The role of NLRP3 in regulating gingival epithelial cell responses evoked by Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) has myriads of virulence factors among which leukotoxin provides A. actinomycetemcomitans with the advantage to thrive in the surrounding hostile environment and evade host immune defences. The NLRP3 inflammasome has been associated with periodontal disease development. However, our understanding of the involvement of caspase-1, caspase-4, and NLRP3 in the release of IL-1ß and other inflammatory mediators from gingival epithelial cells during a A. actinomycetemcomitans infection is limited. The aim of this study was to investigate how the inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 regulate the immune response of gingival epithelial cells during a A. actinomycetemcomitans infection. Human gingival epithelial cells (Ca9-22) deficient in NLRP3, caspase-1 or caspase-4 were created using CRISPR/Cas9. Gingival epithelial cells were stimulated with the A. actinomycetemcomitans low-leukotoxic strain NCTC9710 or the highly leukotoxic JP2 strain HK 165 for 6, 12 and 24 h. The results showed that the JP2 strain HK1651 induced higher IL-1ß and IL-1RA release and mediated more epithelial cell death compared to the NCTC9710 strain. These findings were found to be capsase-1, caspase-4 and NLRP3-dependant. A targeted protein analysis of inflammation-related proteins showed that the expression of 37 proteins were identified as being significantly altered after HK1651 infection compared to unstimulated Cas9 and NLRP3-deficient cells. Of the 37 proteins, 23 of these inflammation-related proteins released by NLRP3-deficient cells differed significantly compared to Cas9 cells after infection. This suggests that NLRP3 has a broad effect on the inflammatory response in gingival epithelial cells.

Aggregatibacter actinomycetemcomitans colonization and persistence in a primate model.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.

Host Chromatin Regulators Required for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Activity in Saccharomyces cerevisiae Model.

Cytolethal distending toxin (CDT) is a bacterial genotoxin that causes host cell cycle arrest and death. We previously employed a Saccharomyces cerevisiae model with inducible expression of the CDT catalytic subunit from Aggregatibacter actinomycetemcomitans, AaCdtB, and showed that a wide variety of host factors play a role in facilitating the activity of CdtB. Our observation that a yeast H2B mutant defective in chromatin condensation was partially resistant to CdtB implies that chromatin structure may affect CDT function. In this study, we identified host chromatin regulatory genes required for CdtB cytotoxicity. We found that the deletion of HTZ1 or certain subunits of SWR, INO80, and SIR complexes increased cellular resistance to CdtB. We hypothesized that CdtB may interact with Htz1 or the chromatin, but immunoprecipitation experiments failed to detect physical interaction between CdtB and Htz1 or the chromatin. However, we observed reduced nuclear localization of CdtB in several mutants, suggesting that impaired nuclear translocation may, at least partly, explain the mechanisms of CdtB resistance. In addition, mutations in chromatin regulatory genes induce changes in the global gene expression profile, and these may indirectly affect CdtB toxicity. Our results suggest that decreased expression of endoplasmic reticulum (ER)-Golgi transport-related genes that may be involved in CdtB transport and/or increased expression of DNA repair genes may contribute to CdtB resistance. These results suggest that the functions of chromatin regulators may contribute to the activity of CDT in host cells.

Commensal and pathogenic biofilms differently modulate peri-implant oral mucosa in an organotypic model.

The impact of oral commensal and pathogenic bacteria on peri-implant mucosa is not well understood, despite the high prevalence of peri-implant infections. Hence, we investigated responses of the peri-implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri-implant mucosa-biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri-implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin-6 (IL-6), interleukin-8 (CXCL8), and monocyte chemoattractant protein-1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor-alpha (TNF-α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri-implant mucosa-biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri-implant disease.

Antibacterial activity of viable and heat-killed probiotic strains against oral pathogens.

