An in vitro and in vivo study on the intensity of adhesion and colonization by Staphylococcus epidermidis and Pseudomonas aeruginosa on originally synthesized biomaterials with different chemical composition and modified surfaces and their effect on expression of TNF-α, ß-defensin 2 and IL-10 in tissues.

UNLABELLED: The aim of this study was to determine adhesion and colonization of bacteria on the surface of originally synthesized glass-ceramic biomaterials and their effect on inflammation reactions in tissues surrounding the implant. MATERIALS AND METHODS: Biomaterial discs were contaminated with bacterial suspensions of 10, 10(2), and 10(3) colony forming units (CFU)/mL (P. aeruginosa ATCC 27853 and S. epidermidis ATCC 12228), and after 2 hours of cultivation, the intensity of bacterial adhesion was determined. For in vivo tests, the samples were contaminated with 102 and 103 CFU/mL cultivated at 37°C for 2 h to ensure bacterial adhesion. Contaminated biomaterial samples were implanted in the interscapular area of chinchilla rabbits for 2 and 4 weeks. The biomaterials were removed, and using plate count and sonification methods, bacterial colonization on the surface of biomaterials was determined. Moreover, the expression of TNF-α, ß-defensin 2, and IL-10 in the surrounding tissues was assessed by using immunohistochemistry methods. RESULTS: P. aeruginosa more intensively colonized biomaterials in the in vivo study as compared with S. epidermidis. Il-10 is a regulatory cytokine, which reduces the intensity of inflammatory cell activity, thus reducing nonspecific resistance of the organism. CONCLUSIONS: The expression of TNF-α and IL-10 was not affected by short (2 and 4 weeks) biomaterial implantation. Pronounced cytokine expression in tissues around implanted biomaterials contaminated with P. aeruginosa was observed.

Treponema denticola suppresses expression of human {beta}-defensin-3 in gingival epithelial cells through inhibition of the toll-like receptor 2 axis.

We reported previously that Treponema denticola, one of the periodontal pathogens, suppresses the expression of human beta-defensins (HBDs) in human gingival epithelial cells. To identify the mechanisms involved in this suppression, immortalized and normal human gingival epithelial cells were infected with live or heat-killed T. denticola for 24 h, and then the expression of HBDs was examined by real-time RT-PCR. Live T. denticola suppressed the expression of HBD-3 substantially and also suppressed the expression of HBD-1 and HBD-2. However, heat-killed bacteria did not produce a suppressive effect but instead slightly upregulated the levels of HBD-2 and HBD-3. In contrast to live T. denticola, which reduced the activation of mitogen-activated protein kinase (MAPK) and NF-kappaB within an hour of infection, heat-killed bacteria did not show any inhibitory effect on the MAPK and NF-kappaB signaling pathways. Knockdown of Toll-like receptor 2 (TLR2) via RNA interference abolished the suppressive effect of T. denticola on the expression of HBD-3. Heat-killed T. denticola but not live bacteria could activate TLR2 in CHO/CD14/TLR2 reporter cells, suggesting that T. denticola contains a heat-labile inhibitor(s) of TLR2 in addition to ligands recognized by TLR2. Indeed, live T. denticola was able to inhibit TLR2 activation by Pam(3)CSK. In conclusion, T. denticola suppressed the expression of HBD-3 by inhibiting the TLR2 axis in gingival epithelial cells. These results may provide new insight into the pathogenesis of periodontitis caused by T. denticola.

The immune response of oral epithelial cells induced by single-species and complex naturally formed biofilms.

