Tomomi Hashizume-Takizawa

Porphyromonas gingivalis infection enhances Th17 responses for development of atherosclerosis

OBJECTIVES Porphyromonas gingivalis has been shown to associate with the development of atherosclerosis. Recent studies indicate that IL-17-producing T helper 17 (Th17) cells have been correlated with the emergence of atherosclerosis. Therefore, we investigated whether the Th17 cell response and expression of Th17-related molecules, in contrast with Th1- and Treg cells, are enhanced by P. gingivalis-challenge in Apolipoprotein E knockout (ApoE KO) mice. DESIGN Five mice were intravenously injected with P. gingivalis three times a week for 3 weeks and killed at 15 weeks of age. The proximal aorta lesion area, flow cytometry analysis and IL-17, IL-10, IFN-γ, and IL-1β levels in splenic cultures, and expression of Th17-related molecules in spleen and hearts were examined. RESULTS P. gingivalis-challenge showed notable accumulation of atherosclerotic plaques by Oil Red O-staining in ApoE KO mice. Intracellular cytokine staining revealed that significantly elevated CD4(+) interleukin (IL)-17A(+) T cells and slightly increased CD4(+) Foxp3(+) T cells was recognized in spleen cells of P. gingivalis-challenged mice compared with those from non-infected mice. P. gingivalis-challenge significantly increased IL-17 and IL-1β production and RORγt expression in splenic cells. Furthermore, the expression of Th17-related genes such as IL-6, TGF-β, RORγt and STAT3 were elevated in splenic cells as well as heart tissue of P. gingivalis-challenged mice. CONCLUSION These results suggest that P. gingivalis infection may enhance pro-inflammatory Th17 cell responses in lesion areas and spleen, thereby accelerating atherosclerosis.

Sublingual Vaccine with GroEL Attenuates Atherosclerosis

Autoimmune responses to heat-shock protein 60 (HSP60) contribute to the progression of atherosclerosis, whereas immunization with HSP60 may induce atheroprotective responses. We assessed the capacity of an atheroprotective vaccine that targeted a recombinant HSP60 from Porphyromonas gingivalis (rGroEL) to induce a protective mucosal immune response. Female apolipoprotein E-deficient spontaneously hyperlipidemic (Apoeshl) mice received sublingual delivery of rGroEL prior to P. gingivalis 381 injection. The animals were euthanized 16 weeks later. Sublingual immunization with rGroEL induced significant rGroEL-specific serum IgG responses. Antigen-specific cells isolated from spleen produced significantly high levels of IL-10 and IFN-γ after antigen re-stimulation in vitro. Flow cytometric analysis indicated that the frequencies of both IL-10+ and IFN-γ+ CD4+ Foxp3+ cells increased significantly in submandibular glands (SMG). Furthermore, sublingual immunization with rGroEL significantly reduced atherosclerosis lesion formation in the aortic sinus and decreased serum CRP, MCP-1, and ox-LDL levels. These findings suggest that sublingual immunization with rGroEL is associated with the increase of IFNγ+ or IL-10+ Foxp3+ cells in SMG and a systemic humoral response, which could be an effective strategy for the prevention of naturally occurring or P. gingivalis-accelerated atherosclerosis.

Aggregatibacter actinomycetemcomitans induces Th17 cells in atherosclerotic lesions.

Th17 cells have been linked to the pathogenesis of several chronic inflammatory and autoimmune diseases. However, the role of Th17 cells and IL-17 in atherosclerosis remains poorly understood. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) bacteremia accelerated atherosclerosis accompanied by inflammation in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. In this study, we investigated whether Aa promotes the Th17 inducing pathway in Aa-challenged Apoe(shl) mice. Mice were intravenously injected with live Aa HK1651 or vehicles. Time-course analysis of splenic IL-17(+)CD4(+) cell frequencies, the proximal aorta lesion area, serum IL-17, IL-6, TGF-β and IL-1β levels, the mRNA expression of Th17-related molecules such as IL-1β, IL-6, IL17RA, STAT3, IL-21, IL-23, TGF-β and RORγt, Th17-related microRNA levels and the levels of AIM-2, Mincle and NLRP3 were examined. Challenge with Aa time dependently induced tropism of Th17 cells in the spleen and increase in atheromatous lesions in the aortic sinus of Apoe(shl) mice. Serum IL-17, IL-6, TGF-β and IL-1β levels were significantly enhanced by Aa. The gene expression of IL-1β, IL-6, IL-17RA, IL-21, IL-23, TGF-β, STAT3, RORγt, AIM-2, Mincle and NLRP3 was also time dependently stimulated in the aorta of Aa-challenged mice. Furthermore, Aa challenge significantly increased the expression of miR-146b and miR-155 in the aorta. Based on the results, it seems that Aa stimulates Th17 induction that affects the progression of Aa-accelerated atherosclerosis.