Probiotics can stabilize gut flora, regulate intestinal immunity and protect the host from enteric diseases; however, their roles in oral health have received little attention compared to their roles in gut health. Nowadays, the prevalence of sugar-sweetened foods and abuse of antibiotics contribute towards dysbiosis of oral microbiota and drug resistance development in oral pathogens, resulting in various intractable oral diseases. We screened the antibacterial activities of viable and heat-killed probiotic strains against the oral pathogens Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. The probiotic strains Lactobacillus salivarius subsp. salicinius AP-32, L. rhamnosus CT-53, L. paracasei ET-66 and Bifidobacterium animalis subsp. lactis CP-9 displayed strong antipathogenic activities, whereas heat-killed AP-32, CT-53 and ET-66 displayed high levels of pathogen inhibition. The antibacterial activities of these probiotics were not associated with their H2 O2 production; L. acidophilus TYCA02 produced high levels of H2 O2 but merely exhibited moderate antibacterial activities. Oral tablets containing probiotics showed positive inhibitory effects against oral pathogens, particularly those containing viable probiotics. Our results indicate that probiotics prevent the growth of oral pathogens and improve oral health, providing insights into the antipathogenic efficacy of different probiotic species and their potential role in functional foods that improve oral health. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study provides insights into the antipathogenic efficacy of different probiotic species and their potential roles in developing functional foods to improve oral health. We showed that the probiotic strains Lactobacillus salivarius subsp. salicinius AP-32, L. rhamnosus CT-53, L. paracasei ET-66 and Bifidobacterium animalis subsp. lactis CP-9 have great potential for use in the development of functional foods to improve oral health. Since active probiotics may provide strong and long-term protection, the development of functional food products should favour the use of viable bacteria.

A bacterial genotoxin causes virus reactivation and genomic instability in Epstein-Barr virus infected epithelial cells pointing to a role of co-infection in viral oncogenesis.

We have addressed the role of bacterial co-infection in viral oncogenesis using as model Epstein-Barr virus (EBV), a human herpesvirus that causes lymphoid malignancies and epithelial cancers. Infection of EBV carrying epithelial cells with the common oral pathogenic Gram-negative bacterium Aggregatibacter actinomycetemcomitans (Aa) triggered reactivation of the productive virus cycle. Using isogenic Aa strains that differ in the production of the cytolethal distending toxin (CDT) and purified catalytically active or inactive toxin, we found that the CDT acts via induction of DNA double strand breaks and activation of the Ataxia Telangectasia Mutated (ATM) kinase. Exposure of EBV-negative epithelial cells to the virus in the presence of sub-lethal doses of CDT was accompanied by the accumulation of latently infected cells exhibiting multiple signs of genomic instability. These findings illustrate a scenario where co-infection with certain bacterial species may favor the establishment of a microenvironment conducive to the EBV-induced malignant transformation of epithelial cells.

Aggregatibacter actinomycetemcomitans infection causes DNA double-strand breaks in host cells.

Periodontal disease, an inflammatory disease, is caused by infection with periodontal pathogens. Long-term periodontal disease increases the risk of oral carcinogenesis. Similar to other peptic cancers, oral carcinogenesis also requires multiple genome instabilities; however, the risk factors related to the accumulation of genome instabilities are poorly understood. Here, we suggested that specific periodontal pathogens may increase the risk of genome instability. Accordingly, we screened several periodontal pathogens based on the ability to induce DNA double-strand breaks (DSBs) in host cells. We found that Aggregatibacter actinomycetemcomitans Y4 infection induced DSB formation in host cells. To assess whether DSB formation induced by infection with A. actinomycetemcomitans occurred through apoptotic chromosome fragmentation, cells were treated with a caspase inhibitor, Z-VAD-FMK. DSB accumulation induced by infection with A. actinomycetemcomitans was observed, even in the presence of Z-VAD-FMK, suggesting that this breakage occurred independently of apoptosis. These results suggested that some periodontal pathogens can increase the risk of genome instabilities in host cells and subsequently increase the risk of carcinogenesis.

Antimicrobial activity of red wine and oenological extracts against periodontal pathogens in a validated oral biofilm model.

