Henrik Dommisch

Expression of defensins in gingiva and their role in periodontal health and disease

Oral epithelium is a stratified squamous epithelium that functions as the barrier between the outside environment and the host. In the oral cavity, epithelial tissues are constantly exposed to a variety of bacteria, but most individuals maintain healthy homeostasis. Epithelial cells contribute to the innate host response, and antimicrobial peptide expression in all human epithelia, including oral epithelia, is an important part of this epithelial function. These antimicrobial peptides have a broad spectrum of activity against both Gram-negative and Gram-positive bacteria as well as against yeast and viruses. In humans these antimicrobial peptides include defensins and a cathelicidin family member LL-37 in skin and oral mucosa and other epithelia. The human defensins include the alpha-defensins of intestinal and neutrophil origin, and the beta-defensins of skin and oral mucosa and other epithelia. Present studies have identified specific signaling routes that pathogens and commensals take in stimulating these innate immune responses, and this may open the way for development of new therapeutic agents for periodontal diseases.

Protease-Activated Receptor 2 Mediates Human Beta-Defensin 2 and CC Chemokine Ligand 20 mRNA Expression in Response to Proteases Secreted by Porphyromonas gingivalis

ABSTRACT The oral pathogen Porphyromonas gingivalis secretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3α/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed to P. gingivalis cell-free culture supernatant or purified RgpB. hBD-2 mRNA in GECs was induced in response to supernatant and purified RgpB from P. gingivalis (P = 0.02 and P = 0.016, respectively). This effect was abrogated by the protease inhibitor tosyl-l-lysine chloromethyl ketone (TLCK) (P < 0.05). In response to P. gingivalis supernatant and to purified RgpB, the hBD-2 mRNA expression was significantly decreased in PAR-2 gene knockdown cells, whereas no change was detected in PAR-1 gene knockdown cells. CCL20 mRNA expression also increased in response to the supernatant of P. gingivalis, and this effect was blocked by the protease inhibitor, TLCK (P = 0.05 and P = 0.024, respectively), and was blocked in PAR-2 gene knockdown cells. Our data indicate that hBD-2 and CCL20 mRNA up-regulation by P. gingivalis supernatant and purified RgpB was mediated via PAR-2, but not via PAR-1, and that proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity.

A 3′ UTR transition within DEFB1 is associated with chronic and aggressive periodontitis

Periodontal diseases are complex inflammatory diseases and affect up to 20% of the worldwide population. An unbalanced reaction of the immune system toward microbial pathogens is considered as the key factor in the development of periodontitis. Defensins have a strong antimicrobial function and are important contributors of the immune system toward maintaining health. Here, we present the first systematic association study of DEFB1. Using a haplotype-tagging single nucleotide polymorphism (SNP) approach, including described promoter SNPs of DEFB1, we investigated the associations of the selected variants in a large population (N=1337 cases and 2887 ethnically matched controls). The 3′ untranslated region SNP, rs1047031, showed the most significant association signal for homozygous carriers of the rare A allele (P=0.002) with an increased genetic risk of 1.3 (95% confidence interval: 1.11–1.57). The association was consistent with the specific periodontitis forms: chronic periodontitis (odds ratio=2.2 (95% confidence interval: 1.16–4.35), P=0.02), and aggressive periodontitis (odds ratio=1.3 (95% confidence interval 1.04–1.68), P=0.02). Sequencing of regulatory and exonic regions of DEFB1 identified no other associated variant, pointing toward rs1047031 as likely being the causative variant. Prediction of microRNA targets identified a potential microRNA-binding site at the position of rs1047031.

