Ki Bum Ahn

Lipoprotein in the cell wall of Staphylococcus aureus is a major inducer of nitric oxide production in murine macrophages.

Staphylococcus aureus is a Gram-positive bacterium that causes inflammation at infection sites by inducing various inflammatory mediators such as nitric oxide (NO). To identify the staphylococcal virulence factors contributing to NO production, we compared the ability of ethanol-killed wild-type S. aureus and mutant strains lacking lipoteichoic acid (ΔltaS), lipoproteins (Δlgt), or d-alanine (ΔdltA) to stimulate NO production in a murine macrophage cell line, RAW 264.7, and the primary macrophages derived from C57BL/6 mice. Wild-type, ΔltaS, and ΔdltA strains induced NO production in a dose-dependent manner but this response was not observed when the cells were stimulated with the Δlgt strain. Moreover, purified lipoproteins triggered NO production in macrophages. Coincident with NO induction, the wild-type, ΔltaS, and ΔdltA strains induced expression of inducible NO synthase (iNOS) at both mRNA and protein levels whereas Δlgt failed to induce iNOS protein or mRNA. Transient transfection followed by a reporter gene assay and Western blotting experiments demonstrated that wild-type, ΔltaS, and ΔdltA strains, but not the Δlgt strain, induced substantial activation of NF-κB and STAT1 phosphorylation, both of which are known to be crucial for iNOS expression. Moreover, wild-type, ΔltaS, and ΔdltA strains increased Toll-like receptor 2 (TLR2) activation, which is known to mediate S. aureus-induced innate immunity, whereas the Δlgt strain did not. Collectively, these results suggest that lipoproteins in the cell wall of S. aureus play a major role in the induction of NO production in murine macrophages through activation of the TLR2 receptor.

Bacterial flagellin induces IL-6 expression in human basophils

Binding of allergen to IgE on basophils positively affects allergic inflammation by releasing inflammatory mediators. Recently, basophils were shown to express pattern-recognition receptors, such as toll-like receptors (TLRs), for recognizing microbe-associated molecular patterns (MAMPs) that are independent of allergen-IgE binding. In this study, we investigated whether MAMP alone can induce IL-6 production in a human basophil cell line, KU812. Stimulation with flagellin in the absence of allergen-IgE association induced IL-6 expression in KU812 cells, while stimulation with lipoteichoic acid, peptidoglycan, or poly I:C did not under the same condition. Flagellin-induced IL-6 expression was also observed in human primary basophils. Flow cytometric analysis showed that KU812 cells expressed flagellin-recognizing TLR5 both on the cell surface and in the cytoplasm while TLR2 and TLR3 were observed only in the cytoplasm. We further demonstrated that although flagellin augmented the phosphorylation of mitogen-activated protein kinases including p38 kinase, ERK, and JNK, flagellin-induced IL-6 production was attenuated by inhibitors for p38 kinase and ERK, but not by JNK inhibitors. In addition, flagellin enhanced phosphorylation of signaling molecules including CREB, PKCδ, and AKT. The inhibitors for PKA and PKC also showed inhibitory effects. Interestingly, flagellin-induced IL-6 production was further enhanced by pretreatment with inhibitors for PI3K, implying that PI3K negatively affects the flagellin-induced IL-6 production. Furthermore, DNA binding activities of NF-κB, AP-1, and CREB, which play pivotal roles in the induction of IL-6 gene expression, were increased by flagellin. These results suggest that flagellin alone is sufficient to induce IL-6 gene expression via TLR5 signaling pathways in human basophils.

Muramyl dipeptide potentiates staphylococcal lipoteichoic acid induction of cyclooxygenase-2 expression in macrophages.

Gram-positive bacteria contain lipoteichoic acid (LTA) and peptidoglycan (PGN) layers, both of which are considered as major virulence factors associated with inflammation. Cyclooxygenase-2 (COX-2) plays an important role in the inflammation by generating prostaglandins at infections. Since LTA and PGN are thought to cooperate in the establishment of inflammation, we examined the ability of staphylococcal LTA (Sa.LTA) to induce COX-2 expression in the presence of muramyl dipeptide (MDP), which is the minimal structural unit of PGN required for inflammation, in macrophages. While MDP failed to induce COX-2 expression, Sa.LTA alone was sufficient to induce COX-2 production. Treatment with MDP enhanced Sa.LTA-induced COX-2 and prostaglandin E2 production. The cooperative effect between Sa.LTA and MDP was not observed in COX-2 expression by macrophages derived from Toll-like receptor 2 (TLR2)- or nucleotide-binding oligomerization domain 2 (NOD2)-deficient mice. In addition, MDP enhanced Sa.LTA-induced activation of the transcription factors NF-κB and CRE, which are known to modulate COX-2 gene transcription. Conclusively, these results suggest that MDP and Sa.LTA cooperatively induce inflammatory response by overproducing COX-2 through NOD2 and TLR2.