BACKGROUND: Previous research findings support an antimicrobial effect of polyphenols against a variety of pathogens, but there is no evidence of this effect against periodontal pathogens in complex biofilms. The purpose of this study was to evaluate the antimicrobial activity of red wine and oenological extracts, rich in polyphenols, against the periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum and total bacteria growing in an in vitro oral biofilm static model. METHODS: A previously validated biofilm model, including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was developed on sterile hydroxyapatite discs. Red wine (and dealcoholized wine), and two polyphenols-rich extracts (from wine and grape seeds) were applied to 72 h biofilms by dipping the discs during 1 and 5 min in the wine solutions and during 30 s and 1 min in the oenological extracts. Resulting biofilms were analyzed by confocal laser scanning microscopy and viable bacteria (colony forming units/mL) were measured by quantitative polymerase chain reaction combined with propidium monoazide. A generalized linear model was constructed to determine the effect of the tested products on the viable bacterial counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, as well on the total number of viable bacteria. RESULTS: The results showed that red wine and dealcoholized red wine caused reduction in viability of total bacteria within the biofilm, with statistically significant reductions in the number of viable P. gingivalis after 1 min (p = 0.008) and in A. actinomycetemcomitans after 5 min of exposure (p = 0.011) with red wine. No evidence of relevant antibacterial effect was observed with the oenological extracts, with statistically significant reductions of F. nucleatum after 30 s of exposure to both oenological extracts (p = 0.001). CONCLUSIONS: Although moderate, the antimicrobial impact observed in the total bacterial counts and counts of A. actinomycetemcomitans, P. gingivalis and F. nucleatum, encourage further investigations on the potential use of these natural products in the prevention and treatment of periodontal diseases.

Increased citrullination and expression of peptidylarginine deiminases independently of P. gingivalis and A. actinomycetemcomitans in gingival tissue of patients with periodontitis.

BACKGROUND: A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. METHODS: Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. RESULTS: Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. CONCLUSION: Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.

Effects of bacterial physiological states and bacterial species on host-microbe interactions.

This study investigated how the physiological states of Aggregatibacter actinomycetemcomitans (Aa) and Streptococcus mitis affect their intracellular invasion capabilities and the resulting host cell responses. The physiological states included two forms of planktonic states, floating or sedimented (by centrifugation) and the biofilm state (with centrifugation). Confluent epithelial Ca9-22 cells were challenged with floating or sedimented planktonic cultures, or with 24-h biofilms for 3 h. The results show that intracellular invasion efficiencies were clearly affected by the bacterial physiological states. For both bacterial species, the sedimented-cells displayed 2-10 times higher invasion efficiency than the floating-cells (p < 0.05). The invasion efficiency of Aa biofilms was three fold lower than sedimented cells, whereas those of S. mitis biofilms were similar to sedimented cells. Unlike invasion, the metabolic activities of Ca9-22 were unaffected by different bacterial physiological states. However, Aa biofilms induced higher IL-1ß expression than planktonic cultures. In conclusion, different bacterial physiological states can affect the outcomes of (in vitro) host-microbe interaction in different ways.

Quorum sensing of Streptococcus mutans is activated by Aggregatibacter actinomycetemcomitans and by the periodontal microbiome.

BACKGROUND: The oral cavity is inhabited by complex microbial communities forming biofilms that can cause caries and periodontitis. Cell-cell communication might play an important role in modulating the physiologies of individual species, but evidence so far is limited. RESULTS: Here we demonstrate that a pathogen of the oral cavity, Aggregatibacter actinomycetemcomitans (A. act.), triggers expression of the quorum sensing (QS) regulon of Streptococcus mutans, a well-studied model organism for cariogenic streptococci, in dual-species biofilms grown on artificial saliva. The gene for the synthesis of the QS signal XIP is essential for this interaction. Transcriptome sequencing of biofilms revealed that S. mutans up-regulated the complete QS regulon (transformasome and mutacins) in the presence of A. act. and down-regulated oxidative stress related genes. A.act. required the presence of S. mutans for growth. Fimbriae and toxins were its most highly expressed genes and up-regulation of anaerobic metabolism, chaperones and iron acquisition genes was observed in co-culture. Metatranscriptomes from periodontal pockets showed highly variable levels of S. mutans and low levels of A. act.. Transcripts of the alternative sigma-factor SigX, the key regulator of QS in S. mutans, were significantly enriched in periodontal pockets compared to single cultures (log2 4.159, FDR ≤0.001, and expression of mutacin related genes and transformasome components could be detected. CONCLUSION: The data show that the complete QS regulon of S. mutans can be induced by an unrelated oral pathogen and S. mutans may be competent in oral biofilms in vivo.