INTRODUCTION: In the oral cavity, the surfaces are constantly exposed to a complex variety of microorganisms organized in biofilms. As part of a sophisticated local immune response, gingival epithelial cells (GECs) express antimicrobial peptides, such as human beta-defensin-2 (hBD-2), ribonuclease 7 (RNAase-7), and psoriasin (PSO), and pro-inflammatory mediators, such as interleukin-8 (IL-8) and 5-lipoxygenase (5-LO). The aim of the present study was to test whether GECs show a differential immune response to single-species biofilms compared with multi-species biofilms. METHODS: GECs were cultured from biopsies derived from three different healthy donors (n = 3). To obtain naturally formed biofilm (NFB), polymer disks were attached to prostheses and carried intraorally for 12, 24, 36, and 48 h. In addition, single-species biofilms (SSB; Streptococcus mutans and Streptococcus mitis) were cultured on polymer disks in vitro (12, 24, 36, and 48 h). The messenger RNA (mRNA) expression of hBD-2, RNAase-7, PSO, IL-8, 5-LO, and glycerylaldehyde-3-phosphate dehydrogenase was analysed using semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: In GECs, the hBD-2 mRNA expression was significantly upregulated in response to S. mitis-biofilm stimulation compared with S. mutans-biofilm stimulation (P < 0.0001). In contrast, the RNAase-7 mRNA expression was significantly higher in GECs when responding to both S. mutans biofilms and naturally formed biofilms compared with S. mitis biofilms (P < 0.0001 and P < 0.001, respectively). The IL-8 and 5-LO mRNA was significantly upregulated in response to S. mutans biofilms (P < 0.0001 and P = 0.0002, respectively). CONCLUSION: This in vitro study found biofilm-dependent expression of antimicrobial peptides and inflammatory mediators in GECs.

Modulation of cytokine and beta-defensin 2 expressions in human gingival fibroblasts infected with Chlamydia pneumoniae.

Human beta-defensin 2 is an antimicrobial peptide that is produced by several epithelial cells after stimulation with micro-organisms and inflammatory mediators. Gram-negative bacteria, which are typically detected in periodontal pockets in periodontitis, elicit a stronger antibacterial peptide response of human beta-defensin 2 by epithelial cells. In this study, we investigated whether Chlamydia pneumoniae is able both to enter and grow in human gingival fibroblasts (HGF), to modify the production of cytokines, and is involved in regulation of beta-defensin 2 expression. Gingival fibroblasts discarded from periodontal procedures on healthy young individuals were infected with viable C. pneumoniae or with heat- or ultraviolet-inactivated organisms at a multiplicity of infection of 4 inclusion-forming units per cell. Our results demonstrate that after 48 h of incubation with viable C. pneumoniae, gingival fibroblasts showed a proliferative response as seen by both colorimetric assay and direct cell count (40% and 45%, respectively). Moreover, cells incubated with viable or ultraviolet light-inactivated C. pneumoniae organisms showed an increase in the levels of interleukin-6, interleukin-10 and human beta-defensin 2 in a time-dependent fashion, while the cells infected with heat-killed bacteria did not show a significant production either of the cytokines or beta-defensin 2 at any time. In conclusion, we demonstrate the correlation between multiplication of C. pneumoniae in human gingival fibroblasts and release of interleukin-6, interleukin-10 and up-regulation of beta-defensin 2, suggesting that gingival fibroblasts may be a periodontium niche for obligate intracellular C. pneumoniae and may play a role in innate gingival immune system and inflammatory response mechanisms of periodontitis.

Comparison of IL-1ß, TNF-α, hBD-2, and hBD-3 Expression in the Dental Pulp of Smokers Versus Nonsmokers.

INTRODUCTION: To date, the endodontic literature lacks research on the effect of smoking on cytokine and defensin expression in the dental pulp. Therefore, the aim of this study was to investigate the expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, human beta defensin (hBD)-2 and hBD-3 in the dental pulp of smokers and compare them with nonsmokers. We hypothesized that cytokine and defensin expression would be reduced in smokers as compared with nonsmokers. METHODS: Thirty-two smokers and 37 nonsmokers with endodontic pulpal diagnoses of normal, symptomatic irreversible pulpitis and asymptomatic irreversible pulpitis were included in this cross-sectional study. Samples from pulp chambers were collected and stored in phosphate-buffered saline at -80°C. Luminex was used to measure IL-1ß and TNF-α levels. The levels of hBD-2 and hBD-3 were measured using enzyme-linked immunosorbent assay. Marker levels were normalized to protein concentrations and data were analyzed using Kruskal-Wallis test, Mann-Whitney U test, and 2-way analysis of variance (α = 0.05). RESULTS: Pulpal concentrations of TNF-α and hBD-2 were significantly lower among smokers (P < .01), whereas no significant difference was observed for IL-1ß, or hBD-3. Two-way analysis of covariance revealed that smoking status (P < .001), not endodontic diagnosis (pulpal status), significantly affected TNF-α and hBD-2 levels. CONCLUSIONS: This study reported that smokers are immunologically deficient in TNF-α and hBD-2, suggesting that dental pulps of smokers possess limited defense mechanisms, affecting their endodontic prognosis and indicating a cause for their reported inferior outcome.

