Humoral immune response to Aggregatibacter actinomycetemcomitans leukotoxin

Brage M, Holmlund A, Johansson A. Humoral immune response toAggregatibacter actinomycetemcomitans leukotoxin. J Periodont Res 2011; 46: 170–175. © 2010 John Wiley & Sons A/S Background and Objective: Periodontal disease is an inflammatory condition caused by bacterial infections that result in loss of the tooth supporting tissue. The periodontal pathogens produce virulence factors with capacity to affect the host immune response. Aggregatibacter actinomycetemcomitans is a periodontal pathogen that produces a leukotoxin that specifically affects human leukocytes. The aims of the present study were to examine the presence and function of systemic antibodies to the leukotoxin. Material and Methods: One hundred and ninety‐seven middle‐aged (57 ± 5 years) Swedes with well‐documented periodontal status and medical factors related to cardiovascular diseases were studied. These data have been published previously. The serum samples were examined for the presence of leukotoxin antibodies by western blot and the capacity to neutralize leukotoxicity in an activity assay with leukotoxin and cultured leukemic cells. Results: The results showed a high prevalence (57%) of antibodies against A. actinomycetemcomitans leukotoxin in the analyzed population. These antibodies were correlated with leukotoxin neutralizing capacity as well as with the ELISA titers of A. actinomycetemcomitans‐specific IgA and IgG. In addition, high levels of leukotoxin antibodies were correlated with increasing age, but not with periodontal disease parameters or cardiovascular risk factors. Conclusion: Systemic antibodies against A. actionmycetemcomitans leukotoxin were common in this adult Swedish population. These antibodies might contribute to limit the systemic effects of the infection.     

Epigallocatechin gallate inhibits leukotoxin release by Aggregatibacter actinomycetemcomitans by promoting association with the bacterial membrane

The oral pathogen, Aggregatibacter actinomycetemcomitans, produces a number of virulence factors, including a leukotoxin (LtxA), which specifically kills human white blood cells, to provide a colonization advantage to the bacterium. Strains of A. actinomycetemcomitans that produce more LtxA have been more closely linked to disease, indicating that this toxin plays a key role in pathogenesis of the bacterium. Disruption of the activity of LtxA thus represents a promising approach to reducing the pathogenicity of the bacterium. Catechins are polyphenolic molecules derived from plants, which have shown potent antibacterial and antitoxin activities. We have previously shown that galloylated catechins are able to prevent LtxA delivery to host cells by altering the toxin's secondary structure and preventing binding to cholesterol on the host cell membrane. Here, we have investigated how one particular galloylated catechin, epigallocatechin gallate (EGCg), affects A. actinomycetemcomitans growth and toxin secretion. Our results demonstrate that EGCg, at micromolar concentrations, inhibits A. actinomycetemcomitans growth, as has been reported for other bacterial species. At subinhibitory concentrations, EGCg promotes LtxA production, but the toxicity of the bacterial supernatant against human immune cells is reduced. The results of our biophysical studies indicate that this seemingly contradictory result is caused by an EGCg‐mediated enhancement of LtxA affinity for the bacterial cell surface. Together, these results demonstrate the potential of EGCg in the treatment of virulent A. actinomycetemcomitans infections.     

The conserved carboxyl domain of MorC,an inner membrane protein of Aggregatibacter actinomycetemcomitans,is essential for membrane function

Morphogenesis protein C (MorC) of Aggregatibacter actinomycetemcomitans is important for maintaining the membrane morphology and integrity of the cell envelope of this oral pathogen. The MorC sequence and operon organization were found to be conserved in Gammaproteobacteria, based on a bioinformatic analysis of 435 sequences from representative organisms. Functional conservation of MorC was investigated using an A. actinomycetemcomitans morC mutant as a model system to express MorC homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. The A. actinomycetemcomitans strains expressing the homologous proteins were assessed for sensitivity to bile salts, leukotoxin secretion, autoaggregation and membrane morphology. MorC from the most closely related organism (H. influenzae) was functionally identical to MorC from A. actinomycetemcomitans. However, the genes from more distantly related organisms restored some but not all A. actinomycetemcomitans mutant phenotypes. In addition, deletion mutagenesis indicated that the most conserved portion of the protein, the C‐terminus DUF490 domain, was necessary to maintain the integrity of the membrane. Deletion of the last 10 amino acids of this domain of the A. actinomycetemcomitans MorC protein was sufficient to disrupt membrane stability and leukotoxin secretion. The data suggest that the MorC sequence is functionally conserved across Gammaproteobacteria and the C‐terminus of the protein is essential for maintaining membrane physiology.     