Oral microbe-host interactions: influence of ß-glucans on gene expression of inflammatory cytokines and metabolome profile.

BACKGROUND: The aim of this study was to evaluate the effects of ß-glucan on the expression of inflammatory mediators and metabolomic profile of oral cells [keratinocytes (OBA-9) and fibroblasts (HGF-1) in a dual-chamber model] infected by Aggregatibacter actinomycetemcomitans. The periodontopathogen was applied and allowed to cross the top layer of cells (OBA-9) to reach the bottom layer of cells (HGF-1) and induce the synthesis of immune factors and cytokines in the host cells. ß-glucan (10 µg/mL or 20 µg/mL) were added, and the transcriptional factors and metabolites produced were quantified in the remaining cell layers and supernatant. RESULTS: The relative expression of interleukin (IL)-1-α and IL-18 genes in HGF-1 decreased with 10 µg/mL or 20 µg/mL of ß-glucan, where as the expression of PTGS-2 decreased only with 10 µg/mL. The expression of IL-1-α increased with 20 µg/mL and that of IL-18 increased with 10 µg/mL in OBA-9; the expression of BCL 2, EP 300, and PTGS-2 decreased with the higher dose of ß-glucan. The production of the metabolite 4-aminobutyric acid presented lower concentrations under 20 µg/mL, whereas the concentrations of 2-deoxytetronic acid NIST and oxalic acid decreased at both concentrations used. Acetophenone, benzoic acid, and pinitol presented reduced concentrations only when treated with 10 µg/mL of ß-glucan. CONCLUSIONS: Treatment with ß-glucans positively modulated the immune response and production of metabolites.

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro.

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P < 0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20-50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.

Inner-membrane protein MorC is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans.

Fimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans are essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner-membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To investigate further the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the WT fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies and the presence of fimbriae. When the identical plasmid was transformed into a morC mutant strain, the bacterium did not display any of the phenotypes of fimbriated strains. Extension of these studies to a naturally fimbriated clinical strain showed that the resulting morC mutant maintained the characteristic colony morphology of fimbriated strains. There was, however, a reduction in the secretion of fimbrial subunits, and fewer fimbriae were observed on the surface of the mutant strain. Furthermore, the morC mutant of the fimbriated strain displayed a significantly altered biofilm microcolony architecture, while maintaining a similar biofilm mass to the parent strain. These results suggest that MorC influences fimbrial secretion and microcolony formation in A. actinomycetemcomitans.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Maresin 1 Biosynthesis and Proresolving Anti-infective Functions with Human-Localized Aggressive Periodontitis Leukocytes.

Localized aggressive periodontitis (LAP) is a distinct form of early-onset periodontitis linked to periodontal infection with uncontrolled inflammation and leukocyte-mediated tissue destruction. The resolution of inflammation is an active process orchestrated by specialized proresolving lipid mediators (SPMs). Since the level of the Maresin pathway marker 14-hydroxy-docosahexaenoic acid (14-HDHA) was lower in activated peripheral blood from LAP patients, we investigated the Maresin 1 (MaR1) biosynthetic pathway in these subjects and its role in regulating phagocyte functions. Macrophages from LAP patients had a lower level of expression of 12-lipoxygenase (∼30%) and reduced MaR1 (LAP versus healthy controls [HC], 87.8 ± 50 pg/10(6) cells versus 239.1 ± 32 pg/10(6) cells). Phagocytosis by LAP macrophages was reduced ∼40% compared to that of HC, and killing of periodontal pathogens, including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were similarly reduced. LAP neutrophils also displayed slower kinetics (∼30%) and decreased maximal phagocytosis (∼20% lower) with these pathogens than those of HC. The administration of MaR1 at 1 nM enhanced phagocytosis (31 to 65% increase), intracellular antimicrobial reactive oxygen species production (26 to 71% increase), bacterial killing of these periodontal pathogens (22 to 38% reduction of bacterial titers), and restored impairment of LAP phagocytes. Together, these results suggest that therapeutics targeting the Maresin pathway have clinical utility in treating LAP and other oral diseases associated with infection, inflammation, and altered phagocyte functions.

