Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss.

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss.

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2-/- ) and wild-type (Tfr2+/+ ) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2+/+ and Tfr2-/- mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.

Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-ß1/Smad signaling pathway.

It has been known that periodontal ligament-associated protein-1 (PLAP-1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP-1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP-1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP-1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP-1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF-ß1)/Smad3 in inflammatory environments. We further found the KO of PLAP-1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF-ß1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP-1/Asporin KO mice was promoted through TGF-ß1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP-1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP-1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF-ß1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis.

Exploring the role of ITGB6: fibrosis, cancer, and other diseases.

Integrin ß6 (ITGB6), a member of the integrin family of proteins, is only present in epithelial tissues and frequently associates with integrin subunit αv to form transmembrane heterodimers named integrin αvß6. Importantly, ITGB6 determines αvß6 expression and availability. In addition to being engaged in organ fibrosis, ITGB6 is also directly linked to the emergence of cancer, periodontitis, and several potential genetic diseases. Therefore, it is of great significance to study the molecular-biological mechanism of ITGB6, which could provide novel insights for future clinical diagnosis and therapy. This review introduces the structure, distribution, and biological function of ITGB6. This review also expounds on ITGB6-related diseases, detailing the known biological effects of ITGB6.

lncRNA CDKN2B-AS1 regulates collagen expression.

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10- 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10- 25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1 × 10- 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10- 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10- 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.

Macrophage-enriched novel functional long noncoding RNAs LRRC75A-AS1 and GAPLINC regulate polarization and innate immune responses.

INTRODUCTION: Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated. OBJECTIVE: To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses. METHODS: Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation, polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs, M1/M2 marker expression. RESULTS: Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarization in vivo. CONCLUSION: Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.

Periodontitis causally affects the brain cortical structure: A Mendelian randomization study.

OBJECTIVE: To estimate whether genetically proxied periodontitis causally impacts the brain cortical structure using Mendelian randomization (MR). BACKGROUND: Periodontitis is one of the most prevalent inflammatory conditions globally, and emerging evidence has indicated its influences on distal organs, including the brain, whose disorders are always accompanied by magnetic resonance imaging (MRI)-identified brain cortical changes. However, to date, no available evidence has revealed the association between periodontitis and brain cortical structures. METHODS: The instrumental variables (IVs) were adopted from previous genome-wide association study (GWAS) studies and meta-analyses of GWAS studies of periodontitis from 1844 to 5266 cases and 8255 to 12 515 controls. IVs were linked to GWAS summary data of 51 665 patients from the ENIGMA Consortium, assessing the impacts of genetically proxied periodontitis on the surficial area (SA) or the cortical thickness (TH) of the global and 34 MRI-identified functional regions of the brain. Inverse-variance weighted was used as the primary estimate; the MR pleiotropy residual sum and outlier (MR-PRESSO), the MR-Egger intercept test, and leave-one-out analyses were used to examine the potential horizontal pleiotropy. RESULTS: Genetically proxied periodontitis affects the SA of the medial orbitofrontal cortex, the lateral orbitofrontal cortex, the inferior temporal cortex, the entorhinal cortex, and the temporal pole, as well as the TH of the entorhinal. No pleiotropy was detected. CONCLUSIONS: Periodontitis causally influences the brain cortical structures, implying the existence of a periodontal tissue-brain axis.

Potential diagnostic markers and therapeutic targets for periodontitis and Alzheimer's disease based on bioinformatics analysis.

BACKGROUND AND OBJECTIVE: As a chronic inflammatory disease, periodontitis threatens oral health and is a risk factor for Alzheimer's disease (AD). There is growing evidence that these two diseases are closely related. However, current research is still incomplete in understanding the common genes and common mechanisms between periodontitis and AD. In this study, we aimed to identify common genes in periodontitis and AD and analyze the relationship between crucial genes and immune cells to provide new therapeutic targets for clinical treatment. MATERIALS AND METHODS: We evaluated differentially expressed genes (DEGs) specific to periodontitis and AD. Co-expressed genes were identified by obtaining gene expression profile data from the Gene Expression Omnibus (GEO) database. Using the STRING database, protein-protein interaction (PPI) networks were constructed, and essential genes were identified. We also used four algorithms to identify critical genes and constructed regulatory networks. The association of crucial genes with immune cells and potential therapeutic effects was also assessed. RESULTS: PDGFRB, VCAN, TIMP1, CHL1, EFEMP2, and IGFBP5 were obtained as crucial common genes. Immune infiltration analysis showed that Natural killer cells and Myeloid-derived suppressor cells were significantly differentially expressed in patients with PD and AD compared with the normal group. FOXC1 and GATA2 are important TFs for PD and AD. MiR-23a, miR-23b, miR-23a, and miR-23b were associated with AD and PD. Finally, the hub genes retrieved from the DSigDB database indicate multiple drug molecule and drug-target interactions. CONCLUSION: This study reveals commonalities in common hub genes and immune infiltration between periodontitis and AD, and the analysis of six hub genes and immune cells may provide new insights into potential therapeutic directions for the pathogenesis of periodontitis complicated by AD.

