Periodontitis Samples Research Articles

SMDI: An Index for Measuring Subgingival Microbial Dysbiosis

An intuitive, clinically relevant index of microbial dysbiosis as a summary statistic of subgingival microbiome profiles is needed. Here, we describe a subgingival microbial dysbiosis index (SMDI) based on machine learning analysis of published periodontitis/health 16S microbiome data. The raw sequencing data, split into training and test sets, were quality filtered, taxonomically assigned to the species level, and centered log-ratio transformed. The training data set was subject to random forest analysis to identify discriminating species (DS) between periodontitis and health. DS lists, compiled by various “Gini” importance score cutoffs, were used to compute the SMDI for samples in the training and test data sets as the mean centered log-ratio abundance of periodontitis-associated species subtracted by that of health-associated ones. Diagnostic accuracy was assessed with receiver operating characteristic analysis. An SMDI based on 49 DS provided the highest accuracy with areas under the curve of 0.96 and 0.92 in the training and test data sets, respectively, and ranged from −6 (most normobiotic) to 5 (most dysbiotic) with a value around zero discriminating most of the periodontitis and healthy samples. The top periodontitis-associated DS were Treponema denticola, Mogibacterium timidum, Fretibacterium spp., and Tannerella forsythia, while Actinomyces naeslundii and Streptococcus sanguinis were the top health-associated DS. The index was highly reproducible by hypervariable region. Applying the index to additional test data sets in which nitrate had been used to modulate the microbiome demonstrated that nitrate has dysbiosis-lowering properties in vitro and in vivo. Finally, 3 genera (Treponema, Fretibacterium, and Actinomyces) were identified that could be used for calculation of a simplified SMDI with comparable accuracy. In conclusion, we have developed a nonbiased, reproducible, and easy-to-interpret index that can be used to identify patients/sites at risk of periodontitis, to assess the microbial response to treatment, and, importantly, as a quantitative tool in microbiome modulation studies.

Crosstalk between RNA-Binding Proteins and Immune Microenvironment Revealed Two RBP Regulatory Patterns with Distinct Immunophenotypes in Periodontitis.

Periodontitis is an inflammatory disease whose pathogenesis is closely related with immunology. RNA-binding proteins (RBPs) were found to play crucial roles in immunity. Therefore, we aimed to investigate the potential impact of RBPs in the immune microenvironment in periodontitis. The differential expressions of RBPs in periodontitis and healthy samples were determined and were used to construct an RBP-based classifier for periodontitis using logistic regression. The correlations between RBPs and immune characteristics were investigated by the Spearman correlation. Unsupervised clustering was conducted to identify the RBP regulatory patterns. RBP-related genes were identified by WGCNA, while biological distinctions were revealed by GSVA and GO. 24 dysregulated RBPs were identified, from which a 12-RBP classifier was established to distinguish periodontitis with AUC of 0.942. Close protein-protein interactions and expression correlations were observed especially between SPATS2 and ISG20. ISG20 and ESRP1 were found to be highly correlated with immunocyte infiltration, immune signaling activation, and HLA expressions in periodontitis. Two distinct RBP regulatory patterns were identified with different immune and other biological characteristics in periodontitis. Our findings indicate a significant impact of RBPs in shaping the immune microenvironment in periodontitis, which might bring new insights into the understanding of immune mechanisms in the pathogenesis of periodontitis.

Intron 4 VNTR A/B polymorphism of endothelial nitric oxide synthase gene in periodontitis

Nitric Oxide (NO) is an important mediator in the inflammatory and immune systems. The eNOS gene is one of the three isoforms of Nitric Oxide Synthase (NOS), which is responsible for synthesizing NO. Periodontitis is an inflammatory disease in periodontal tissue with genetic involvement. Polymorphism in eNOS gene changes the functional aspect of this gene and is associated with several inflammatory diseases including periodontitis. Aim: To detect Endothelial Nitric Oxide Synthase intron 4 gene polymorphism in Indonesian population with periodontitis. Analysis of the Endothelial Nitric Oxide Synthase (eNOS) intron 4 gene polymorphism was observed by carrying out PCR method followed by electrophoresis for the analysis, without the usage of restriction enzyme. The chi-square test and odds ratio were performed for statistical analysis. In this study, there were 34 samples with AA genotype, 3 samples with AB genotype, and 13 samples with BB genotype in periodontitis group. Whereas in the control group, there were 41 samples with AA genotype and 9 samples with BB genotype. AB genotype was absent in the control group. In periodontitis group, there were 71 A alleles and 29 B alleles, and in the control group, 82 A alleles and 18 B alleles were found. Polymorphic genotypes and alleles were found higher in periodontitis sample (32% and 29%) than healthy controls (18%). The polymorphism of eNOS intron 4 was found in periodontitis patients. There is no significant distribution difference was found between the periodontitis patients and the control group. ENOS intron 4 gene polymorphism does not affect the risk of periodontitis.