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell‐surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function‐associated antigen‐1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of ³³⁴LEEYSKR³⁴⁰, in the N‐terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP‐1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC³³⁶ motif of LtxA (CRAC^336WT). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP‐1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol‐binding site of LtxA may have a potential clinical applicability in controlling infections of repeats‐in‐toxin‐producing organisms.     

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

Aggregatibacter actinomycetemcomitans is a Gram‐negative bacterium associated with localized aggressive periodontitis, as well as other systemic diseases. This organism produces a number of virulence factors, all of which provide some advantage to the bacterium. Several studies have demonstrated that clinical isolates from diseased patients, particularly those of African descent, frequently belong to specific clones of A. actinomycetemcomitans that produce significantly higher amounts of a protein exotoxin belonging to the repeats‐in‐toxin (RTX) family, leukotoxin (LtxA), whereas isolates from healthy patients harbor minimally leukotoxic strains. This finding suggests that LtxA might play a key role in A. actinomycetemcomitans pathogenicity. Because of this correlation, much work over the past 30 years has been focused on understanding the mechanisms by which LtxA interacts with and kills host cells. In this article, we review those findings, highlight the remaining open questions, and demonstrate how knowledge of these mechanisms, particularly the toxin's interactions with lymphocyte function‐associated antigen‐1 (LFA‐1) and cholesterol, enables the design of targeted anti‐LtxA strategies to prevent/treat disease.     

Aggregatibacter actinomycetemcomitans,a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis

Aggregatibacter actinomycetemcomitans is a perio‐pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and preclinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra‐physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non‐hematopoietic and hematopoietic. Non‐hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid‐derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo‐immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast–osteoblast‐mediated bone remodeling, which subsequently leads to net alveolar bone loss.     

A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans

Takashima E, Yamada H, Yajima A, Shiomi K, ōmura S, Kiyoshi K. A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01211.x. © 2009 John Wiley & Sons A/S Background and Objective: Quinol peroxidase (QPO) catalyzes peroxidase activity using quinol in the respiratory chain as a substrate. Quinol peroxidase is essential for the secretion of leukotoxin (LtxA), which destroys leukocytes and erythrocytes in humans and is one of the major virulence factors of Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis. In the present study, we aimed to find a highly potent QPO inhibitor to attenuate the virulence of A. actinomycetemcomitans. Material and Methods: For screening of QPO inhibitors, QPO activity was measured kinetically by SpectraMax Plus with 96‐well UV plates. Three hundred compounds in the Kitasato Institute for Life Sciences Chemical Library were screened. Secretion of LtxA in the culture supernatant was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Cytotoxicity against human promyelocytic leukemia cell line (HL‐60) cells from the culture supernatant was measured by Trypan Blue exclusion test. Results: The present study characterized ascofuranone as a highly potent inhibitor of QPO (K_i = 9.557 ± 0.865 nm ). Ascofuranone inhibited secretion of LtxA by A. actinomycetemcomitans in a dose‐dependent manner, making A. actinomycetemcomitans less pathogenic to HL‐60 cells. Conclusion: Quinol peroxidase inhibitors are promising candidates as alternative drugs for the treatment and prevention of the onset of localized aggressive periodontitis. Using ascofuranone as a seed compound, further study of QPO inhibitors could provide novel chemotherapeutic strategies for controlling localized aggressive periodontitis.     

Actinobacillus actinomycetemcomitans induces lethal effects on the macrophage‐like human cell line U937

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte‐macrophage‐like cell line U937 using the trypan blue exclusion test and WST‐1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol‐12‐myristate 13‐acetate (PMA)‐induced U937 cells than uninduced U937 cells. Morphological changes in PMA‐induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 °C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST‐1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.     

Frequency of 530‐bp deletion in Actinobacillus actinomycetemcomitans leukotoxin promoter region

Actinobacillus actinomycetemcomitans strains showing a 530‐bp deletion in the promoter region of the leukotoxin gene operon elaborate high amounts of leukotoxin that may play a role in the pathogenesis of periodontal disease. This study used polymerase chain reaction detection to determine the occurrence of the 530‐bp deletion in 94 A. actinomycetemcomitans strains from individuals of various ethnic backgrounds. Eleven blacks and one Hispanic subject but no Caucasian or Asian subjects showed the 530‐bp deletion in the leukotoxin promoter region, suggesting that the deletion is mainly a characteristic of individuals of African descent. A. actinomycetemcomitans strains exhibiting a deletion in the leukotoxin promotor region occurred both in individuals having severe periodontitis and in adolescents revealing no evidence of destructive periodontal disease.     