Periodontitis and thyroid function: A bidirectional Mendelian randomization study.

BACKGROUND AND OBJECTIVE: Previous studies suggest interaction between periodontitis and thyroid function, while the causality has not yet been established. We applied the Mendelian randomization (MR) method to assess bidirectional causal association between periodontitis and thyroid-related traits, including free thyroxine (FT4), thyroid stimulating hormone (TSH), hypothyroidism, hyperthyroidism and autoimmune thyroid disease (AITD). METHODS: Genetic instruments were extracted from large-scale genome-wide association studies on normal-range FT4 (N = 49 269) and TSH (N = 54 288) levels, TSH in full range (N = 119 715); hypothyroidism (discovery/replication cohorts: N = 53 423/334 316), hyperthyroidism (discovery/replication cohorts: N = 51 823/257 552), AITD (N = 755 406) and periodontitis (N = 45 563). Here, the inverse variance weighted (IVW) method was applied as the primary analysis, and robustness of results were assessed by several pleiotropic-robust methods. Results were adjusted for Bonferroni correction thresholds with significant p < .004 (0.05/13) and suggestive p between .004 and .05. RESULTS: The IVW analysis revealed a suggestively causal linkage between genetic predisposition to periodontitis and the increased risk of hypothyroidism (discovery cohort: odds ratio [OR] = 1.24, 95% confidence interval [CI] = 1.05-1.46, p = .012; replication cohort: OR = 1.06, 95% CI = 1.01-1.11, p = .011). No evidence was found for supporting the impact of periodontitis on hyperthyroidism and AITD risks (associated p ≥ .209), as well as thyroid-related traits on periodontitis risk (associated p ≥ .105). These findings were robust and consistent through sensitivity analysis with other MR models. CONCLUSION: This bidirectional MR reveals periodontitis should not be attributed to variations in thyroid function but it has potential causal effect on hypothyroidism risk, which provides a better understanding of the relationship between periodontitis and thyroid function, and potential evidence for the clinical intervention of hypothyroidism. Further investigations are warranted to elucidate the nature and underlying mechanisms of this finding.

Identifying genes for regulating osteogenic differentiation of human periodontal ligament stem cells in inflammatory environments by bioinformatics analysis.

BACKGROUND AND OBJECTIVES: Periodontitis is an immuno-inflammatory disease caused by dental plaque biofilms and inflammations. The regeneration of bone tissue in inflammatory environment is of great significance for the treatment of periodontal disease, but the specific molecular mechanism of bone formation in periodontitis still needs further exploration. The objective of this study was to identify key osteogenesis-related genes (ORGs) in periodontitis. METHODS: We used two datasets from the Gene Expression Omnibus (GEO) database to find differentially expressed mRNAs and miRNAs, further performed Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Then we predicted the downstream genes of the differentially expressed miRNAs (DEMs) by the TargetScan database and established a miRNA-mRNA regulatory network. Finally, the osteogenic mechanism of periodontitis was explored through quantitative real-time PCR (qRT-PCR) by inducing inflammatory environment and osteogenic differentiation of hPDLSCs. RESULTS: Through differential expression analysis and prediction of downstream target genes of DEMs, we created a miRNA-mRNA regulatory network consisting of 29 DEMs and 11 differentially expressed osteogenesis-related genes (DEORGs). In addition, the qRT-PCR results demonstrated that BTBD3, PLAT, AKAP12, SGK1, and GLCE expression levels were significantly upregulated, while those of TIMP3, ZCCHC14, LIN7A, DNAH6, NNT, and ITGA6 were downregulated under the dual effects of inflammatory stimulation and osteogenic induction. CONCLUSION: DEORGs might be important factors in the osteogenic phase of periodontitis, and the miRNA-mRNA network may shed light on the clarification of the role and mechanism of osteogenesis in periodontitis and contribute to the development of novel therapeutic strategies.