Genetic evidence for PLASMINOGEN as a shared genetic risk factor of coronary artery disease and periodontitis

Background—Genetic studies demonstrated the presence of risk alleles in the genes ANRIL and CAMTA1/VAMP3 that are shared between coronary artery disease (CAD) and periodontitis. We aimed to identify further shared genetic risk factors to better understand conjoint disease mechanisms. Methods and Results—In-depth genotyping of 46 published CAD risk loci of genome-wide significance in the worldwide largest case–control sample of the severe early-onset phenotype aggressive periodontitis (AgP) with the Illumina Immunochip (600 German AgP cases, 1448 controls) and the Affymetrix 500K array set (283 German AgP cases and 972 controls) highlighted ANRIL as the major risk gene and revealed further associations with AgP for the gene PLASMINOGEN (PLG; rs4252120: P=5.9×10−5; odds ratio, 1.27; 95% confidence interval, 1.3–1.4 [adjusted for smoking and sex]; 818 cases; 5309 controls). Subsequent combined analyses of several genome-wide data sets of CAD and AgP suggested TGFBRAP1 to be associated with AgP (rs2679895: P=0.0016; odds ratio, 1.27 [95% confidence interval, 1.1–1.5]; 703 cases; 2.143 controls) and CAD (P=0.0003; odds ratio, 0.84 [95% confidence interval, 0.8–0.9]; n=4117 cases; 5824 controls). The study further provides evidence that in addition to PLG, the currently known shared susceptibility loci of CAD and periodontitis, ANRIL and CAMTA1/VAMP3, are subjected to transforming growth factor-&bgr; regulation. Conclusions—PLG is the third replicated shared genetic risk factor of atherosclerosis and periodontitis. All known shared risk genes of CAD and periodontitis are members of transforming growth factor-&bgr; signaling.

CDKN2BAS is associated with periodontitis in different European populations and is activated by bacterial infection

Epidemiological studies have indicated a relationship between coronary heart disease (CHD) and periodontitis. Recently, CDKN2BAS was reported as a shared genetic risk factor of CHD and aggressive periodontitis (AgP), but the causative variant has remained unknown. To identify and validate risk variants in different European populations, we first explored 150 kb of the genetic region of CDKN2BAS including the adjacent genes CDKN2A and CDKN2B, covering 51 tagging single nucleotide polymorphisms (tagSNPs) in AgP and chronic periodontitis (CP) in individuals of Dutch origin (n=313). In a second step, we tested the significant SNP associations in an independent AgP and CP population of German origin (n=1264). For the tagSNPs rs1360590, rs3217992, and rs518394, we could validate the associations with AgP before and after adjustment for the covariates smoking, gender and diabetes, with SNP rs3217992 being the most significant (OR 1.48, 95% CI 1.19 to 1.85; p=0.0004). We further showed in vivo gene expression of CDKN2BAS, CDKN2A, CDKN2B, and CDK4 in healthy and inflamed gingival epithelium (GE) and connective tissue (CT), and detected a significantly higher expression of CDKN2BAS in healthy CT compared to GE (p=0.004). After 24 h of stimulation with Porphyromonas gingivalis in Streptococcus gordonii pre-treated gingival fibroblast (HGF) and cultured gingival epithelial cells (GECs), we observed a 25-fold and fourfold increase of CDKN2BAS gene expression in HGFs (p=0.003) and GECs (p=0.004), respectively. Considering the global importance of CDKN2BAS in the disease risk of CHD, this observation supports the theory of inflammatory components in the disease physiology of CHD.

The stage of native biofilm formation determines the gene expression of human β‐defensin‐2, psoriasin, ribonuclease 7 and inflammatory mediators: a novel approach for stimulation of keratinocytes with in situ formed biofilms

