DNA-based adaptive immunity protect host from infection-associated periodontal bone resorption via recognition of Porphyromonas gingivalis virulence component.

BACKGROUND: Porphyromonas gingivalis (Pg) is one of a constellation of oral organisms associated with human chronic periodontitis. While adaptive immunity to periodontal pathogen proteins has been investigated and is an important component of periodontal bone resorption, the effect of periodontal pathogen DNA in eliciting systemic and mucosal antibody and modulating immune responses has not been investigated. METHODS: Rowett rats were locally injected with whole genomic Pg DNA in alum. Escherichia coli (Ec) genomic DNA, Fusobacterium nucleatum (Fn) genomic DNA, and saline/alum injected rats served as controls. After various time points, serum IgG and salivary IgA antibody to Ec, Fn or Pg were detected by ELISA. Serum and salivary antibody reactions with Pg surface antigens were determined by Western blot analyses and the specific antigen was identified by mass spectrometry. Effects of genomic DNA immunization on Pg bacterial colonization and experimental periodontal bone resorption were also evaluated. RESULTS: Sera from Pg DNA, Ec DNA and Fn DNA-injected rats did not react with Ec or Fn bacteria. Serum IgG antibody levels to Pg and Pg surface extracts were significantly higher in animals immunized with Pg DNA as compared to the control groups. Rats injected with Pg DNA demonstrated a strong serum IgG and salivary IgA antibody reaction solely to Pg fimbrillin (41kDa), the major protein component of Pg fimbriae. In the Pg DNA-immunized group, the numbers of Pg bacteria in oral cavity and the extent of periodontal bone resorption were significantly reduced after Pg infection. CONCLUSIONS: This study suggests that infected hosts may select specific genes from whole genomic DNA of the periodontal pathogen for transcription and presentation. The results indicate that the unique gene selected can initiate a host protective immune response to the parent bacterium.

Porphyromonas gingivalis infection-associated periodontal bone resorption is dependent on receptor activator of NF-κB ligand.

Porphyromonas gingivalis is one of the oral microorganisms associated with human chronic periodontitis. The purpose of this study is to determine the role of the receptor activator of nuclear factor-κB ligand (RANKL) in P. gingivalis infection-associated periodontal bone resorption. Inbred female Rowett rats were infected orally on four consecutive days (days 0 to 3) with 1 × 10(9) P. gingivalis bacteria (strain ATCC 33277). Separate groups of rats also received an injection of anti-RANKL antibody, osteoprotegerin fusion protein (OPG-Fc), or a control fusion protein (L6-Fc) into gingival papillae in addition to P. gingivalis infection. Robust serum IgG and salivary IgA antibody (P < 0.01) and T cell proliferation (P < 0.05) responses to P. gingivalis were detected at day 7 and peaked at day 28 in P. gingivalis-infected rats. Both the concentration of soluble RANKL (sRANKL) in rat gingival tissues (P < 0.01) and periodontal bone resorption (P < 0.05) were significantly elevated at day 28 in the P. gingivalis-infected group compared to levels in the uninfected group. Correspondingly, RANKL-expressing T and B cells in rat gingival tissues were significantly increased at day 28 in the P. gingivalis-infected group compared to the levels in the uninfected group (P < 0.01). Injection of anti-RANKL antibody (P < 0.05) or OPG-Fc (P < 0.01), but not L6-Fc, into rat gingival papillae after P. gingivalis infection resulted in significantly reduced periodontal bone resorption. This study suggests that P. gingivalis infection-associated periodontal bone resorption is RANKL dependent and is accompanied by increased local infiltration of RANKL-expressing T and B cells.

Development of an in vitro alternative assay method for vaginal irritation.

The vaginal mucosa is commonly exposed to chemicals and therapeutic agents that may result in irritation and/or inflammation. In addition to acute effects, vaginal irritation and inflammation can make women more susceptible to infections such as HIV-1 and herpes simplex virus 2 (HSV-2). Hence, the vaginal irritation potential of feminine care formulations and vaginally administered therapeutic agents is a significant public health concern. Traditionally, testing of such materials has been performed using the rabbit vaginal irritation (RVI) assay. In the current study, we investigated whether the organotypic, highly differentiated EpiVaginal™ tissue could be used as a non-animal alternative to the RVI test. The EpiVaginal tissue was exposed to a single application of ingredients commonly found in feminine hygiene products and the effects on tissue viability (MTT assay), barrier disruption (measured by transepithelial electrical resistance, TEER and sodium fluorescein (NaFl) leakage), and inflammatory cytokine release (interleukin (IL)-1α, IL-1ß, IL-6, and IL-8) patterns were examined. When compared to untreated controls, two irritating ingredients, nonoxynol 9 and benzalkonium chloride, reduced tissue viability to <40% and TEER to <60% while increasing NaFl leakage by 11-24% and IL-1α and IL-1ß release by >100%. Four other non-irritating materials had minimal effects on these parameters. Assay reproducibility was confirmed by testing the chemicals using three different tissue production lots and by using tissues reconstructed from cells obtained from three different donors. Coefficients of variation between tissue lots reconstructed with cells obtained from the same donor or lots reconstructed with cells obtained from different donors were less than 10% and 12%, respectively. In conclusion, decreases in tissue viability and barrier function and increases in IL-1α and IL-1ß release appear to be useful endpoints for preclinical screening of topically applied chemicals and formulations for their vaginal irritation potential.

Periodontal bacterial DNA suppresses the immune response to mutans streptococcal glucosyltransferase.

Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.