Genetic analysis of impaired healing responses after periodontal therapy in type 2 diabetes: Clinical and in vivo studies.

OBJECTIVE: This study aims to investigate the mechanisms underlying the impaired healing response by diabetes after periodontal therapy. BACKGROUND: Outcomes of periodontal therapy in patients with diabetes are impaired compared with those in patients without diabetes. However, the mechanisms underlying impaired healing response to periodontal therapy have not been sufficiently investigated. MATERIALS AND METHODS: Zucker diabetic fatty (ZDF) and lean (ZL) rats underwent experimental periodontitis by ligating the mandibular molars for one week. The gingiva at the ligated sites was harvested one day after ligature removal, and gene expression was comprehensively analyzed using RNA-Seq. In patients with and without type 2 diabetes (T2D), the corresponding gene expression was quantified in the gingiva of the shallow sulcus and residual periodontal pocket after non-surgical periodontal therapy. RESULTS: Ligation-induced bone resorption and its recovery after ligature removal were significantly impaired in the ZDF group than in the ZL group. The RNA-Seq analysis revealed 252 differentially expressed genes. Pathway analysis demonstrated the enrichment of downregulated genes involved in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. PPARα and PPARγ were decreased in mRNA level and immunohistochemistry in the ZDF group than in the ZL group. In clinical, probing depth reduction was significantly less in the T2D group than control. Significantly downregulated expression of PPARα and PPARγ were detected in the residual periodontal pocket of the T2D group compared with those of the control group, but not in the shallow sulcus between the groups. CONCLUSIONS: Downregulated PPAR subtypes expression may involve the impaired healing of periodontal tissues by diabetes.

MicroRNA-671-5p regulates the inflammatory response of periodontal ligament stem cells via the DUSP8/p38 MAPK pathway.

BACKGROUND: MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. METHODS AND RESULTS: HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. CONCLUSIONS: microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

A genome-wide association study meta-analysis in a European sample of stage III/IV grade C periodontitis patients ≤35 years of age identifies new risk loci.

AIM: Few genome-wide association studies (GWAS) have been conducted for severe forms of periodontitis (stage III/IV grade C), and the number of known risk genes is scarce. To identify further genetic risk variants to improve the understanding of the disease aetiology, a GWAS meta-analysis in cases with a diagnosis at ≤35 years of age was performed. MATERIALS AND METHODS: Genotypes from German, Dutch and Spanish GWAS studies of III/IV-C periodontitis diagnosed at age ≤35 years were imputed using TopMed. After quality control, a meta-analysis was conducted on 8,666,460 variants in 1306 cases and 7817 controls with METAL. Variants were prioritized using FUMA for gene-based tests, functional annotation and a transcriptome-wide association study integrating eQTL data. RESULTS: The study identified a novel genome-wide significant association in the FCER1G gene (p = 1.0 × 10-9 ), which was previously suggestively associated with III/IV-C periodontitis. Six additional genes showed suggestive association with p < 10-5 , including the known risk gene SIGLEC5. HMCN2 showed the second strongest association in this study (p = 6.1 × 10-8 ). CONCLUSIONS: This study expands the set of known genetic loci for severe periodontitis with an age of onset ≤35 years. The putative functions ascribed to the associated genes highlight the significance of oral barrier tissue stability, wound healing and tissue regeneration in the aetiology of these periodontitis forms and suggest the importance of tissue regeneration in maintaining oral health.

Complement C3 as a potential drug target in periodontitis: Evidence from the cis-Mendelian randomization approach.