Isolation and identification methods of Rothia species in oral cavities

Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable bacteria number on BHI-Y agar in the oral cavities of all subjects, respectively. It was indicated that among oral Rothia species, R. mucilaginosa is most predominant in the oral cavity of humans. A novel selective medium, ORSM, was useful for isolating each oral Rothia species.

A Feasible Enzyme-Linked Immunosorbent Assay System Using Monoclonal and Polyclonal Antibodies Against Glucosyltransferase-B from Streptococcus mutans

Streptococcus mutans has been considered the principal etiological agent of dental caries in humans. S. mutans can secrete three kinds of glucosyltransferases (GTFs). One of these, GTF-B, which synthesizes water-insoluble glucans from sucrose, has been considered to be one of the most important factors of cariogenic dental plaque formation. Therefore, determination of whether GTF-B is present in plaque and saliva samples may contribute to the evaluation of individual virulence potential (caries risk). The aim of this study was to develop a feasible enzyme-linked immunosorbent assay (ELISA) for the routine quantification of GTF-B in plaque-derived cultures and clinical samples, and to apply this assay to an epidemiological study. To determine the presence of GTF-B in plaque samples, a sandwich-ELISA was devised, consisting of mouse monoclonal and rabbit polyclonal antibodies against GTF-B and a horseradish peroxidase-conjugated anti-rabbit antibody. The developed ELISA allowed for quantification of the amounts of purified GTF-B with satisfactory sensitivity and specificity; this method was not affected by other components such as plaque and saliva. Plaque samples from healthy volunteers were examined using this ELISA method and microbial analysis to apply the assay to an epidemiological study. A correlation was observed between the amount of extracted GTF-B and S. mutans levels as determined by ELISA and cultivated with Mitis Salivarius Bacitracin agar plates derived from plaque samples, although there were some exceptions. In this regard, this ELISA system has the advantage of estimating both the individual numbers of S. mutans and the productivity of GTF-B, namely, the cariogenic potential of S. mutans simultaneously. These results indicate that this ELISA method is a useful tool for the diagnosis of caries risk.

Pseudopropionibacterium rubrum sp. nov., a novel red-pigmented species isolated from human gingival sulcus: Novel Pseudopropionibacterium

In this study, sStrain SK‐1T, a novel gram‐positive, pleomorphic, rod‐shaped, non‐spore forming, non‐motile organism, designated SK‐1T, was isolated from human gingival sulcus and found to produce acetic acid, propionic acid, lactic acid, and succinic acid as end products of glucose fermentation. Strain SK‐1T is most closely related to Pseudopropionibacterium (Propionibacterium) propionicum with sequence homologies of the 16S rRNA and RNA polymerase β subunit (rpoB) genes of 96.6% and 93.1%, respectively. The genomic DNA G + C content of the isolate was 61.8 mol%. On the basis of the sequence data of the 16S rRNA and housekeeping (rpoB) genes, a novel taxon is here proposed, Pseudopropionibacterium rubrum sp. nov. (type strain SK‐1T = JCM 31317T = DSM 100122T). The 16S rRNA and rpoB gene sequences of strain SK‐1T have been deposited in the DDBJ under the accession numbers LC002971 and LC102236, respectively.

Toll-like receptor 5 is not essential for the promotion of secretory immunoglobulin A antibody responses to flagellated bacteria.

Toll‐like receptor 5 recognizes bacterial flagellin, plays a critical role in innate immunity, and contributes to flagellin‐specific humoral immunity. Further, TLR5‐expressing dendritic cells play an important role in IgA synthesis in the intestine; however, the contribution of TLR5 to antigen (Ag)‐specific mucosal immunity remains unclear. Thus, whether TLR5 is essential for the induction of intestinal secretory (S)IgA antibody (Ab) responses against flagellin and bacterial Ags attached to the bacterial surface in response to an oral flagellated bacterium, Salmonella, was explored in this study. Our results indicate that when TLR5 knockout (TLR5−/−) mice are orally immunized with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella‐Tox C), tetanus toxoid (TT)‐ and flagellin (FliC)‐specific systemic IgG and intestinal SIgA Abs are elicited. The numbers of TT‐specific IgG Ab‐forming cells (AFCs) in the spleen and IgA AFCs in the lamina propria (LP) of TLR5−/− mice were comparable to those in wild‐type mice. rSalmonella‐Tox C was equally disseminated in TLR5−/− mice, TLR5−/− mice lacking Peyers patches (PPs), and wild‐type mice. In contrast, TLR5−/− PP‐null mice failed to induce TT‐ and FliC‐specific SIgA Abs in the intestine and showed significantly reduced numbers of TT‐specific IgA AFCs in the LP. These results suggest that TLR5 is dispensable for the induction of flagellin and surface Ag‐specific systemic and mucosal immunity against oral flagellated bacteria. Rather, pathogen recognition, which occurs in PPs, is a prerequisite for the induction of mucosal immunity against flagellated bacteria.