Molecular mimicry of Aggregatibacter actinomycetemcomitans with β2 glycoprotein I

Introduction: β2‐Glycoprotein I (β2GPI) is important in the suppression of coagulation, and antibodies against TLRVYK peptides on the β2GPI molecule are related to thrombosis. According to the Swiss‐Prot database, Aggregatibacter actinomycetemcomitans leukotoxin c has sequences (SIRVYK) that are homologous to the TLRVYK peptides. The aim of this study was to investigate the effects of A. actinomycetemcomitans infection on the antibody response against SIRVYK peptides in patients with periodontitis. Methods: Serum immunoglobulin G (IgG) antibody and IgG subclass antibody titers against SIRVYK or TLRVYK peptides were measured by enzyme‐linked immunosorbent assay in 46 patients with aggressive periodontitis (eight with localized disease, 38 with generalized disease), 28 patients with chronic periodontitis, and 20 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction. Results: The level of anti‐SIRVYK antibodies was significantly higher in patients who were A. actinomycetemcomitans‐positive than in A. actinomycetemcomitans‐negative patients (P < 0.05) in the chronic periodontitis group. A similar trend was found in the antibody response to TLRVYK peptide; however, no statistically significant difference was seen between A. actinomycetemcomitans‐positive and ‐negative patients. The A. actinomycetemcomitans‐positive patients displayed significantly higher levels of anti‐SIRVYK IgG2 and IgG3 antibodies than A. actinomycetemcomitans‐negative patients (P < 0.05 and P < 0.05, respectively). The level of IgG2 was highest among the four IgG subclasses and it predominantly increased in patients who were A. actinomycetemcomitans‐positive. Anti‐TLRVYK antibody levels were significantly correlated with anti‐SIRVYK IgG antibody levels. Conclusion: The results suggest that A. actinomycetemcomitans infection may elicit anti‐SIRVYK IgG antibodies and modify the anti‐TLRVYK antibody response in patients with periodontitis by molecular mimicry with β2GPI.     

Host–microbial co-evolution in periodontitis associated with Aggregatibacter actinomycetemcomitans infection

Periodontitis is caused by bacterial biofilms composed of more than 800 bacterial species. Not all biofilm bacteria are equally pathogenic; Aggregatibacter actinomycetemcomitans is recognized as one of the most virulent periodontopathic bacteria. A. actinomycetemcomitans is also associated with systemic diseases, including cardiovascular disease and brain abscesses. Molecular mimicry between A. actinomycetemcomitans and human β2-glycoprotein I may be associated with systemic diseases such as threatened preterm labor (TPL), preterm birth (PB), and Buerger's disease. Patients with certain genetic neutrophil dysfunction disorders (e.g., Kostmann and Papillon-Lefevre syndromes) also suffer from severe periodontitis. These patients are frequently infected with A. actinomycetemcomitans, suggesting that an impaired immune system might be relevant in the pathogenesis of A. actinomycetemcomitans-associated periodontitis. Virulence of A. actinomycetemcomitans differs amongst clones. JP2 is a highly pathogenic A. actinomycetemcomitans clone that has a deletion in the leukotoxin promoter, resulting in the production of large amounts of leukotoxin. Dissemination of the JP2 clone is skewed to certain regions and ethnic groups. JP2 has not been reported in East Asia, e.g., Japan, Korea, and China. However, an A. actinomycetemcomitans clone with an insertion sequence in the leukotoxin promoter was reported as a highly pathogenic clone in Japanese periodontitis patients. This evidence suggests that host–microbial co-evolution might exist in A. actinomycetemcomitans-associated periodontitis, and elucidation of the co-evolution of A. actinomycetemcomitans and its host might enable molecular diagnosis and treatment of A. actinomycetemcomitans-associated periodontitis.     

Microbiological and immunological characteristics of young Moroccan patients with aggressive periodontitis with and without detectable Aggregatibacter actinomycetemcomitans JP2 infection

Cross‐sectional and longitudinal studies identify the JP2 clone of Aggregatibacter actinomycetemcomitans as an aetiological agent of aggressive periodontitis (AgP) in adolescents of northwest African descent. To gain information on why a significant part of Moroccan adolescents show clinical signs of periodontal disease in the absence of this pathogen we performed comprehensive mapping of the subgingival microbiota of eight young Moroccans, four of whom were diagnosed with clinical signs of AgP. The analysis was carried out by sequencing and phylogenetic analysis of a total of 2717 cloned polymerase chain reaction amplicons of the phylogenetically informative 16S ribosomal RNA gene. The analyses revealed a total of 173 bacterial taxa of which 39% were previously undetected. The JP2 clone constituted a minor proportion of the complex subgingival microbiota in patients with active disease. Rather than identifying alternative aetiologies to AgP, the recorded infection history of the subjects combined with remarkably high concentrations of antibodies against the A. actinomycetemcomitans leukotoxin suggest that disease activity was terminated in some patients with AgP as a result of elimination of the JP2 clone. This study provides information on the microbial context of the JP2 clone activity in a JP2‐susceptible population and suggests that such individuals may develop immunity to AgP.     

