participants age-matched with those of the EG group were recruited. GCF samples were analyzed for 39 mediators of inflammatory/immune responses and tissue remodeling using commercially available bead-based multiplex immunoassays. The differences in GI and mediator expression among groups were determined at a 95% confidence level (p ≤ 0.05) by a 2-way analysis of variance (ANOVA) with a post-hoc Tukey's test. RESULTS: Our findings showed that EG had a greater gingival index than NG and was healthy (p < 0.01 of all comparisons). Furthermore, EG showed significantly higher levels of MPO (p < 0.001), CCL3 (p < 0.05), and IL-1B (p < 0.001) than NG. In contrast, NG had increased levels of MIF (p < 0.05), Fractalkine (p < 0.001), angiogenin (p < 0.05), C3a (p < 0.001), BMP-2 (p < 0.001), OPN (p < 0.05), RANKL (p < 0.001), and MMP-13 (p < 0.001) than EG. CONCLUSIONS: Consistent with the findings from chronic (NG) versus acute (EG) inflammatory lesions, these data reveal that NG displays greater immune regulation, angiogenesis, and bone remodeling compared to EG.

Macrophage migration inhibitory factor regulates specific innate immune sensor responses in gingival epithelial cells.

BACKGROUND: The gingival epithelium protects periodontal tissues and the alveolar bone by maintaining a steady state of regulated inflammatory surveillance, also known as healthy homeostasis. Accordingly, the repertoire of receptors present within the gingival epithelium showcases its ability to recognize microbial colonization and contribute to bacterial sensing. Macrophage migration inhibitory factor (MIF) is one of many cytokines that are expressed in this protective state and is involved in neutrophil regulation. However, its role in the maintenance of healthy gingival tissue has not been described. METHODS: Gingival tissues from wild-type (WT) and Mif knock-out (KO) mice were stained for neutrophils and three key neutrophil chemoattractants: MIF, Gro-α/CXCL1, and Gro-ß/CXCL2 in the junctional epithelium (JE). In addition, gene silencing studies were performed using gingival epithelial cells (GECs) to examine the role of MIF on transcription of key bacterial recognition receptors Toll-like receptors (TLR)-1, -2, -4, -6, -9 and interleukin-1 receptors (IL-1R1 and IL-1R2) in response to oral bacterial stimulation. RESULTS: WT murine gingival tissues demonstrated high expression of MIF in the JE. In Mif KO mice, despite the significant reduction of Gro-α/CXCL1 and Gro-ß/CXCL2, there was a slight increase in neutrophils. Gene silencing experiments showed that MIF down-regulated the mRNA expression of TLR4, IL-1R1, and IL-1R2 in GEC, in addition to decreasing secreted IL-8/CXCL8 in response to bacteria. CONCLUSIONS: MIF regulates the expression of TLR4, IL-1Rs, and IL-8/CXCL8, components that are all involved in maintaining oral health. Our data demonstrate that MIF is a significant contributor to the maintenance of healthy oral homeostasis.

Comparison of the sealing ability of various bioceramic materials for endodontic surgery.

OBJECTIVES: Endosequence Bioceramic Root Repair Material (BC-RRM) is used in endodontic microsurgery. It is available as a paste and a putty. However, no studies to date have examined the sealing ability of these forms alone or in combination as root-end filling materials. Hence, this study aimed to compare the sealing properties of these 2 forms of BC-RRM. MATERIALS AND METHODS: Forty-two extracted upper anterior teeth were divided into 3 experimental groups, a positive and negative control. After the root canal treatment, the root ends were resected, retroprepared and retrofilled with either putty, paste + putty or mineral trioxide aggregate (MTA). The teeth were mounted in tubes so the apical 3 mm was submerged in Brain Heart Infusion (BHI) broth. The coronal portions of the canals were inoculated with Enterococcus faecalis and BHI broth and incubated for 30 days. The broth in the tubes was analyzed for colony forming units to check for leakage of bacteria from the canal. The teeth from the groups were sectioned and analyzed using scanning electron microscopy (SEM). The Kruskal-Wallis test and analysis of variance were used to analyze the data with a significance level p < 0.05. RESULTS: The BC-RRM and MTA groups showed similar sealing ability. The positive control showed leakage in all samples. The SEM imaging showed the presence of bacteria in all experimental groups at the material-tooth interface. CONCLUSIONS: No significant differences were noted in the experimental groups, providing sufficient evidence that any combination could be effectively used during endodontic microsurgery.