Coaggregation and biofilm growth of Granulicatella spp. with Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans.

BACKGROUND: Members of fastidious Granulicatella and Aggregatibacter genera belong to normal oral flora bacteria that can cause serious infections, such as infective endocarditis. Aggregatibacter actinomycetemcomitans has long been implicated in aggressive periodontitis, whereas DNA-based methods only recently showed an association between Granulicatella spp. and dental diseases. As bacterial coaggregation is a key phenomenon in the development of oral and nonoral multispecies bacterial communities it would be of interest knowing coaggregation pattern of Granulicatella species with A. actinomycetemcomitans in comparison with the multipotent coaggregator Fusobacterium nucleatum. The aim was to investigate coaggregation and biofilm formation of Granulicatella elegans and Granulicatella adiacens with A. actinomycetemcomitans and F. nucleatum strains. RESULTS: F. nucleatum exhibited significantly (p < 0.05) higher autoaggregation than all other test species, followed by A. actinomycetemcomitans SA269 and G. elegans. A. actinomycetemcomitans CU1060 and G. adiacens did not autoaggregate. G. elegans with F. nucleatum exhibited significantly (p < 0.05) higher coaggregation than most others, but failed to grow as biofilm together or separately. With F. nucleatum as partner, A. actinomycetemcomitans strains SA269, a rough-colony wild-type strain, and CU1060, a spontaneous smooth-colony laboratory variant, and G. adiacens were the next in coaggregation efficiency. These dual species combinations also were able to grow as biofilms. While both G. elegans and G. adiacens coaggregated with A. actinomycetemcomitans strain SA269, but not with CU1060, they grew as biofilms with both A. actinomycetemcomitans strains. CONCLUSIONS: G. elegans failed to form biofilm with F. nucleatum despite the strongest coaggregation with it. The ability of Granulicatella spp. to coaggregate and/or form biofilms with F. nucleatum and A. actinomycetemcomitans strains suggests that Granulicatella spp. have the potential to integrate into dental plaque biofilms.

Interleukin-8 induces DNA synthesis, migration and down-regulation of cleaved caspase-3 in cultured human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC). MATERIAL AND METHODS: DNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit. RESULTS: IL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC. CONCLUSION: IL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells.

FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

BACKGROUND: Periodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation. METHODS: Murine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR). RESULTS: FTY720 dose-dependently inhibited IL-1ß, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts. CONCLUSION: FTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

Effect of Aggregatibacter actinomycetemcomitans from Aggressive Periodontitis patients on Streptococcus mutans.

OBJECTIVES: To determine in vivo association between Aggregatibacter actinomycetemcomitans (Aa) and Streptococcus mutans (Sm) in aggressive periodontitis patients (AgP) and the in vitro influence on Sm of saliva and of Aa strains isolated from individual Aa-positive patients. MATERIALS AND METHODS: Clinical indices and saliva samples were taken from 30 AgP patients. Aa and mutans streptococci levels were determined. Antibacterial effect of saliva from 12 Aa-positive patients, and their individual Aa strain, was checked turbidimetrically in vitro on Sm. RESULTS: Aa salivary level was inversely correlated with levels of mutans streptococci and directly correlated with pockets of ≥7 mm. During exponential growth phase: (i) All Aa-positive and Aa-negative saliva samples showed no significant influence on Sm growth. (ii) Each individually isolated Aa strain presented significant inhibitory effect on Sm growth. During stationary growth phase, all the above demonstrated an inhibitory effect on Sm growth, with significantly greater influence of Aa individual strains. CONCLUSION: Saliva of each AgP Aa-positive subject had an inhibitory effect on Sm growth, which is most likely derived from Aa bacterial physiology. This research raises the possibility that suppression of Aa due to periodontal treatment may increase Sm levels and hence caries incidence.