AIM: Evidence from a Phase IIa trial showed that a complement C3-targeted drug reduced gingival inflammation in patients with gingivitis. Using drug-target Mendelian randomization (MR), we investigated whether genetically proxied C3 inhibition alters the risk of periodontitis. MATERIALS AND METHODS: We used multiple 'cis' instruments from the vicinity of the encoding loci of C3. Instrument selection was restricted to the drug target encoding loci (chromosome 19; 6,677,715-6,730,573 (GRCh37/hg19)). We selected three uncorrelated single-nucleotide polymorphisms (rs141552034, rs145406915, rs11569479) that were associated with serum C3 levels (p value <1 × 10-4 ) from a genome-wide association study (GWAS) of 5368 European descent individuals. We extracted association statistics from a GWAS of 17,353 clinical periodontitis cases and 28,210 European controls. Wald ratios were combined using inverse-variance weighted meta-analysis to estimate the odds ratio (OR) of the genetically proxied inhibition of C3 in relation to periodontitis. RESULTS: MR analysis revealed that the inhibition of C3 reduces the odds of periodontitis (OR 0.91 per 1 standard deviation reduction in C3; 95% confidence interval 0.87-0.96, p value = .0003). CONCLUSIONS: Findings from our MR analysis suggest a potential protective effect of C3 blockade against periodontitis.

Neutrophil extracellular traps in rheumatoid arthritis and periodontitis: Contribution of PADI4 gene polymorphisms.

AIM: We sought to investigate the release of neutrophil extracellular traps (NETs) in neutrophils from individuals with rheumatoid arthritis (RA) and controls and compare the presence of NETs in gingival tissues according to periodontal status. Also, the association between single nucleotide polymorphisms (SNPs) of the peptidyl arginine deaminase type 4 (PADI4) gene and the GTG haplotype with RA, periodontitis and NETs was evaluated in vitro. MATERIALS AND METHODS: Peripheral neutrophils were isolated by density gradient, and NET concentration was determined by the PicoGreen method. Immunofluorescence was studied to identify NETs by co-localization of myeloperoxidase (MPO)-citrullinated histone H3 (H3Cit). Genotyping for SNPs (PADI4_89; PADI4_90; PADI4_92; and PADI4_104) was performed in 87 individuals with RA and 111 controls. RESULTS: The release of NETs in vitro was significantly higher in individuals with RA and periodontitis and when stimulated with Porphyromonas gingivalis. Gingival tissues from subjects with RA and periodontitis revealed increased numbers of MPO-H3Cit-positive cells. Individuals with the GTG haplotype showed a higher release of NETs in vitro and worse periodontal parameters. CONCLUSIONS: The release of NETs by circulating neutrophils is associated with RA and periodontitis and is influenced by the presence of the GTG haplotype.

Exploring the causal relationship between periodontitis and gut microbiome: Unveiling the oral-gut and gut-oral axes through bidirectional Mendelian randomization.

AIM: This Mendelian randomization (MR) study was performed to explore the potential bidirectional causal relationship between the gut microbiome (GM) and periodontitis. MATERIALS AND METHODS: We used genetic instruments from the genome-wide association study of European descent for periodontitis from the GeneLifestyle Interactions in Dental Endpoints (GLIDE) consortium (17,353 cases and 28,210 controls) and the FinnGen consortium (4434 cases and 259,234 controls) to investigate the causal relationship with GM (the MiBioGen consortium, 18,340 samples), and vice versa. Several MR techniques, which include inverse variance weighting (IVW), MR-Egger, weighted median, simple mode and weighted mode approaches, were employed to investigate the causal relationship between the exposures and the outcomes. Cochran's Q-test was performed to detect heterogeneity. The MR-Egger regression intercept and MR pleiotropy residual sum and outlier test (MR-PRESSO) were conducted to test potential horizontal pleiotropy. Leave-one-out sensitivity analyses were used to assess the stabilities of single nucleotide polymorphisms (SNPs). Finally, the IVW results from the two databases were analysed using meta-analysis. RESULTS: We confirmed three potential causal relationships between GM taxa and periodontitis at the genus level. Among them, the genera Alistipes and Holdemanella were genetically associated with an increased risk of periodontitis. In reverse, periodontitis may lead to a decreased abundance of the genus Ruminococcaceae UCG014. CONCLUSIONS: The demonstration of a causal link between GM and periodontitis provides compelling evidence, highlighting the interconnectivity and interdependence of the gut-oral and oral-gut axes.

The transcriptomic response to cannabidiol of Treponema denticola, a phytocannabinoid-resistant periodontal pathogen.