A gene delivery approach for antimicrobials: expression of defensins.

INTRODUCTION: Peptide antibiotics as new therapeutic agents are becoming a popular option to investigate due to their broad bacterial target selectivity and limited resistance problems. Although attractive, these new drug candidates have several limitations including low potency and delivery issues which face all peptides/proteins. METHODS: In this study, we designed a plasmid expression system for human beta defensin 3. This sequence was cloned from a human epithelial lung cell into a CMV driven expression cassette. This expression plasmid was then evaluated for its ability to produce human-beta defensin 3 with the use of the non-viral transfection agent, polyethylenimine (PEI). RESULTS: The results indicate the expression cassette was transcriptionally active in HEK 293 cells, as measured by RT-PCR and that a beta defensin peptide was produced by the cells as confirmed by Western blot. The biological activity of the peptide was confirmed against both gram negative E. coli and gram positive Bacillus species using in vitro screening. CONCLUSION: Both the cultured media as well as the transfected cell lysate demonstrated biological activity demonstrating the peptide is also secreted.

The expression of human ß-defensins (hBD-1, hBD-2, hBD-3, hBD-4) in gingival epithelia.

OBJECTIVE: The aim of this study was to analyze and compare the expression patterns of human ß-defensin-4 (hBD-4) with those of hBD-1, hBD-2, and hBD-3 in healthy and chronically inflamed gingival tissues. MATERIALS AND METHODS: A total of 96 gingival samples were collected, comprising 46 biopsies from chronic periodontitis (CP) patients and 50 from individuals with healthy tissue. Of these, 21CP and 21 healthy samples were used to examine the protein expression of the hBDs by immunohistochemistry. The remaining 25CP and 29 healthy tissue samples were subjected to real-time reverse transcription polymerase chain reaction to quantify the mRNA expression of the hBDs. RESULTS: All four types of ß-defensin peptides were confined to the gingival epithelia. The percentage of samples positive for hBD-4 mRNA was significantly lower than the percentages for the other three ß-defensins, both in healthy (p=0.003) and in inflamed (p=0.030) gingiva. However, in the case of the relative mRNA expression levels, that of hBD-4 was significantly higher than the levels for hBD-2 (healthy group: p<0.01; CP group: p<0.01) and hBD-3 (healthy group: p=0.004; CP group: p<0.01). CONCLUSIONS: Our study demonstrates differential expression of hBD-1, hBD-2, hBD-3, and hBD-4 in healthy and CP gingiva.

Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells.

BACKGROUND: To investigate the possible role of beta-defensins in gingival health and periodontal disease, we examined the effect of several stimuli on the expression of interleukin-8 (IL-8), human beta-defensin-1, -2, -3, and -4 (hBD) in primary human diseased gingival epithelial (HGE) cell cultures from periodontitis patients by quantitative TaqMan reverse transcription polymerase chain reaction (RT-PCR). METHODS: Several strains of the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were added to the cells, as well as the oral commensal bacteria Fusobacterium nucleatum and Escherichia coli. The induction by the proinflammatory stimuli phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF-alpha) was also tested. RESULTS: In addition to the published observations (PMA induces hBD-2 and -4; TNF-alpha induces hBD-2 and -3), it was found that PMA can upregulate hBD-1 and hBD-3, whereas TNF-alpha can induce hBD-4. The commensal bacteria were significant inducers of hBD-2, hBD-3, and IL-8. The pathogen P. gingivalis induced hBD-1 and hBD-3 at different time points than the commensals, but no induction of IL-8 and hBD-2 could be observed. These data fit with the chemokine paralysis theory. A correlation was found between the pathogenicity of different serotypes of A. actinomycetemcomitans and the induction profiles of defensins and IL-8. CONCLUSION: The results suggest that a correlation can be found in diseased oral epithelium between the defensin profiles that are induced and the pathogenicity of the oral bacterial strains.