M6A regulator-mediated RNA methylation modification patterns are involved in immune microenvironment regulation of periodontitis.

The role of epigenetic regulation in immunity is emerging, especially for RNA N6‐methyladenosine (m6A) modification. However, little is known about the role of m6A in the regulation of the immune microenvironment of periodontitis. Thus, we aim to investigate the impact of m6A modification in periodontitis immune microenvironment. The RNA modification patterns mediated by 23 m6A‐regulators were systematically evaluated in 310 periodontitis samples. The impact of m6A modification on immune microenvironment characteristics was explored, including infiltrating immunocytes, immune reaction gene‐sets and HLAs (human leukocyte antigen) gene. m6A phenotype‐related immune genes were also identified. 17 m6A regulators were dysregulated and a 15‐m6A regulator signature can well distinguish periodontitis and control samples. ALKBH5 and FMR1 are closely related to infiltrating monocyte abundance. ELAVL1 and CBLL1 are significant regulators in immune reaction of TNF_Family_Members_Receptors and Cytokine. The expression of HLA‐B and HLA‐DOA is affected by ALKBH5 and LRPPRC. 3 distinct RNA modification patterns mediated by 23 m6A regulators were identified. They differ from immunocyte abundance, immune reaction and HLA gene. 1631 m6A phenotype‐related genes and 70 m6A‐mediated immune genes were identified, and the biological functions of these were explored. Our finding demonstrated the m6A modification plays a crucial role in the diversity and complexity of the immune microenvironment of periodontitis.

Quantitative proteomic analysis in symptomatic and asymptomatic apical periodontitis.

To quantitatively and qualitatively compare the host proteomic profile in samples of symptomatic and asymptomatic apical periodontitis (AP) using nano-liquid chromatography-electron spray tandem mass spectrometry. Samples were obtained from 18 patients with radiographically evident AP, divided into symptomatic and asymptomatic groups (nine per group) according to clinical characteristics. After sample collection, protein extraction, purification and quantification of the samples were performed, which were analysed by reverse-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in expression of proteins between the groups were calculated using the Monte Carlo algorithm, considering P<0.05 for down-regulated proteins and 1-P>0.95 for up-regulated proteins. Proteins were identified with the embedded ion accounting algorithm in the software and a search of the Homo sapiens UniProt database. A total of 853 individual human proteins were identified. In the quantitative analysis, common proteins to both groups accounted for 143 proteins. Differences in expression between groups resulted in 51 up-regulated proteins (1-P>0.95) in the symptomatic group, including alpha-1-antitrypsin, protein S100-A8, myeloperoxidase, peroxiredoxin and lactotransferrin. This group also had 43 down-regulated proteins (P<0.05), comprising immunoglobulin, neutrophil defensin, pyruvate kinase and alpha-enolase. The qualitative analysis considered only the exclusive proteins of each group. For the symptomatic group, 318 complete proteins and 29 fragments were identified, such as dedicator of cytokinesis protein, intersectin, prostaglandin, phospholipase DDHD2 and superoxide dismutase. For the asymptomatic group, 326 complete proteins and 37 fragments were identified, including azurocidin, C-reactive protein, collagen alpha, cathepsin, heat shock and laminin. Quantitative differences in the expression of common proteins in cases of symptomatic and asymptomatic AP were found, which were mostly related to host immune response in both groups. Exclusive proteins in the symptomatic group were mainly related to the host response to the presence of viruses in endodontic infections, oxidative stress and proteolytic enzymes. The results provide a basis for a better understanding of cellular and molecular pathways involved in AP, establishing specific proteomic profiles for symptomatic and asymptomatic conditions.

