Youngnim Choi

The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer

BackgroundAlthough mice have long served as an animal model for periodontitis, information on the composition of their indigenous oral microbiota is limited. The aim of the current study was to characterize mouse oral bacterial flora by applying extensive parallel pyrosequencing using the latest model pyrosequencer, a Roche/454 Genome Sequencer FLX Titanium. In addition, the effect of Toll-like receptor (TLR) 2 deficiency on oral microbiota was evaluated.ResultsEight oral bacterial communities of wild-type (n = 4) and TLR2 knock-out (n = 4) C57BL/6 mice were characterized by analyzing 80,046 reads of 16S rRNA genes obtained by pyrosequencing. Excluding the PCR primers, the average length of each sequencing product was 443 bp. The average species richness of the murine oral bacterial communities was estimated to be about 200, but the communities were dominated by only two main phyla and several species. Therefore, the bacterial communities were relatively simple. The bacterial composition of the murine oral microbiota was significantly different from that of humans, and the lack of TLR2 had a negligible effect on the murine oral microbiota.ConclusionPyrosequencing using the Roche/454 FLX Titanium successfully characterized mouse oral bacterial communities. The relatively simple oral bacterial communities of mice were not affected by TLR2 deficiency. These findings will provide a basis for future studies on the role of periodontal pathogens in the murine model of periodontitis.

Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8+ T cells

Host immune response is known to contribute to the progression of periodontitis, and alveolar bone destruction in periodontitis is associated with enhanced osteoclast activity. Therefore, we evaluated the roles of activated lymphocyte subsets in osteoclastogenesis. Osteoclast precursors were co‐cultured with activated lymphocytes (B, CD4+ T, CD8+ T) in the presence of either macrophage colony‐stimulating factor (M‐CSF) alone or M‐CSF plus soluble receptor activator of NF‐κB ligand (sRANKL), and subsequent differentiation into active osteoclasts was evaluatedby a resorption assay. The activated B and CD4+ cells, but not CD8+ T cells, induced osteoclast differentiation in the presence of M‐CSF alone. In the presence of M‐CSF and sRANKL, B cells induced the formation of small but highly active osteoclasts and increased resorption, while CD8+ T cells profoundly suppressed osteoclastogenesis. Co‐culture using an insert wellor supernatant suggested that both B and CD8+ T cells acted on osteoclasts mostly via soluble proteins. Activated B cells expressed many osteoclastogenic factors including RANKL, TNF‐α, IL‐6, MIP‐1α, and MCP‐3. CD8+ T cells expressed a substantial amount of osteoprotegerin (OPG) along with RANKL. However, blocking antibody to OPG did not reverse the suppression by CD8+ T cells, suggesting that other factor(s) are involved. Taken together, activated B cells promoted osteoclastogenesis, while CD8+ T cells inhibited the osteoclast formation via direct interaction. The results imply the importance of lymphocyte subpopulations in the development of periodontitis.

The Triacylated ATP Binding Cluster Transporter Substrate-binding Lipoprotein of Staphylococcus aureus Functions as a Native Ligand for Toll-like Receptor 2

Some synthetic lipopeptides, in addition to native lipoproteins derived from both Gram-negative bacteria and mycoplasmas, are known to activate TLR2 (Toll-like receptor 2). However, the native lipoproteins inherent to Gram-positive bacteria, which function as TLR2 ligands, have not been characterized. Here, we have purified a native lipoprotein to homogeneity from Staphylococcus aureus to study as a native TLR2 ligand. The purified 33-kDa lipoprotein was capable of stimulating TLR2 and was identified as a triacylated SitC lipoprotein, which belongs to a family of ATP binding cluster (ABC) transporter substrate-binding proteins. Analyses of the SitC-mediated production of cytokine using mouse peritoneal macrophages revealed that the SitC protein (3 nm) induced the production of tumor necrosis factor-α and interleukin-6. Moreover, analysis of knock-out mice showed that SitC required TLR2 and MyD88, but not TLR1 or TLR6, for the induction of cytokines. In addition to the S. aureus SitC lipoprotein, we purified two other native ABC transporter substrate-binding lipoproteins from Bacillus subtilis and Micrococcus luteus, which were both shown to stimulate TLR2. These results demonstrate that S. aureus SitC lipoprotein is triacylated and that the ABC transporter substrate-binding lipoproteins of Gram-positive bacteria function as native ligands for TLR2.