BACKGROUND/AIMS Antimicrobial peptides such as human beta-defensin-2 (hBD-2), psoriasin (PSO), and ribonuclease 7 (RNase 7) play an important role in innate immunity. The aim of the present study was to test the hypothesis that epithelial cells show a differential gene expression pattern of antimicrobial peptides (hBD-2, PSO, RNase 7) and inflammatory mediators such as interleukin-8 (IL-8) and 5-lipoxygenase (5-LO) in response to different stages of naturally formed biofilms. METHODS Epithelial cells were cultured from biopsies obtained from five healthy individuals. Native bacterial biofilms were taken from the same subjects that donated the gingival biopsies. To obtain different stages of biofilm formation, polymer disks were attached to prostheses and carried intraorally for 1, 3, 5, and 9 days. The expression of genes for hBD-2, PSO, RNase 7, 5-LO, and IL-8 was examined using semi-quantitative reverse transcription-polymerase chain reaction. The bacterial composition of the individual biofilms was defined using a microarray system (Parocheck), which showed the presence of 20 different bacterial species that are associated with plaque formation. RESULTS The expression of the messenger RNAs of hBD-2, RNase 7, and 5-LO was upregulated as a result of the exposure to early biofilm stages, whereas the gene expression of IL-8 was increased in response to matured biofilms. Inter-individual differences in the innate immune response were observed. CONCLUSION The results of the present study showed a time-dependent messenger RNA expression of antimicrobial peptides (hBD-2, RNase 7), 5-LO, and IL-8 in oral epithelial cells responding to different stages of biofilm formation.

Fluorescence‐controlled Er:YAG laser for caries removal in permanent teeth: a randomized clinical trial

The aim of this randomized clinical study was to compare the efficacy of a fluorescence-controlled erbium-loaded yttrium aluminum garnet (Er:YAG) laser with conventional bur treatment for caries therapy in adults. Twenty-six patients with 102 carious lesions were treated using either the Er:YAG laser, at threshold levels of 7, 8, 9, and 10 [U], or rotary burs. Both techniques were applied to each lesion at separate locations. After treatment, dentine samples were obtained using a carbide bur. The viable counts of Streptococcus mutans (SM) and lactobacilli (LB) [expressed as colony-forming units (log10 CFUs)], treatment time, pain, vibration, and sound intensity were determined. The median numbers of CFUs for SM and LB were not statistically different between laser and bur treatment at threshold levels 7 and 8 [U]. At threshold levels 9 and 10 [U], the median number of CFUs for LB [1.11 (range: 0.00-2.04)] were significantly higher following laser treatment than following bur treatment [0.30 (range: 0.00-0.60)]. The results indicate that treatment with a fluorescence-controlled Er:YAG laser at threshold levels of 7 and 8 removed caries to a level similar to that achieved using conventional bur treatment, with clinically irrelevant amounts of remaining bacteria. Although more time consuming, laser treatment provided higher patient comfort than bur treatment.

Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease

BACKGROUND AND AIMS The scope of this working group was to review (1) ecological interactions at the dental biofilm in health and disease, (2) the role of microbial communities in the pathogenesis of periodontitis and caries, and (3) the innate host response in caries and periodontal diseases. RESULTS AND CONCLUSIONS A health-associated biofilm includes genera such as Neisseria, Streptococcus, Actinomyces, Veillonella and Granulicatella. Microorganisms associated with both caries and periodontal diseases are metabolically highly specialized and organized as multispecies microbial biofilms. Progression of these diseases involves multiple microbial interactions driven by different stressors. In caries, the exposure of dental biofilms to dietary sugars and their fermentation to organic acids results in increasing proportions of acidogenic and aciduric species. In gingivitis, plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species. The natural mucosal barriers and saliva are the main innate defence mechanisms against soft tissue bacterial invasion. Similarly, enamel and dentin are important hard tissue barriers to the caries process. Given that the present state of knowledge suggests that the aetiologies of caries and periodontal diseases are mutually independent, the elements of innate immunity that appear to contribute to resistance to both are somewhat coincidental.

Neutrophil extracellular trap formation in supragingival biofilms.

BACKGROUND Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins. METHODS Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation. RESULTS Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation. CONCLUSIONS Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter-patient variability suggests NET formation as a potential factor influencing the individual course of disease.

Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria.

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.