Coculture method for in vitro cultivation of uncultured oral bacteria.

Purpose:The purpose of the study is to culture uncultured oral bacteria with helper strains using the coculture method from the subgingival plaque samples of chronic periodontitis patients.Materials and Methods:The samples were processed and inoculated on a blood agar medium enriched with hemin and Vitamin K. A helper strain Propionibacterium acnes (ATCC 6919) was cross-streaked across the inoculums to facilitate coculture. The plates were then incubated for 7 days with subsequent subculturing and further incubation.Results:Satellite colonies around helper strain showed one colony type of Porphyromonas gingivalis, one was of nonpigmented Prevotella, three were of Fusobacterium nucleatum and five isolates remained unidentified.Conclusions:Coculture could be used effectively as one of the methods in the isolation and in vitro cultivation of oral bacteria. Incubation using the anaerobic jar technique was found to be economical and efficient for the growth of anaerobic oral bacteria.

Autophagy-mediated regulation patterns contribute to the alterations of the immune microenvironment in periodontitis.

The relationship between autophagy and immunity has been thoroughly investigated. However, little is known about the role of autophagy in shaping the immune-microenvironment of periodontitis. Thus, we aim to explore the impact of autophagy on the immune-microenvironment of periodontitis. The expression distinctions of autophagy genes between healthy and periodontitis samples have been investigated. The connections between autophagy and immune characteristics including infiltrating immunocyte, immune reaction and human leukocyte antigen (HLA) gene were evaluated. The distinct autophagy-mediated expression patterns were identified and immune characteristics under distinct patterns were revealed. Autophagy phenotype-related genes were identified. 16 autophagy genes were dysregulated and a ten-autophagy classifier was constructed that can well distinguish periodontitis and healthy samples. Immune characteristics were closely related to autophagy: higher expression of EDEM1 positively relates to infiltrating activated B cell; NCKAP1 negatively relates to monocyte; CXCR4 enhances BCR Signaling Pathway and PEX3 decreases the activity of TNF Family Members Receptors; positive expression correlation of EDEM1-HLADOB and negative correlation of RAB11A-HLADOB were observed. Two distinct autophagy expression patterns were identified which demonstrated different immune characteristics. 4309 autophagy phenotype-related genes were identified, and 219 of them were related to immunity, whose biological functions were found to be involved in immunocyte regulations. Our study revealed the strong impact of autophagy on the immune-microenvironment of periodontitis and brought new insights into the understanding of the pathogenesis of periodontitis.

Cellular expression of DNA damage/repair and reactive oxygen/nitrogen species in human periodontitis and peri-implantitis lesions.

Aim of the studyTo evaluate differences in the cellular expression of DNA damage/repair and reactive oxygen/nitrogen species between human periodontitis and peri‐implantitis lesions.Material and methods40 patients presenting with generalized severe periodontitis and 40 patients with severe peri‐implantitis were included. Soft tissue biopsies were collected from diseased sites in conjunction with surgical therapy and prepared for histological analysis. Four regions of interest were identified: the pocket epithelium (PE), the infiltrated connective tissue (ICT), which was divided into one inner area facing the PE (ICT‐1) and one outer area (ICT‐2). A non‐infiltrated connective tissue area (NCT) lateral of the ICT was also selected.ResultsIt was demonstrated that the ICT of peri‐implantitis specimens was considerably larger and contained significantly larger area proportions and densities of CD68‐, MPO‐ and iNOS‐positive cells than that of periodontitis samples. Cellular densities were overall higher in the inner ICT zone lateral of the PE (ICT‐1) than in the outer ICT compartment (ICT‐2). While the NCT area lateral of the ICT comprised significantly larger proportions and densities of y‐H2AX‐, iNOS‐, NOX2‐, MPO‐ and PAD4/MPO‐positive cells in peri‐implantitis than in periodontitis sites, a reverse difference was noted for the area proportion and density of 8‐OHdG‐positive cells in the PE.ConclusionsIt is suggested that peri‐implantitis lesions are associated with an enhanced and upregulated host response and contain larger numbers of neutrophils, macrophages and iNOS‐positive cells than periodontitis lesions.