Pandemic-Related Instructor Talk: How New Instructors Supported Students at the Onset of the COVID-19 Pandemic.

At the same time that COVID-19 cases in the United States first began to increase, fellows in a mentored teaching apprenticeship for postdoctoral scientists began to teach undergraduate seminars. The fellows suddenly needed to support students emotionally and switch to online instruction. They were encouraged to acknowledge and address the pandemic during each class and decided to do so. In this case study, we examined the language fellows used in response to this encouragement, hypothesizing that they would engage in a variety of pandemic-related instructor talk, i.e., language that instructors use in the classroom that is not directly tied to educational content. We analyzed transcripts from 17 2-hour undergraduate biology seminar courses and found 167 instances of pandemic-related instructor talk. We used grounded theory to identify categories that emerged from these quotations: Positive coping mechanisms and self-care; Adjusting to online learning; Compassionate instruction; Personal impacts; COVID-19 and society; Dreaming; and Biology of COVID-19. Talk in these categories may help build relationships among instructors and students. The category about quickly Adjusting to online learning is unique, in that it is unlikely that there will be another time that will require simultaneous and rapid national movement to online instruction. In addition, four of the seven categories are direct consequences of COVID-19 specifically, and thus are unique to this time. Analyzing pandemic-related instructor talk has shed light on how new instructors navigated the trials of teaching in 2020.

Maintaining homeostatic control of periodontal epithelial tissue.

Years of coevolution with resident microbes has made them an essential component of health. Yet, little is known about oral commensal bacteria's contribution to and role in the maintenance of oral health and homeostasis. Commensal bacteria are speculated to play a host protective role in the maintenance of health. In this review, we describe and provide examples of the coordinate regulation that occurs between oral commensal bacteria and the host innate immune response to modulate and maintain oral homeostasis.

Clinically Healthy Human Gingival Tissues Show Significant Inter-individual Variability in GCF Chemokine Expression and Subgingival Plaque Microbial Composition.

Aim: Clinically healthy gingival tissue is maintained through controlled regulation of host defense mechanisms against plaque biofilm overgrowth. One key component is the transit of neutrophils from the vasculature into gingival tissue where the expression of different neutrophil chemokines are tightly regulated. This cross-sectional study examines the inter-individual variability in chemokine profiles within gingival crevicular fluid (GCF) in relation to the subgingival bacterial community in a state of gingival health. Methods: Gingival crevicular fluid and subgingival plaque samples were collected from mesiobuccal surfaces of all six Ramfjord teeth of 20 systemically healthy individuals (14.55 ± 1.67 years). A multiplex immunoassay was carried out to quantify the expression of 40 different chemokines in the healthy gingival tissue. Neutrophils were assessed indirectly by myeloperoxidase (MPO) in GCF using traditional ELISA. Characterization of healthy subgingival plaque was conducted with the Illumina Miseq targeting the 16S rRNA gene. Results: In health, there are distinct variations within individual gingival crevicular fluid chemokine expression profiles, as well as in the concentration of neutrophils, that divided the participants into high or low chemokine expressing groups. Specifically, key differences were identified within MIF (2683.54 ± 985.82 pg per 30-s sample), IL-8/CXCL8 (170.98 ± 176.96 pg per 30-s sample), Gro-α/CXCL1 (160.42 ± 94.21 pg per 30-s sample), ENA-78/CXCL5 (137.76 ± 76.02 pg per 30-s sample), IL-1ß (51.39 ± 37.23 pg per 30-s sample), TNF-α (1.76 ± 1.79 pg per 30-s sample), and IFN-γ (0.92 ± 0.54 pg per 30-s sample). Of these identified chemokines, the highest correlation was associated between IL-8/CXCL8 and neutrophils (r = 0.54, p = 0.014). Furthermore, species characterization of healthy subgingival plaque revealed significant inter-individual variability that identified two unique groups unrelated to the previously identified chemokine groups. Conclusion: The lack of concordance between the microbial composition and chemokine profile during health may be a reflection of the unique microbial composition of each individual coupled with variations within their host response, emphasizing the vast complexity of the defense mechanisms in place to maintain gingival health.