Toll-Like Receptor 2 and NALP2 Mediate Induction of Human Beta-Defensins by Fusobacterium nucleatum in Gingival Epithelial Cells

ABSTRACT We previously reported that infection by Fusobacterium nucleatum strongly induced the expression of both human beta-defensin 2 (HBD-2) and HBD-3 by gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptors (PRRs) and regulatory mechanisms involved in the induction of HBD-2 and -3 expression by F. nucleatum in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with live or heat-killed F. nucleatum, and the expression of HBDs and interleukin-8 (IL-8) was examined by real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Live, but not heat-killed, F. nucleatum invaded HOK-16B cells, as seen by confocal microscopy and flow cytometry. Live F. nucleatum induced both HBD-2 and -3 efficiently, whereas heat-killed bacteria induced only HBD-3 at a reduced level. Knockdown of NACHT-LRR- and pyrin domain-containing protein 2 (NALP2), the most abundant intracellular PRR in HOK-16B cells, by RNA interference (RNAi) significantly reduced the induction of HBD-3 but not HBD-2 and IL-8. In addition, knockdown of Toll-like receptor 2 (TLR2) by RNAi reduced the upregulation of HBD-2 and -3 but not IL-8. Heat-killed F. nucleatum was hindered in its ability to activate TLR2 and JNK signaling pathways. Theses data show that TLR2 and NALP2 mediate the induction of HBDs by F. nucleatum in gingival epithelial cells, but thresholds for the two HBD genes are different.

Interaction of Fas Ligand and Fas Expressed on Osteoclast Precursors Increases Osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1β and TNF-α, and the activation of NF-κB. Functional blocking of IL-1β and TNF-α using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.

Bacterial invasion and persistence: critical events in the pathogenesis of periodontitis?

Periodontitis is chronic inflammation of the periodontium caused by the hosts inflammatory response to plaque biofilm, which destroys tooth-supporting soft and hard tissues. Periodontitis is a complex disease that involves interactions among three main features - microbial challenge, the host immune response, and environmental and genetic risk factors - in its pathogenesis. Although periodontitis has been regarded as the result of hyperimmune or hyperinflammatory responses to plaque bacteria, recent studies indicate that periodontal pathogens are rather poor activators and/or suppressors of the host immune response. This raises the question of how periodontal pathogens cause inflammation. To resolve this issue, in the present review we propose that bacterial invasion into gingival tissue is a key event in the initiation of periodontitis and that the persistence of these bacteria within host tissue results in chronic inflammation. In support of this hypothesis, we present the ways in which microbial, environmental and genetic risk factors contribute to bacterial invasion. It is hoped that the current model will instigate active discussion and new research to complete the puzzle of this complex disease process.

Treponema denticola Suppresses Expression of Human β-Defensin-3 in Gingival Epithelial Cells through Inhibition of the Toll-Like Receptor 2 Axis

ABSTRACT We reported previously that Treponema denticola, one of the periodontal pathogens, suppresses the expression of human β-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-κB within an hour of infection, heat-killed bacteria did not show any inhibitory effect on the MAPK and NF-κB 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 Pam3CSK. 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.

Receptor activator of NF-κB ligand enhances the activity of macrophages as antigen presenting cells

Receptor activator of NFκB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFα, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-γ, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.

B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis

Host immune response has been considered as an important disease-modifying factor of periodontitis, however, which immune cell(s) or factor(s) are involved in the destruction of periodontium remains unclear. Previously, we reported that osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+)T cells. We present new data that B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis. Purified murine B cells were activated with anti-IgD mAb, IL-4, and anti-CD40 mAb, in the absence (B(Th2)) or presence of Th1 cytokines, either IL-2 (B(IL-2)) or IFN-γ (B(IFN-γ)). Each activated B cell population was co-cultured with RAW264.7 cells in the presence of soluble receptor activator of NF-κB ligand (sRANKL), and the effect on osteoclastic differentiation was evaluated. While B(Th2)increased osteoclastogenesis, B(IL-2)and B(IFN-γ)suppressed it profoundly. To verify the mediating molecule(s), we analyzed cytokine profiles of the activated B cells. Compared to B(Th2), B(IL-2)expressed increased amount of IFN-γ and B(IFN-γ)expressed decreased amounts of IL-4, IL-5, and IL-10. IFN-γ was a key negative regulator of osteoclastic differentiation, and mediated the inhibition by B(IL-2). These results suggest that Th1 cytokines may have new important roles in resistance to periodontitis, acting directly on osteoclasts or indirectly through B cells.

Treponema denticola suppresses expression of human β-defensin-2 in gingival epithelial cells through inhibition of TNFα production and TLR2 activation

We previously reported that Treponema denticola, a periodontal pathogen, suppressed the expression of human β-defensins (HBDs) and IL-8 in human gingival epithelial cells. To clarify the receptor(s) involved in the suppression of HBD-2, immortalized gingival epithelial (HOK-16B) cells were infected with live or heat-killed T. denticola for 24 h, and the expression of HBD-2 was examined by real-time RT-PCR. Live T. denticola, but not heat-killed bacteria, suppressed the expression of HBD-2 about 40%. Time courses of suppression revealed that T. denticola suppressed HBD-2 expression only at late time points, which was accompanied with the suppression of TNFα production. Neutralization of TNFα with an antibody abrogated the suppressive effect of T. denticola on HBD-2. Accordingly, heat-killed T. denticola did not suppress TNFα production. Knock-down of toll-like receptor (TLR) 2 via RNA interference reversed the suppressive effect of T. denticola on the expression of HBD-3, but not on the production of TNFα. Collectively, T. denticola suppresses the expression of HBD-2 in gingival epithelial cells by inhibiting the TLR2 axis and TNFα production, which may contribute to the pathogenesis of periodontitis by T. denticola.