Identification of immune-related lncRNAs in periodontitis reveals regulation network of gene-lncRNA-pathway-immunocyte

BackgroundLong noncoding RNAs (lncRNAs), which is essential in regulating multiple biological functions, have been found to have pivotal roles in immune regulation. Since immune reaction and immunocytes are the key part in periodontitis progression, we aim to investigate the underlying lncRNA-immunity regulatory network of periodontitis. Methods: A series of bioinformatic algorithms were used to identify immune-related lncRNAs in periodontitis. Infiltrating immunocyte were calculated by MCP-count. Pathway activity were estimated by the GSVA. The relationships between immune-related lncRNA and periodontitis features were investigated including immune gene categories, perturbated lncRNAs, immunocytes and pathways. Immune-related periodontitis subtypes were identified by ConsensusClusterPlus algorithm. Immunocytes related gene-lncRNA modules were identified by WGCNA. Results: An integrated algorithm and pipeline to identify immune-related lncRNAs was developed and 1059 immune-related lncRNAs in 14 immune categories were identified, 291 of them were perturbated in periodontitis. An independent validation set verified the robustness of immune-related lncRNAs. A higher proportion of immune-related lncRNAs are correlated with immunocyte infiltration. Pathways associated with immune-related lncRNAs were also revealed. Two distinct immune-related periodontitis subtypes were identified according to perturbated immune-related lncRNAs with different immune and clinical characteristics, in which subtype-1 has a higher infiltrated immunocytes, higher immune reaction scores and more chronic periodontitis samples. Immunocytes and clinical phenotypes matching their gene-lncRNA modules, and their functions were annotated. Conclusions: Our study systematically investigated periodontitis immune-related lncRNAs and have taken a glimpse of the underlying mechanism of periodontitis from gene-lncRNA-immunocyte networks, which can not only inspire researchers but also help in periodontitis related immune researches.

Comparative Metatranscriptomics of Periodontitis Supports a Common Polymicrobial Shift in Metabolic Function and Identifies Novel Putative Disease-Associated ncRNAs.

Periodontitis is an inflammatory disease that deteriorates bone supporting teeth afflicting ∼743 million people worldwide. Bacterial communities associated with disease have been classified into red, orange, purple, blue, green, and yellow complexes based on their roles in the periodontal pocket. Previous metagenomic and metatranscriptomics analyses suggest a common shift in metabolic signatures in disease vs. healthy communities with up-regulated processes including pyruvate fermentation, histidine degradation, amino acid metabolism, TonB-dependent receptors. In this work, we examine existing metatranscriptome datasets to identify the commonly differentially expressed transcripts and potential underlying RNA regulatory mechanisms behind the metabolic shifts. Raw RNA-seq reads from three studies (including 49 healthy and 48 periodontitis samples) were assembled into transcripts de novo. Analyses revealed 859 differentially expressed (DE) transcripts, 675 more- and 174 less-expressed. Only ∼20% of the DE transcripts originate from the pathogenic red/orange complexes, and ∼50% originate from organisms unaffiliated with a complex. Comparison of expression profiles revealed variations among disease samples; while specific metabolic processes are commonly up-regulated, the underlying organisms are diverse both within and across disease associated communities. Surveying DE transcripts for known ncRNAs from the Rfam database identified a large number of tRNAs and tmRNAs as well as riboswitches (FMN, glycine, lysine, and SAM) in more prevalent transcripts and the cobalamin riboswitch in both more and less prevalent transcripts. In silico discovery identified many putative ncRNAs in DE transcripts. We report 15 such putative ncRNAs having promising covariation in the predicted secondary structure and interesting genomic context. Seven of these are antisense of ribosomal proteins that are novel and may involve maintaining ribosomal protein stoichiometry during the disease associated metabolic shift. Our findings describe the role of organisms previously unaffiliated with disease and identify the commonality in progression of disease across three metatranscriptomic studies. We find that although the communities are diverse between individuals, the switch in metabolic signatures characteristic of disease is typically achieved through the contributions of several community members. Furthermore, we identify many ncRNAs (both known and putative) which may facilitate the metabolic shifts associated with periodontitis.