Genotype-specific regulation of oral innate immunity by T2R38 taste receptor.

The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group.

Intracellular calcium in signaling human beta-defensin-2 expression in oral epithelial cells.

Expression of human beta-defensins is correlated with differentiation in the oral epithelium, consistent with their function as part of the epithelial antimicrobial barrier. Because calcium is a known regulator of epithelial differentiation, we tested the hypothesis that calcium concentration mediates beta-defensin expression. Gingival epithelial cells were cultured in medium containing low calcium concentration (0.03 mM), then either changed to high extracellular calcium concentrations or stimulated with thapsigargin to release intracellular calcium stores in the presence or absence of BAPTA-AM, a calcium chelator. Human beta-defensin-2 (hBD-2) mRNA expression was rapidly induced by thapsigargin, and more slowly induced by high extracellular calcium. Induction of hBD-2 peptide was confirmed by immunofluorescence. BAPTA-AM inhibited hBD-2 induction by both thapsigargin and calcium in a dose-dependent fashion. In addition, BAPTA-AM inhibited hBD-2 induction by a bacterial stimulant. Collectively, these findings demonstrate that intracellular calcium is a critical mediator of hBD-2 expression. Abbreviations used in this study are: BAPTA-AM, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis (acetoxymethyl ester); DMSO, dimethylsulfoxide; F. nucleatum, Fusobacterium nucleatum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBDs, human beta-defensins; HGECs, human gingival epithelial cells; MAP, mitogen-activated protein; and RT-PCR, reverse-transcriptase/polymerase chain-reaction.

Health- and disease-associated species clusters in complex natural biofilms determine the innate immune response in oral epithelial cells during biofilm maturation.

The aim of the present study was to verify our hypothesis concerning the differential induction of various antimicrobial and immunomodulatory responses in oral epithelial cells by diverse bacterial species clusters. For this purpose, oral biofilms between 1 and 14 days of maturation (36 volunteers) were co-incubated with gingival epithelial cells. Subsequently, human ß-defensin (hBD)-2, hBD-3, LL-37, interleukin (IL)-1ß, IL-6, IL-8 and IL-10 mRNA expression profiles were quantified by quantitative reverse transcription PCR. The correlation between bacterial species and the host innate immune response was determined by relating these results to existing 16S rRNA phylogenetic analysis by amplicon sequencing (Langfeldt et al. 2014. PLoS One 9: e87449). Data were analysed by multiple factor analysis. Transcription of hBD-2 and hBD-3 was significantly associated with the abundance of species of the Prevotella cluster and the absence of species of the Streptococcus cluster. IL-1ß, -6, -8 and -10 mRNA syntheses were significant correlated with Leptotrichia species [Leptotrichia 302H02 (0.448, P < 0.0001), Leptotrichia nbw822e09c1 (0.214, P = 0.008) and Leptotrichia wadei (0.218, P = 0.007)] of the Prevotella cluster. In the third dimension IL-10 and members of the Prevotella cluster were negatively correlated, whereas hBD-3 and IL-1ß, IL-6 and IL-8 were positive correlated to axis 3, like members of the Proteobacteria cluster. In conclusion, distinct species of health- and disease-associated bacterial clusters induce antibacterial or immunomodulatory reactions in oral epithelial cells during early stages of bacteria-host interactions.

The induction expression of human ß-defensins in gingival epithelial cells and fibroblasts.