IgE in the absence of allergen induces the expression of monocyte chemoattractant protein-1 in the rat basophilic cell-line RBL-2H3

Recently, basophils have been suggested to produce inflammatory mediators in response to IgE in the absence of allergens. Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the initiation of inflammatory responses by recruiting various immune cells to the site of allergic inflammation. In the present study, we examined whether IgE under allergen-free conditions could stimulate basophils and lead to the production of MCP-1. Exposure of the rat basophilic cell-line RBL-2H3 to IgE without allergen resulted in a dose- and time-dependent induction of MCP-1 expression at both the mRNA and protein level. Although allergen was not necessary for IgE-induced MCP-1 expression, it was essential for degranulation as determined by β-hexosaminidase release assay. IgE enhanced phosphorylation of MAP kinases including ERK, p38 kinase, and JNK. However, IgE-induced MCP-1 expression was attenuated by inhibitors for JNK and PKC. Concomitantly, IgE induced activation of AP-1, which is an important transcription factor for MCP-1 gene expression in RBL-2H3 cells. Taken together, our results suggest that IgE alone is sufficient to stimulate basophils to increase expression of MCP-1, which in turn might contribute to the inflammatory response.

Status of group B streptococcal vaccine development

Streptococcus agalactiae (group B streptococcus, GBS) is a leading causal organism of neonatal invasive diseases and severe infections in the elderly. Despite significant advances in the diagnosis and treatment of GBS infections and improvement in personal hygiene standards, this pathogen is still a global health concern. Thus, an effective vaccine against GBS would augment existing strategies to substantially decrease GBS infection. In 2014, World Health Organization convened the first meeting for consultation on GBS vaccine development, focusing on the GBS maternal immunization program, which was aimed at reducing infections in neonates and young infants worldwide. Here, we review the history of GBS infections, the current vaccine candidates, and the current status of immunogenicity assays used to evaluate the clinical efficacy of GBS vaccines.

Streptococcus gordonii induces nitric oxide production through its lipoproteins stimulating Toll-like receptor 2 in murine macrophages

HighlightsLipoprotein‐deficient S. gordonii induces less NO production than the wild‐type in macrophages.Lipoprotein‐deficient S. gordonii weakly induces NF‐&kgr;B activation, IFN‐&bgr; expression and STAT1 phosphorylation.Lipoprotein of S. gordonii is a major TLR2 ligand inducing NO production in macrophages. ABSTRACT Streptococcus gordonii, a Gram‐positive commensal in the oral cavity, is an opportunistic pathogen that can cause endodontic and systemic infections resulting in infective endocarditis. Lipoteichoic acid (LTA) and lipoprotein are major virulence factors of Gram‐positive bacteria that are preferentially recognized by Toll‐like receptor 2 (TLR2) on immune cells. In the present study, we investigated the effect of S. gordonii LTA and lipoprotein on the production of the representative inflammatory mediator nitric oxide (NO) by the mouse macrophages. Heat‐killed S. gordonii wild‐type and an LTA‐deficient mutant (&Dgr;ltaS) but not a lipoprotein‐deficient mutant (&Dgr;lgt) induced NO production in mouse primary macrophages and the cell line, RAW 264.7. S. gordonii wild‐type and &Dgr;ltaS also induced the expression of inducible NO synthase (iNOS) at the mRNA and protein levels. In contrast, the &Dgr;lgt mutant showed little effect under the same condition. Furthermore, S. gordonii wild‐type and &Dgr;ltaS induced NF‐&kgr;B activation, STAT1 phosphorylation, and IFN‐&bgr; expression, which are important for the induction of iNOS gene expression, with little activation by &Dgr;lgt. S. gordonii wild‐type and &Dgr;ltaS showed an increased adherence and internalization to RAW 264.7 cells compared to &Dgr;lgt. In addition, S. gordonii wild‐type and &Dgr;ltaS, but not &Dgr;lgt, substantially increased TLR2 activation while none of these induced NO production in TLR2‐deficient macrophages. Triton X‐114‐extracted lipoproteins from S. gordonii were sufficient to induce NO production. Collectively, we suggest that lipoprotein is an essential cell wall component of S. gordonii to induce NO production in macrophages through TLR2 triggering NF‐&kgr;B and STAT1 activation.

Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells

HighlightsS. gordonii wild‐type, but not lipoprotein‐deficient mutant, induces IL‐8 production in human PDL cells.Lipoproteins isolated from S. gordonii induce IL‐8 production via TLR2 in human PDL cells.S. gordonii induces IL‐8 production through p38 kinase, while S. gordonii lipoproteins signal through p38 kinase, ERK, and JNK.S. gordonii lipoproteins cooperate with other bacterial components in the induction of IL‐8 in human PDL cells. &NA; Streptococcus gordonii, a Gram‐positive oral bacterium, is a life‐threatening pathogen that causes infective endocarditis. It is frequently isolated from the periapical lesions of patients with apical periodontitis and has thus been implicated in inflammatory responses. However, little is known about the virulence factors of S. gordonii responsible for the induction of inflammatory responses in the periapical areas. Here, we investigated the role of S. gordonii cell wall‐associated virulence factors on interleukin (IL)‐8 induction in human periodontal ligament (PDL) cells using ethanol‐inactivated wild‐type S. gordonii, a lipoteichoic acid (LTA)‐deficient mutant (&Dgr;ltaS), and a lipoprotein‐deficient mutant (&Dgr;lgt). Wild‐type S. gordonii induced IL‐8 expression at both the protein and mRNA levels in human PDL cells in a dose‐ and time‐dependent manner. A transient transfection and reporter gene assay demonstrated that wild‐type S. gordonii activated Toll‐like receptor 2 (TLR2). Additionally, IL‐8 production induced by wild‐type S. gordonii was substantially inhibited by anti‐TLR2‐neutralizing antibodies. Both wild‐type S. gordonii and the &Dgr;ltaS mutant induced IL‐8 production; however, this response was not observed when cells were stimulated with the &Dgr;lgt mutant. Interestingly, lipoproteins purified from S. gordonii induced IL‐8 production, whereas purified LTA did not. In addition, purified lipoproteins stimulated TLR2 more potently than LTA. Furthermore, S. gordonii‐induced IL‐8 expression was specifically inhibited by blocking p38 kinase, while lipoprotein‐induced IL‐8 expression was inhibited by blocking p38 kinase, ERK, or JNK. Of particular note, exogenous addition of purified S. gordonii lipoproteins enhanced &Dgr;lgt‐induced IL‐8 production in human PDL cells to an extent similar to that induced by the wild‐type strain. Collectively, these results suggest that lipoproteins are an important component of S. gordonii for the induction of IL‐8 production in human PDL cells through TLR2 activation. Therefore, lipoproteins potentially contribute to inflammatory apical periodontitis.

Serine-rich Repeat Adhesin Gordonii Surface Protein B is Important for Streptococcus gordonii Biofilm Formation

INTRODUCTION Streptococcus gordonii is a predominant member of the oral microflora and has been isolated from root canals of teeth with refractory apical periodontitis. Biofilm formation is important for various dental diseases, and S. gordonii is involved in dental biofilm formation as an early colonizer. Although serine-rich repeat (SRR) adhesins of S. gordonii such as gordonii surface protein B (GspB) are associated with bacterial colonization, the role of GspB in biofilm formation is not clearly understood. In the present study, we investigated the effect of S. gordonii GspB on biofilm formation using wild-type and GspB-deficient mutant S. gordonii strains. METHODS Confocal microscopy and crystal violet assay were used to determine biofilm formation. Bacterial growth was examined by measuring optical density with spectrometry. Bacterial adherence and biofilm on the culture plate and human dentin slices were visualized with a scanning electron microscope. RESULTS The GspB-deficient S. gordonii mutant strain was less potent than the wild-type strain in biofilm formation. Of note, there was no difference in the bacterial growth rate between the mutant and wild-type strains. Differences in biofilm-forming ability between the wild-type and mutant strains were more distinct in the sucrose-supplemented media. Furthermore, the GspB-deficient mutant exhibited attenuated formation of aggregates on the surface of the culture plate and human dentin slices. CONCLUSIONS These results suggest that GspB is important for S. gordonii biofilm formation, which may contribute to the development of dental biofilm-associated diseases.