Histological evaluation of peri-implant mucosal and gingival tissues in peri-implantitis, peri-implant mucositis and periodontitis patients: a cross-sectional clinical study

Objective: Aim of present study was to evaluate gingival tissue samples obtained from healthy and diseased sites of teeth and dental implants in terms of hypoxia and collagenase activity.Methods: Four study groups were created as Group-1; healthy individuals (H), Group-2; periodontitis patients with stage 3 grade B (P), Group-3; patients with peri-implant mucositis. Group-4; patients with peri-implantitis (P-IMP). Plaque index (PI), gingival index (GI) and probing pocket depth (PPD) were recorded. Gingival and peri-implant mucosal biopsies were obtained. Fibroblast and inflammatory cells were counted. Hypoxia-inducible factor (HIF)-1α, prolyl hydroxylase (PH), matrix metalloproteinase (MMP)-8, tissue inhibitor of MMPs (TIMP)-1, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) levels were determined via immunohistochemistry.Results: Healthy controls had highest fibroblast cell counts and lowest inflammatory cell counts compared to other groups. Peri-implantitis and periodontitis samples had similar fibroblast and inflammatory cell counts, while peri-implant mucositis had higher fibroblast cells and lowered inflammatory cells compared to periodontitis and peri-implantitis samples. HIF-1α, COX-2 and iNOS levels were lowest in healthy controls and increased in other groups. Peri-implant mucositis samples had significantly lower expressions of HIF-1α, COX-2 and iNOS compared to peri-implantitis and periodontitis groups. PH expressions were lower in periodontitis and peri-implantitis groups compared to healthy controls and peri-implant mucositis groups. MMP-8 levels were lower in healthy group compared to other groups while levels were similar in periodontitis, peri-implant mucositis and peri-implantitis groups. TIMP levels were similar in groups.Conclusion: Periodontitis, peri-implantitis, and peri-implant mucositis samples exhibited higher inflammation and lower fibroblast cell counts and tend to have increased tissue collagenase activity, hypoxia and inflammation compared to healthy samples.

Identification of novel key lncRNAs involved in periodontitis by weighted gene co-expression network analysis.

Periodontitis is a multifactorial disease that can lead to the progressive destruction of dental support tissue. However, the detailed mechanisms and specific biomarkers involved in periodontitis remain to be further studied. Recently, long non-coding RNAs (lncRNAs) have been found to play a more important role than other types of RNAs. In our study, we analysed the expression of lncRNAs in periodontitis by analysing GSE16134. We identified highly correlated genes by analysing GSE16134 with weighted gene co-expression network analysis (WGCNA) and identified 50 hub lncRNAs that were dysregulated. Then, we used the Linear Models for Microarray Data (Limma) package to identify the hub lncRNAs that were differentially expressed (DElncRNAs). The ceRNA co-expression network data were obtained from several sites, including miRcode, and were used to assess the potential WGCNA function of hub DElncRNAs in periodontitis. Besides, we divided the samples into LBX2-AS1 high and low expression group by the expression level of LBX2-AS1 and calculated DEG by Limma package. Furthermore, we performed GO function, KEGG pathway and GSEA enrichment of DEGs. In the analysis, we identified 50 hub lncRNAs that may play important roles in periodontitis. Then, we used the Limma package to identify 3 hub DElncRNAs (LINC00687, LBX2-AS1 and LINC01566). We elucidated the potential function of the hub DElncRNA LBX2-AS1 in periodontitis by constructing a co-expression network of lncRNA-miRNA-mRNA interactions. Totally, 573 DEGs (354 up- and 219 downregulated) in periodontitis samples were identified. DEGs were enriched in different GO terms and pathways, such as neutrophil degranulation, neutrophil activation, neutrophil activation involved in immune response, neutrophil-mediated immunity, antigen processing and presentation, JAK-STAT signalling pathway, natural killer cell-mediated cytotoxicity, EGFR tyrosine kinase inhibitor resistance, phosphatidylinositol signalling system and Vascular Endothelial Growth Factor (VEGF) signalling pathway. In our study, we found that 3 hub DElncRNAs (LINC00687, LBX2-AS1 and LINC01566) may be involved in the pathogenesis of periodontitis based on WGCNA and Limma analysis. Our study aimed to elucidate the mechanisms involved in periodontitis at the genetic and epigenetic levels by constructing a ceRNA network associated with lncRNA. Besides, identification DEGs of differential LBX2-AS1 and functional annotation showed that LBX2-AS1 might be associated with periodontitis.