OBJECTIVE: The gingival epithelial cells and fibroblasts can produce antimicrobial peptides when stimulated by inflammatory cytokines. The purpose of the present study was to test whether gingival keratinocytes and gingival fibroblasts respond differently to inflammatory cytokine activation. This will enable us to understand the chronic inflammatory response in the process of periodontal disease. DESIGN: Gingival keratinocytes and fibroblasts were isolated and treated with different concentrations of IL-1ß and quantitative real-time PCR was performed to evaluate the induced expressions of hBD-1, hBD-2 and hBD-3. The induced response was compared between the gingival epithelial cells and fibroblasts. The inhibitors of p38 protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were applied to explore the molecular mechanism during the induction of hBDs in both cells. RESULTS: The results showed that the hBDs expressions were found to be induced by different concentrations of IL-1ß, but with several differences between gingival epithelial cells and fibroblasts. The hBDs mRNA expression in gingival fibroblasts was more sensitive compared with keratinocytes to different concentrations of IL-1ß. The hBD-1 and hBD-3 expressions in these two cells were down-regulated by IL-1ß and hBD-2 expression was up-regulated. The inflammatory cytokine IL-1ß had dual effect on hBDs expression. CONCLUSIONS: The gingival epithelial cells and fibroblasts respond differently to the inflammatory cytokine IL-1ß which indicated different roles played by the two cells in the host defense. The dual effect of IL-1ß on hBDs expression may contribute to the defensins down-regulation in periodontal disease.

A novel organotypic dento-epithelial culture model: effect of Fusobacterium nucleatum biofilm on B-defensin-2, -3, and LL-37 expression.

BACKGROUND: In the present study, the expression and localization of three epithelial peptides (human ß-defensin [hBD]-2 and -3, and cathelicidin [LL-37]) are studied in an organotypic dento-epithelial (OD-E) model exposed to Fusobacterium nucleatum (Fn) biofilm. METHODS: Biofilm of Fn ATCC 25586 or AHN 9508 were produced by culturing each strain on semipermeable membranes. The OD-E model was constructed by seeding keratinocytes on fibroblast-containing collagen gels and by placing dentin pieces on the top. A 3-day-old biofilm was placed on the top of the OD-E and the coculture was incubated for 5 hours or 24 hours. Production of epithelial antimicrobial peptides was determined immunohistochemically. RESULTS: After 5 hours of incubation, the biofilm of each Fn strain stimulated expression of hBD-2 and -3. hBD-2 was localized on superficial layers and hBD-3 on basal cell layers of the epithelium and dento-epithelial junctions, whereas LL-37 was only weakly expressed. After 24 hours, hBD-2 expression was extended toward basal cell layers of the epithelium. In contrast, hBD-3 expression extended toward superficial layers of the epithelium. In the case of Fn AHN 9508 biofilm, LL-37 was localized in the cell layers of the dento-epithelial junction. CONCLUSION: In our OD-E model, epithelial antimicrobial peptide responses to Fn biofilms have distinct regulation and localization characteristics, resembling those known to occur in the gingival epithelium in vivo.

ß-Defensin-4 (HBD-4) is expressed in chondrocytes derived from normal and osteoarthritic cartilage encapsulated in PEGDA scaffold.

Defensins are antibiotic peptides involved in host defense mechanisms, wound healing and tissue repair. Furthermore, they seem to play an important role in protection mechanisms in articular joints. The aim of this study was to investigate ß-defensin-4 expression in chondrocytes taken from articular cartilage of knees of patients with osteoarthritis (OA) compared to normal cartilage, in vivo in explanted tissue, and in vitro in chondrocytes encapsulated in construct PEGDA hydrogels. The present investigation was conducted to try and elucidate the possible use of ß-defensin-4 as a relevant marker for the eventual use of successive scaffold allografts, and to provide new insights for hydrogel PEGDA scaffold efficacy in re-differentiation or repair of OA chondrocytes in vitro. Articular cartilage specimens from OA cartilage and normal cartilage were assessed by histology, histochemistry, immunohistochemistry and Western blot analysis. The results showed strong ß-defensin-4 immunoexpression in explanted tissue from OA cartilage and weak ß-defensin-4 expression in control cartilage. The chondrocytes from OA cartilage after 4 weeks of culture in PEGDA hydrogels showed the formation of new hyaline cartilage and a decreased expression of ß-defensin-4 immunostaining comparable to that of control cartilage. Our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repair of cartilage lesions in patients with OA using ß-defensin-4 as a relevant marker.