AIM: The use of cannabis, which contains multiple antimicrobials, may be a risk factor for periodontitis. We hypothesized that multiple oral spirochetes would be phytocannabinoid-resistant and that cannabidiol (CBD) would act as an environmental stressor to which Treponema denticola would respond transcriptionally, thereby providing first insights into spirochetal survival strategies. MATERIALS AND METHODS: Oral spirochete growth was monitored spectrophotometrically in the presence and absence of physiologically relevant phytocannabinoid doses, the transcriptional response to phytocannabinoid exposure determined by RNAseq, specific gene activity fluxes verified using qRT-PCR and orthologues among fully sequenced oral spirochetes identified. RESULTS: Multiple strains of oral treponemes were resistant to CBD (0.1-10 µg/mL), while T. denticola ATCC 35405 was resistant to all phytocannabinoids tested (CBD, cannabinol [CBN], tetrahydrocannabinol [THC]). A total of 392 T. denticola ATCC 35405 genes were found to be CBD-responsive by RNAseq. A selected subset of these genes was independently verified by qRT-PCR. Genes found to be differentially activated by both methods included several involved in transcriptional regulation and toxin control. Suppressed genes included several involved in chemotaxis and proteolysis. CONCLUSIONS: Oral spirochetes, unlike some other periodontal bacteria, are resistant to physiological doses of phytocannabinoids. Investigation of CBD-induced transcriptomic changes provided insight into the resistance mechanisms of this important periodontal pathogen. These findings should be considered in the context of the reported enhanced susceptibility to periodontitis in cannabis users.

Arrestin beta-2 deficiency exacerbates periodontal inflammation by mediating activating transcription factor 6 activation and abnormal remodelling of the extracellular matrix.

AIM: To investigate the specific role of arrestin beta-2 (ARRB2) in the progression of periodontitis and the underlying mechanisms. MATERIALS AND METHODS: Single-cell RNA sequencing data were used to analyse gene expression in periodontal tissues from healthy controls and patients with periodontitis. Real-time quantitative polymerase chain reaction, Western blotting and immunohistochemical staining were performed to detect the expression of ARRB2. Furthermore, a ligature-induced periodontitis model was created. Using radiographic and histological methods, RNA sequencing and luciferase assay, the role of ARRB2 in periodontitis and the underlying mechanisms were explored. Finally, the therapeutic effect of melatonin, an inhibitor of activating transcription factor 6 (ATF6), on periodontitis in mice was assessed in both in vivo and in vitro experiments. RESULTS: ARRB2 expression was up-regulated in inflammatory periodontal tissue. In the ligature-induced mouse model, Arrb2 knockout exacerbated alveolar bone loss (ABL) and extracellular matrix (ECM) degradation. ARRB2 exerted a negative regulatory effect on ATF6, an essential targeted gene. Melatonin ameliorated ABL and an imbalance in ECM remodelling in Arrb2-deficient periodontitis mice. CONCLUSIONS: ARRB2 mediates ECM remodelling via inhibition of the ATF6 signalling pathway, which ultimately exerts a protective effect on periodontal tissues.

Periodontal disease in patients with WHIM syndrome.

AIM: WHIM (warts, hypogammaglobulinaemia, infections and myelokathexis) syndrome is a rare combined primary immunodeficiency disease caused by gain-of-function (GOF) mutations in the chemokine receptor CXCR4 and includes severe neutropenia as a common feature. Neutropenia is a known risk factor for periodontitis; however, a detailed periodontal evaluation of a WHIM syndrome cohort is lacking. This study aimed to establish the evidence base for the periodontal status of patients with WHIM syndrome. MATERIALS AND METHODS: Twenty-two adult WHIM syndrome patients and 22 age- and gender-matched healthy volunteers (HVs) were evaluated through a comprehensive medical and periodontal examination. A mouse model of WHIM syndrome was assessed for susceptibility to naturally progressing or inducible periodontitis. RESULTS: Fourteen patients with WHIM syndrome (63.6%) and one HV (4.5%) were diagnosed with Stage III/IV periodontitis. No WHIM patient presented with the early onset, dramatic clinical phenotypes typically associated with genetic forms of neutropenia. Age, but not the specific CXCR4 mutation or absolute neutrophil count, was associated with periodontitis severity in the WHIM cohort. Mice with a Cxcr4 GOF mutation did not exhibit increased alveolar bone loss in spontaneous or ligature-induced periodontitis. CONCLUSIONS: Overall, WHIM syndrome patients presented with an increased severity of periodontitis despite past and ongoing neutrophil mobilization treatments. GOF mutations in CXCR4 may be a risk factor for periodontitis in humans.