Multilevel modeling of gingival bleeding on probing in young adult carriers of non-JP2-like strains of Aggregatibacter actinomycetemcomitans

The influence of Aggregatibacter actinomycetemcomitans on inflammation in subjects with gingivitis has not been studied in great detail. Seventeen healthy young adults with plaque-induced gingivitis or localized mild chronic periodontitis harboring cultivable numbers of A. actinomycetemcomitans were thoroughly examined. Samples of subgingival plaque were obtained from mesial surfaces of all teeth present. In addition, 12 oral mucosal surfaces and unstimulated saliva were sampled. Species identity, presence of the leukotoxin gene, and absence of a specific 530 b deletion in the leukotoxin promoter region indicating non-JP2-like strains were assessed by polymerase chain reaction. Based on a multilevel random intercept model adjusted for probing depth, age, and smoking status, the odds of bleeding on probing was increased by a factor of 1.89 (1.09–3.29, p = 0.024) if, in addition to plaque, A. actinomycetemcomitans could be recovered from the site. At a site without visible supragingival plaque but with cultivable numbers of subgingival A. actinomycetemcomitans the odds ratio of bleeding on probing was 3.37 (0.86–13.2, p = 0.081). Simulating variance partition coefficients revealed that between 1–2% (a clean, shallow site without A. actinomycetemcomitans; a deep site covered by plaque containing A. actinomycetemcomitans) and 6–7% (a moderately deep site with neither visible plaque nor cultivable A. actinomycetemcomitans) of the residual variance was attributable to differences between subjects. The present cross-sectional study indicates that non-JP2-like strains of A. actinomycetemcomitans may enhance gingival bleeding tendency even in the absence of clinically visible supragingival plaque.     

The in vivo T helper type 17 and regulatory T cell immune responses to Aggregatibacter actinomycetemcomitans

The periodontal pathogen Aggregatibacter actinomycetemcomitans is known to elicit a systemic immune response in the infected host, and occasionally causes non‐oral infections. Detailed information on its immunopathological responses and the involvement of bacterial virulence factors remains to be elucidated. The aim of this study was to assess the systemic immune response to A. actinomycetemcomitans oral infection. We used an animal model that simulates systemic dissemination of the bacteria by injecting live wild‐type (WT) D7S‐1 and a double knockout mutant of leukotoxin and cytolethal distending toxin (ΔltxΔcdt) A. actinomycetemcomitans strains in rat oral mucosa. Draining lymph nodes were examined for regulatory T (Treg) and T helper type 17 (Th17) cell subsets and their associated mediators. An increase in the proportion of Th17 cells and a decrease in Treg cells over the experimental period of 3 weeks were similarly observed for rats challenged with WT and ΔltxΔcdt. Significant upregulation and downregulation of proinflammatory cytokines in the Th17 gene pathway was noted, as well as several qualitative differences between WT and ΔltxΔcdt. Furthermore, we observed differential fold regulation in key genes associated with a proinflammatory response in ΔltxΔcdt‐inoculated rats relative to D7S‐1 group. This suggests that although the knockout of these two virulence factors (ΔltxΔcdt) may suppress certain proinflammatory genes, it causes similar over‐expression of other genes compared with D7S‐1, indicating a common factor that still remains in the pathogenicity of A. actinomycetemcomitans.     

Interaction of oral bacteria with gingival epithelial cell multilayers

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air–liquid interface in low‐calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐6 and IL‐8 secretion; and F. nucleatum stimulated production of IL‐1β and TNF‐α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.     

Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA‐1 expressing immune cells

Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function‐associated antigen 1 (LFA‐1; CD11a/CD18)‐bearing immune cells through a lysosomal‐mediated mechanism. Lysosomes are membrane‐bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH‐sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8–7.5). In the current study, we have probed the effect of LtxA‐induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562‐puro/LFA‐1 cells were stably transfected with CD11a and CD18 cDNA to express LFA‐1 on the cell surface while K562‐puro, which does not express LFA‐1, served as a control. Following treatment with 100 ng ml^?1 LtxA cells were analyzed by live cell imaging in conjunction with time‐lapse confocal microscopy and by flow cytometry. Using a pH‐sensitive indicator (pHrodo^®) we demonstrated that the toxin causes a decrease in the intracellular pH in K562‐puro/LFA‐1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker^® Red DND‐99 staining. These changes were not observed in K562‐puro cells or when heat inactivated toxin was added to K562‐puro/LFA‐1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562‐puro/LFA‐1 cells.     