Human salivary proteins with affinity to lipoteichoic acid of Enterococcus faecalis

Enterococcus faecalis is associated with refractory apical periodontitis and its lipoteichoic acid (Ef.LTA) is considered as a major virulence factor. Although the binding proteins of Ef.LTA may play an important role for mediating infection and immunity in the oral cavity, little is known about Ef.LTA-binding proteins (Ef.LTA-BPs) in saliva. In this study, we identified salivary Ef.LTA-BPs with biotinylated Ef.LTA (Ef.LTA-biotin) through mass spectrometry. The biotinylation of Ef.LTA was confirmed by binding capacity with streptavidin-FITC on CHO/CD14/TLR2 cells. The biological activity of Ef.LTA-biotin was determined based on the induction of nitric oxide and macrophage inflammatory protein-1α in a macrophage cell-line, RAW 264.7. To identify salivary Ef.LTA-BPs, the Ef.LTA-biotin was mixed with a pool of human saliva obtained from nine healthy subjects followed by precipitation with a streptavidin-coated bead. Ef.LTA-BPs were then separated with 12% SDS-PAGE and subjected to the mass spectrometry. Six human salivary Ef.LTA-BPs including short palate lung and nasal epithelium carcinoma-associated protein 2, zymogen granule protein 16 homolog B, hemoglobin subunit α and β, apolipoprotein A-I, and lipocalin-1 were identified with statistical significance (P<0.05). Ef.LTA-BPs were validated with lipocalin-1 using pull-down assay. Hemoglobin inhibited the biofilm formation of E. faecalis whereas lipocalin-1 did not show such effect. Collectively, the identified Ef.LTA-BPs could provide clues for our understanding of the pathogenesis of E. faecalis and host immunity in oral cavity.

Th17 activation by dendritic cells stimulated with gamma-irradiated Streptococcus pneumoniae

Highlightsr‐SP shows modest binding and internalization to BM‐DCs compared with h‐SP or f‐SP.r‐SP induces phenotypic maturation of BM‐DCs less potently than h‐SP or f‐SP.r‐SP induces lower cytokine expression in BM‐DCs compared with h‐SP or f‐SP.r‐SP‐sensitized BM‐DCs preferentially promote Th17 polarization. &NA; Dendritic cells (DCs) play an important role in antigen presentation, which is an essential step for the induction of antigen‐specific adaptive immunity. Inactivated bacterial whole cell vaccines have been widely used to prevent many bacterial infections because they elicit good immunogenicity due to the presence of various antigens and are relatively inexpensive and easy to manufacture. Recently, gamma‐irradiated whole cells of nonencapsulated Streptococcus pneumoniae were developed as a broad‐spectrum and serotype‐independent multivalent vaccine. In the present study, we generated gamma‐irradiated S. pneumoniae (r‐SP) and investigated its capacity to stimulate mouse bone marrow‐derived DCs (BM‐DCs) in comparison with heat‐inactivated and formalin‐inactivated S. pneumoniae (h‐SP and f‐SP, respectively). r‐SP showed an attenuated binding and internalization level to BM‐DCs when compared to h‐SP or f‐SP. r‐SP weakly induced the expression of CD80, CD83, CD86, MHC class I, and PD‐L2 compared with h‐SP or f‐SP. Furthermore, r‐SP less potently induced IL‐6, TNF‐&agr;, and IL‐23 expression than h‐SP or f‐SP but more potently induced IL‐1&bgr; expression than h‐SP or f‐SP in BM‐DCs. Since Th17‐mediated immune responses are known to be important for the protection against pneumococcal infections, r‐SP‐primed DCs were co‐cultured with splenocytes or splenic CD4+ T cells. Interestingly, r‐SP‐sensitized BM‐DCs markedly induced IL‐17A+ CD4+ T cells whereas h‐SP‐ or f‐SP‐sensitized BM‐DCs weakly induced them. Collectively, these results suggest that r‐SP could be an effective pneumococcal vaccine candidate eliciting Th17‐mediated immune responses by stimulation of DCs.