Protective roles of FICZ and aryl hydrocarbon receptor axis on alveolar bone loss and inflammation in experimental periodontitis.

The aryl hydrocarbon receptor (AhR)-ligand axis has been shown to be involved in inflammatory diseases and bone homeostasis. However, the activation of AhR signalling pathway and the possible functions of AhR ligands in periodontitis are underexplored. This study investigated the expression of the AhR target gene cytochrome P450 subfamily B member 1 (CYP1B1) and the functions and mechanisms of the AhR ligand 6 formylindolo[3,2-b]carbazole (FICZ) in periodontitis. CYP1B1 expression was detected in human periodontitis samples, mice with ligature-induced periodontitis and lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells (PDLCs) in vitro. FICZ was administered topically or systemically. The therapeutic functions of FICZ were detected via qPCR, micro-computed tomography and immunohistochemistry. Finally, the mechanisms of AhR signalling in periodontitis were investigated by cell assays. CYP1B1 expression was downregulated in periodontitis. FICZ rescued the alveolar bone loss and mitigated the inflammatory cytokines in periodontitis mice. In vitro, FICZ pre-treatment reduced the LPS-induced inflammation in PDLCs via the increased phosphorylation of STAT3. Additionally, FICZ prompted the mineralization of PDLCs via activation of the Wnt/β-catenin signalling pathway. AhR signalling pathway is suppressed in periodontitis and the AhR ligand FICZ can prevent periodontitis.

CD73-dependent adenosine dampens interleukin-1β-induced CXCL8 production in gingival fibroblasts: Association with heme oxygenase-1 and adenosine monophosphate-activated protein kinase.

During inflammation, stressed or infected cells can release adenosine triphosphate (ATP) to the extracellular medium, which can be hydrolyzed to adenosine by ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and 5'-nucleotidase (CD73). The role of CD73 in the modulation of cytokine release by human gingival fibroblasts (HGFs) remains underexplored. Here, we investigated whether CD73-mediated hydrolysis of extracellular ATP (eATP) could affect interleukin (IL)-1β-induced CXCL8 secretion. The levels of mRNA expression of adenosine receptors, CD39 and CD73 of periodontitis samples were retrieved from a public database. Moreover, HGF mRNA levels were measured by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) after 3, 6, or 24hours of IL-1β stimulation. IL-1β-induced CXCL8 protein levels were measured after pretreatment with 100-µM eATP in the presence or absence of CD73 inhibitor. The effect of eATP degradation to adenosine on CXCL8 levels was investigated using agonist and antagonist of adenosine receptors. Levels of CD39, CD73, and adenosine receptor mRNA were differentially modulated by IL-1β. ATP pretreatment impaired IL-1β-induced CXCL8 secretion and required activation of heme oxygenase-1 (HO-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK). The inhibition of CD73 or the inhibition of adenosine receptors abrogated the ATP effect on CXCL8 secretion. CD73-generated adenosine dampens IL-1β-induced CXCL8 in HGFs and involves HO-1 and pAMPK signaling. These results imply that CD73 is a negative regulator of the inflammatory microenvironment, suggesting that this ectoenzyme could be involved in the generation of deficient CXCL8 gradient in chronic inflammation.

Down-regulation of long non-coding RNA MEG3 suppresses osteogenic differentiation of periodontal ligament stem cells (PDLSCs) through miR-27a-3p/IGF1 axis in periodontitis.

Objective: This study aimed to investigate the roles of long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in periodontitis.Methods: Differentially expressed lncRNAs and mRNAs between periodontitis periodontal ligament tissues and healthy periodontal ligament tissues were selected out using R project. PDLSCs were identified using flow cytometry. Western blot was employed to detect pathway relative proteins. Besides, targeted relationships between lncRNA and miRNA, as well as miRNA and mRNA were verified by dual luciferase reporter gene assay. Osteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining and Alizarin Red Staining (ARS). Markers for osteoblast (Runx2, Osterix, Osteocalcin, Colla1) were detected using western blot.Results: LncRNA MEG3 and IGF1 were both down-regulated, while miR-27a-3p was up-regulated in periodontitis samples compared with healthy samples. Overexpression of MEG3 promoted osteogenic differentiation by enhancing expression of IGF1 yet suppressing expression of miRNA-27a-3p. Meanwhile, the results of ALP and ARS staining indicated that up-regulation of lncRNA MEG3 or IGF1 promoted osteogenic differentiation in PDLSCs, which could be reversed with up-regulation of miRNA-27a-3p.Conclusion: Down-regulation of MEG3 suppressed osteogenic differentiation of PDLSCs through miR-27a-3p/IGF1 axis in periodontitis.