An Ayurvedic herbal extract inhibits oral epithelial cell IL-8 responses to host and bacterial agonists.

BACKGROUND: Natural products constitute a promising class of therapeutics for the treatment of gingivitis and periodontitis as well as the maintenance of oral health. However, the limited understanding behind their potential mechanisms and modes of action have hampered their incorporation into popular western therapeutics. This in vitro study characterizes an Ayurvedic herbal extract mixture, which has been clinically shown to promote gingival health and homeostasis. METHODS: Telomerase immortalized gingival keratinocytes (TIGK) were infected with either Fusobacterium nucleatum cell wall, live F. nucleatum, IL-1ß or TNF-α for 4 hours with and without the herbal extract. The immunomodulatory effects of the extract on host IL-8 production was measured by ELISA. RESULTS: It was found that the Ayurvedic herbal extract mixture inhibited gingival epithelial cell IL-8 expression in response to both bacterial and host cytokine agonists. The herbal extract inhibited IL-8 stimulated by F. nucleatum cell wall, live F. nucleatum, IL-1ß, and TNF-α in a dose-dependent manner that was not a result of host cell death. Furthermore, the extract showed significantly different ID50 doses demonstrating the differential ability to modulate both stimulated and basal IL-8 levels. CONCLUSIONS: In vitro investigation of this herbal extract mixture revealed that it has the ability to modulate gingival epithelial cell IL-8 expression in response to stimulation by bacterial components and host pro-inflammatory signals. This data demonstrates that the reduction in the gingival epithelial cell IL-8 response may in part be responsible for the previously reported ability of the Ayurvedic herbal extract mixture to reduce gingivitis in two separate human clinical studies.

An Ayurvedic Herbal Extract Inhibits Streptococcus mutans Biofilm Formation and Disrupts Preformed Biofilms in vitro.

OBJECTIVE: Sudantha® (SUD), a natural proprietary mixture of herbal extracts that has been incorporated into toothpaste, has been shown in two separate placebo controlled human clinical studies to promote gingival health; and reduce gingival bleeding and plaque formation. However, the herbal based anti-gingivitis mechanisms of Sudantha are not fully understood. The objective of this study was to determine the effect of Sudantha on dental plaque biofilms by investigating its effect on mono-culture biofilms of a primary colonizer, Streptococcus mutans, in vitro. RESULTS: This study found that SUD contributes to the maintenance of oral health through the inhibition of S. mutans biofilm formation. In addition, SUD disrupted preformed S. mutans biofilms after exposure to SUD for 4 hours. Together, this pilot data suggests the inhibition of S. mutans biofilm formation and disruption represents one potential mechanism by which the herbal extract is able to reduce the oral bacterial biofilm resulting in its effective against gingivitis and its potential use in countering biofilm associated oral disease.

Toll-like receptor-2 and -4 responses regulate neutrophil infiltration into the junctional epithelium and significantly contribute to the composition of the oral microbiota.