Aggregatibacter actinomycetemcomitans‐Induced AIM2 Inflammasome Activation Is Suppressed by Xylitol in Differentiated THP‐1 Macrophages

Background: Aggressive periodontitis is characterized by rapid destruction of periodontal tissue caused by Aggregatibacter actinomycetemcomitans. Interleukin (IL)‐1β is a proinflammatory cytokine, and its production is tightly regulated by inflammasome activation. Xylitol, an anticaries agent, is anti‐inflammatory, but its effect on inflammasome activation has not been researched. This study investigates the effect of xylitol on inflammasome activation induced by A. actinomycetemcomitans. Methods: The differentiated THP‐1 macrophages were stimulated by A. actinomycetemcomitans with or without xylitol and the expressions of IL‐1β and inflammasome components were detected by real time PCR, ELISA, confocal microscopy and Immunoblot analysis. The effects of xylitol on the adhesion and invasion of A. actinomycetemcomitans to cells were measured by viable cell count. Results : A. actinomycetemcomitans increased pro IL‐1β synthesis and IL‐1β secretion in a multiplicity of infection‐ and time‐dependent manner. A. actinomycetemcomitans also stimulated caspase‐1 activation. Among inflammasome components, apoptosis‐associated speck‐like protein containing a CARD (ASC) and absent in melanoma 2 (AIM2) proteins were upregulated by A. actinomycetemcomitans infection. When cells were pretreated with xylitol, proIL‐1β and IL‐1β production by A. actinomycetemcomitans infection was significantly decreased. Xylitol also inhibited ASC and AIM2 proteins and formation of ASC puncta. Furthermore, xylitol suppressed internalization of A. actinomycetemcomitans into differentiated THP‐1 macrophages without affecting viability of A. actinomycetemcomitans within cells. Conclusions: A. actinomycetemcomitans induced IL‐1β production and AIM2 inflammasome activation. Xylitol inhibited these effects, possibly by suppressing internalization of A. actinomycetemcomitans into cells. Thus, this study proposes a mechanism for IL‐1β production via inflammasome activation and discusses a possible use for xylitol in periodontal inflammation caused by A. actinomycetemcomitans.     

Aggregatibacter actinomycetemcomitans induces a proatherosclerotic response in human endothelial cells in a three‐dimensional collagen scaffold model

The role of periodontopathogens in inflammatory endothelial dysfunction is not known. This study characterizes a three‐dimensional model with human coronary artery endothelial cells on three‐dimensional (HCAEC‐3D) type I collagen scaffolds to evaluate whether infection with Aggregatibacter actinomycetemcomitans induces a proinflammatory response associated with atherosclerosis. The HCAEC‐3D culture was physicochemically characterized with regard to biocompatibility and barrier function. Then, the culture was infected with A. actinomycetemcomitans strain ATCC 29522 at multiplicities of infection (MOIs) of 1:1, 1:10, and 1:100. Cultures without infection and stimulated with A. actinomycetemcomitans lipopolysaccharide were used as controls. The secretion of soluble factors (IL‐6, IL‐1β, MCP‐1, RANTES, MIP‐1, IL‐8, IL‐1α, and TNF‐α) was evaluated via flow cytometry; TGF‐β1 was evaluated via enzyme‐linked immunosorbent assay (ELISA). The adhesion and migration of fluorescent human THP‐1 monocytes was evaluated. IL‐8, MCP‐1, and IL‐6 secretion increased in a dose‐dependent manner with A. actinomycetemcomitans infection and was significantly greater than that under control treatment. The concentration of TGF‐β1 was significantly higher at MOI 1:100 than in controls. Treatment of the 3D cultures with A. actinomycetemcomitans at different MOIs induced significant differences in the adhesion of monocytes to the endothelium compared to the control without infection. Lastly, conditioned media from 3D cultures treated with A. actinomycetemcomitans induced monocyte migration. The effects of IL‐8, MCP‐1, IL‐6, and TGF‐β1 on the endothelium indicate the ability of A. actinomycetemcomitans to induce an inflammatory response through a mechanism of monocyte adhesion and migration and endothelial dysfunction.