Characterization of macrophage polarization in periodontal disease.

To explore the M1/M2 status of macrophage polarization from healthy, gingivitis, and periodontitis patient samples. Gingival biopsies were collected from 42 individuals (14 gingivitis, 18 periodontitis, and 10 healthy samples) receiving periodontal therapy. Histomorphology analysis was performed with haematoxylin and eosin staining. Immunofluorescence was performed using a combination of CD68 (macrophages), iNOS (M1), and CD206 (M2) in order to acquire changes in macrophage polarization at a single-cell resolution. Macrophages were quantified under microscopy using narrow wavelength filters to detect Alexa 488, Alexa 568, Alexa 633 fluorophores, and Hoechst 33342 to identify cellular DNA content. Gingivitis and periodontitis samples showed higher levels of macrophages compared with healthy samples. Unexpectedly, periodontitis samples displayed lower levels of macrophages dispersed in the stromal tissues compared with gingivitis samples; however, it remained higher than healthy tissues. The polarization of macrophages appears to be reduced in periodontitis and showed similar levels to those observed in healthy tissues. Our study found that gingivitis and periodontitis differ from each other by the levels of macrophage infiltrate, but not by changes in macrophage polarization.

CD73-dependent adenosine dampens IL-1β-induced CXCL8 production in gingival fibroblasts via HO-1 and pAMPK

Abstract During inflammation, stressed or infected cells can release adenosine tri-phosphate (ATP), which can be hydrolyzed to adenosine by ectonucleotidases such as CD39 and CD73. The role of CD73 in the modulation of cytokine release by human gingival fibroblasts (HGF) remains underexplored. Here, we investigated whether CD73-mediated hydrolysis of extracellular ATP (eATP) could affect IL-1β-induced CXCL8 secretion. The mRNA expression of adenosine receptors, CD39, and CD73 of periodontitis samples was retrieved from a public database. HGF mRNA levels were measured after 3, 6 or 24 h of IL-1β stimulation. IL-1β-induced CXCL8 protein levels were measured after pretreatment with 100 μM eATP in the presence of absence of CD73 inhibitor. The effect of eATP degradation to adenosine on CXCL8 levels was investigated using agonist and antagonist of adenosine receptors. The CD39, CD73 and adenosine receptor mRNA were differentially modulated by IL-1β. ATP pretreatment impaired IL-1β-induced CXCL8 secretion and required activation of HO-1 and pAMPK. The inhibition of CD73 or adenosine receptors abrogated the ATP effect on CXCL8 secretion. CD73-generated adenosine dampens IL-1β-induced CXCL8 in HGF via HO-1 and pAMPK. These results imply that CD73 is a negative regulator of the inflammatory microenvironment, suggesting this ectoenzyme should be considered as a target for therapeutic strategies to control periodontal disease.

Array analysis for T-cell associated cytokines in gingival crevicular fluid: Identifying altered profiles associated with periodontal disease status