BACKGROUND: Oral gingival tissue, especially the junctional epithelium (JE), is constantly exposed to sub-gingival plaque. A key component of gingival health is the regulation of the number of neutrophils that migrate into the gingival crevice to counteract its harmful effects. This report investigates the contribution of innate defense receptors, Toll-like receptor (TLR)2, TLR4, and both (TLR2/4) to the maintenance of neutrophil homeostasis in the JE. METHODS: Bacterial composition was analyzed from whole oral swabs collected from 12- to 14-week-old TLR2, TLR4, TLR2/4 double knock-out (KO) mice using a MiSeq platform targeting the V3-V4 region of the 16S ribosomal RNA gene. Mandibles were histologically examined for quantification of neutrophils in the JE and bone loss. Lastly, total bacterial load was quantitated using quantitative real-time PCR. RESULTS: Compared with wild-type, all TLR KO mice displayed significantly increased recruitment of neutrophils (P = 0.0079) into the JE. In addition, TLR4 and TLR2/4 KO mice demonstrated a significant increase in the number of bacteria (P = 0.0022 and P = 0.0152, respectively). Lastly, comparative compositional analyses of the oral microbiome revealed that each KO strain harbored unique microbial communities that are distinct from each other but maintained similar levels of alveolar bone. CONCLUSIONS: Neutrophil migration into healthy mouse JE does not require TLR2 or TLR4. However, a significant increase in the number of neutrophils as well as a significant change in the oral microbial composition in both TLR2 and TLR4 KO mice demonstrate that these TLRs contribute to the homeostatic relationship between bacteria and the host in healthy mice periodontal tissue.

Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, in Porphyromonas gingivalis.

Removal of one acyl chain from bacterial lipid A by deacylase activity is a mechanism used by many pathogenic bacteria to evade the host's Toll-like receptor 4 (TLR4)-mediated innate immune response. In Porphyromonas gingivalis, a periodontal pathogen, lipid A deacylase activity converts a majority of the initially synthesized penta-acylated lipid A, a TLR4 agonist, to tetra-acylated structures, which effectively evade TLR4 sensing by being either inert or antagonistic at TLR4. In this paper, we report successful identification of the gene that encodes the P. gingivalis lipid A deacylase enzyme. This gene, PGN_1123 in P. gingivalis 33277, is highly conserved within P. gingivalis, and putative orthologs are phylogenetically restricted to the Bacteroidetes phylum. Lipid A of ΔPGN_1123 mutants is penta-acylated and devoid of tetra-acylated structures, and the mutant strain provokes a strong TLR4-mediated proinflammatory response, in contrast to the negligible response elicited by wild-type P. gingivalis Heterologous expression of PGN_1123 in Bacteroides thetaiotaomicron promoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCE Periodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population. Porphyromonas gingivalis is a bacterium closely associated with periodontitis, although how and if it is a cause for the disease are not known. It has a formidable capacity to dampen the host's innate immune response, enabling its persistence in diseased sites and triggering microbial dysbiosis in animal models of infection. P. gingivalis is particularly adept at evading the host's TLR4-mediated innate immune response by modifying the structure of lipid A, the TLR4 ligand. In this paper, we report identification of the gene encoding lipid A deacylase, a key enzyme that modifies lipid A to TLR4-evasive structures.

A novel class of lipoprotein lipase-sensitive molecules mediates Toll-like receptor 2 activation by Porphyromonas gingivalis.

Infection by the chronic periodontitis-associated pathogen Porphyromonas gingivalis activates a Toll-like receptor 2 (TLR2) response that triggers inflammation in the host but also promotes bacterial persistence. Our aim was to define ligands on the surfaces of intact P. gingivalis cells that determine its ability to activate TLR2. Molecules previously reported as TLR2 agonists include lipopolysaccharide (LPS), fimbriae, the lipoprotein PG1828, and phosphoceramides. We demonstrate that these molecules do not comprise the major factors responsible for stimulating TLR2 by whole bacterial cells. First, P. gingivalis mutants devoid of the reported protein agonists, PG1828 and fimbriae, activate TLR2 as strongly as the wild type. Second, two-phase extraction of whole bacteria resulted in a preponderance of TLR2 agonist activity partitioning to the hydrophilic phase, demonstrating that phosphoceramides are not a major TLR2 ligand. Third, analysis of LPS revealed that TLR2 activation is independent of lipid A structural variants. Instead, activation of TLR2 and TLR2/TLR1 by LPS is in large part due to copurifying molecules that are sensitive to the action of the enzyme lipoprotein lipase. Strikingly, intact P. gingivalis bacterial cells treated with lipoprotein lipase were attenuated in their ability to activate TLR2. We propose that a novel class of molecules comprised by lipoproteins constitutes the major determinants that confer to P. gingivalis the ability to stimulate TLR2 signaling.