Gene expression of human beta defensins-1 and -2 is significantly reduced in non-inflamed keratinized oral tissue of smokers.

OBJECTIVE: The impact of smoking on the local innate immune response in the oral cavity, and, commonly, on oral health is actively discussed in the scientific literature. The aim of the present study was to evaluate possible effects of smoking on gene expression of human beta-defensin-1 and -2 in human gingival tissue. MATERIAL AND METHODS: Biopsies of keratinized gingival tissues were taken from donors (with written informed consent) undergoing routine surgical treatment. Prior to the sample collection, participants with clinically healthy periodontium were classified as smokers (n=9) or non-smokers (n=9). Gingival tissue was homogenized, and total RNA was extracted and analysed by real-time RT-PCR for human beta-defensins-1-, -2-, and interleukins IL-1ß- and IL-6-, as well as GAPDH-mRNA. The data obtained were analysed for significant differences using the Mann-Whitney-U test. RESULTS: hBD-1- and hBD-2-, as well as IL-1ß- and IL-6-mRNA were detected in all gingival samples. Expression of hBD-1 and -2 was significantly reduced by nearly 2.5-fold (p<0.05; Mann-Whitney) in gingival samples of smokers compared to control group specimens (non-smokers). In contrast, no significant differences of the gene expression of IL-6 and IL-1ß were observed in human gingival tissue of smokers and non-smokers. CONCLUSION: The results presented here suggest that expression of human beta-defensins hBD-1 and -2, and, thus, the basal levels of innate immune defense reactions in the oral cavity are reduced by smoking.

Reduced human beta defensin 3 in individuals with periodontal disease.

The human beta defensin 3 (hBD3) is widely expressed in the oral cavity and exerts strong antibacterial and immunomodulatory activities. Hence, we hypothesized that hBD3 could play a protective role in the maintenance of periodontal homeostasis, and that it could be found in gingival crevicular fluid (GCF) of healthy individuals and those with periodontitis at levels correlating with the degree of periodontal health. By using an ELISA assay to quantify hBD3 in GCF, we demonstrated that the peptide is present at levels easily detectable in the majority of healthy individuals, but it is drastically reduced in GCF from those with periodontitis. Furthermore, hBD3 levels inversely correlate with the severity of the disease and the degree of colonization by combinations of bacterial species with elevated periodontopathogenic potential. Both genetic factors and host/bacterial proteases released in diseased sites may be responsible for the observed low/null hBD3 levels in GCF from individuals with periodontitis.

Role of SIRT1 in heat stress- and lipopolysaccharide-induced immune and defense gene expression in human dental pulp cells.

INTRODUCTION: Although bacterial infection and heat stress are common causes of injury in human dental pulp cells (HDPCs), little is known about the potential defense mechanisms mediating their effects. This study examined the role of SIRT1 in mediating heat stress and lipopolysaccharide (LPS)-induced immune and defense gene expression in HDPCs. METHODS: HDPCs were exposed to heat stress (42°C) for 30 minutes after stimulation with LPS (1 µg/mL) for 48 hours. The expression of defense genes was evaluated by reverse-transcriptase polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. RESULTS: LPS and heat stress synergistically increased the expression of SIRT1 and immune and defense genes such as interleukin (IL)-8, hemeoxygenase-1 (HO-1), and human ß-defensin 2 (hBD-2). Resveratrol enhanced LPS- and heat stress-induced expression of HO-1 and hBD-2 but reduced IL-8 messenger RNA levels. The stimulation of HO-1 and hBD-2 messenger RNA expression by LPS and heat stress was inhibited by sirtinol; SIRT1 small interfering RNA; and inhibitors of p38, ERK, JNK, and nuclear factor κB. CONCLUSIONS: These results show for the first time that SIRT1 mediates the induction of immune and defense gene expression in HDPCs by LPS and heat stress. SIRT1 may play a pivotal role in host immune defense system in HDPCS.