ObjectiveCytokine networks regulate innate and adaptive immune responses, which in turn are recognised to direct the progression or arrest of periodontal disease. This study aimed to compare the profile of seven cytokines, implicated in regulating T-cell networks, in gingival crevicular fluid (GCF) samples with differing classification of periodontal status. MethodsGCF samples were collected from patients with strong clinical evidence for chronic periodontitis, aggressive periodontitis, gingivitis or no gingival inflammation. Cytokines IL-6, IFN-ɣ, IL-4, IL-2, IL-17 A, IL10 and TNFα were measured in each sample using a commercial cytometric bead array assay. Descriptive statistics were used to indicate central tendency, data scatter and analysis of variance for each cytokine concentrations between respective patient groups. Heat maps with dendrograms were produced to visualise hierarchical clustering and trends within the data. ResultsMedian concentrations for all cytokines analysed were highest for gingivitis samples and lowest for aggressive periodontitis samples. The median concentration of IL-6 in gingivitis samples was observed to be 10.5 fold higher (˜17,300 pg/μl) than IL-6 in aggressive periodontitis samples (˜1600 pg/μl). Median concentrations of IL-10, IL-17 A and TNFα were also 2–2.2 fold higher in gingivitis samples compared to aggressive periodontitis. ConclusionsDescriptive statistical analysis noted raised concentrations of IL-6, IL-17 A and TNFα associated with gingivitis; pro-inflammatory cytokines usually associated with periodontal tissue destruction, including bone. Our results would suggest that these cytokines can additionally provide protective roles in preventing progression to advanced forms of periodontal disease. Potential for how these cytokines contribute to providing this role is discussed. Clinical significanceDefining the roles for the many cytokines involved in the pathogenesis of periodontal disease is far from complete. Consequently the results of this study serve to evidence proposals that cytokines can exhibit both pro- and anti-inflammatory effects, which is dependent on the signalling environment within which they exist and the antagonizing or modifying actions of other cytokines. Whilst future research is necessary to explore mechanistic action, our study contributes new knowledge suggesting that IL-6 and IL-17 A can provide roles in stabilising the lesion to limit disease progression, which does not preclude alternative roles in promoting periodontal bone loss in advanced forms of disease progression, which is also documented in the literature.

Detection and Characterization of Prevotella Intermedia and Its In Vitro Susceptibility to Selected Antimicrobial Agents in Chronic Periodontitis and Acute Myocardial Infarction

Background: Periodontal disease has been reported to play a causative role in acute myocardial infarction (AMI), which may add to the various risk factors associated with coronary heart disease. The objective of the present study was to investigate the presence of Prevotella intermedia – an established periodontal pathogen – in subgingival plaque samples of chronic periodontitis and AMI patients in order to identify a possible association, and to evaluate the susceptibility of Prevotella intermedia to nine antimicrobial agents. Methods: After undergoing screening for eligibility, a total of 50 subjects were included in the present study. Twenty patients were diagnosed with AMI and generalized chronic periodontitis (Group I), 20 patients were diagnosed with only AMI (Group II), and 10 subjects were healthy controls (Group III). The isolated Prevotella intermedia strains were tested for susceptibility to bacitracin, chloramphenicol, penicillin G, polymyxin, gentamycin, neomycin, tetracycline, cefotaxime, and cefoxitin using an antibiotic zonescale to determine minimum inhibitory concentrations (MICs). Results: Periodontal pathogens were identified by phenotypic and enzymatic methods. The mean bacterial load of Prevotella intermedia species was higher in Group I compared to Group II and Group III. It was also found that pencillin G, gentamycin, neomycin, tetracycline, cefotaxime, and cefoxitin inhibited 90% of Prevotella intermedia, whereas bacitracin, chloramphenicol, and polymyxin inhibited 80% of Prevotella intermedia. Thus, only 10% of Prevotella intermedia were resistant to these antibiotics. Conclusion: The present study confirms that Prevotella intermedia is associated with chronic periodontitis and AMI.

Immunohistochemical study of CD34 and podoplanin in periodontal disease.

The aim of this study was to evaluate the angiogenesis and lymphangiogenesis in gingival tissue biopsy specimens of individuals with clinically healthy gingiva, chronic gingivitis, and chronic periodontitis (n=30 per clinical condition). Histological sections were stained using hematoxylin and eosin as well as immunohistochemically with hematopoietic progenitor cell antigen CD34 and podoplanin (PDPN) antibodies to evaluate the microvascular count, area, and perimeter of blood and lymphatic vessels, respectively. The results revealed a correlation between the microvascular count of blood and lymphatic vessels (P=0.03; however, in individuals with chronic periodontitis, fewer lymphatic vessels were present than in the clinically healthy gingival tissue (P=0.01), which was not observed in the case of microvascular area and perimeter. Podoplanin labeling was present in the epithelium, and the intensity of labeling was positively correlated to the intensity of the inflammatory infiltrate (P=0.03). In this study, we concluded that an increase in the number of blood and lymphatic vessels was not observed in bouth gingivitis and periodontitis samples. Podoplanin expression is highly associated with an increased inflammatory infiltration suggesting that PDPN might play an additional role in periodontal disease, other than solely as a lymphangiogenesis marker.