Gene expression of human beta-defensins in healthy and inflamed human dental pulps.

Human beta-defensins (hBDs) are antimicrobial peptides that play an important role in the innate host defense against bacterial invasion, contribute to promotion of adaptive immune responses, and show chemotactic activities. The aim of this study was to compare the gene expression of hBD-1, -2, -3, and -4 in healthy teeth and teeth with pulpitis. Samples of healthy and inflamed dental pulps were obtained from extracted third molars and during treatment of teeth with pulpitis. Gene expression was assessed by using reverse transcriptase reaction and real-time polymerase chain reaction. HBD-2 and hBD -3 were only weakly expressed in healthy and inflamed pulps. In contrast, the expression of hBD-1 and hBD -4 was significantly increased in inflamed compared with healthy pulps. These results suggest that hBD-1 and hBD-4 might play a role in the pulpal host defense.

Differential modulation of human {beta}-defensins expression in human gingival epithelia by Porphyromonas gingivalis lipopolysaccharide with tetra- and penta-acylated lipid A structures.

Porphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly involved in the development of chronic periodontitis. It displays a significant amount of lipid A structural heterogeneity, containing both tetra- (LPS(1435/1449) ) and penta-acylated (LPS( 1690)) lipid A structures with opposing effects on E-selectin expression in human endothelial cells. Little is known about how these two isoforms of P. gingivalis LPS could differentially affect host innate immune responses in human gingival epithelia. The present study compares the modulatory effects of P. gingivalis LPS(1435/1449) and LPS(1690) on the expression of human beta-defensins (hBDs) in the reconstituted human gingival epithelium, and examines the involvements of a panel of pattern recognition receptors in the modulatory effects concerned. It is shown that hBD-1, hBD-2 and hBD-3 mRNAs are significantly up-regulated by P. gingivalis LPS(1690), but down-regulated by P. gingivalis LPS( 1435/1449). Toll-like receptor (TLR) 2 and CD14 mRNAs are also differentially regulated, and the modulation of hBD-2 expression may be through the co-operation of both TLR2 and TLR4. This study suggests that P. gingivalis LPS with different lipid A structures could differentially modulate host innate immune responses in human gingival epithelia, which may be a hitherto undescribed novel pathogenic mechanism of P. gingivalis in periodontal pathogenesis.

Differential utilization of nuclear factor-kappaB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria.

INTRODUCTION: Human beta-defensin-2 (hBD-2) is an antimicrobial peptide, induced by bacterial stimuli and inflammation, that plays a role in mucosal and skin innate immune defense. The nuclear factor-kappaB (NF-kappaB) transcription factor family is important in innate and adaptive immune responses to bacteria and proinflammatory cytokines. NF-kappaB operates via the traditional IKKbeta signaling, as well as an alternative pathway utilizing IKKalpha signaling, which is important in keratinocyte differentiation. Our previous studies showed that pathogenic, but not commensal, bacteria used NF-kappaB signaling in hBD-2 induction. The objective of this study was to understand which arm of the NF-kappaB pathway is involved in gingival epithelial cell responses to pathogenic bacteria, including hBD-2 induction. METHODS: Cultured oral epithelial cells were transfected with synthetic small interfering RNAs (siRNAs) specific for various steps in each pathway, namely IKKbeta, TRAF6 and MyD88 in the canonical, and IKKalpha and TRAF3 in the alternative pathway, and subsequently stimulated with various oral bacteria. RESULTS: The hBD-2 induction level was reduced to 21-61% in cells in which the alternative NF-kappaB pathway was blocked and subsequently stimulated with pathogenic bacteria, while cells in which the canonical pathway was blocked showed reduction to 78-99%. Cells stimulated with commensals showed little change in hBD-2 induction level regardless of the siRNA used. Microarray analysis showed that oral epithelia differentially regulated numerous innate immune markers in response to pathogens and commensals. CONCLUSION: Our data suggest a role for the IKKalpha/TRAF3 pathway in NF-kappaB activation by pathogenic bacteria, while commensal bacteria do not utilize either NF-kappaB pathway, for hBD-2 induction.