Jannet Katz

Role of the Phosphatidylinositol 3 Kinase-Akt Pathway in the Regulation of IL-10 and IL-12 by Porphyromonas gingivalis Lipopolysaccharide

Stimulation of the APC by Porphyromonas gingivalis LPS has been shown to result in the production of certain pro- and anti-inflammatory cytokines. However, the signaling pathways that regulate these processes are currently unknown. In the present study, the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in regulating P. gingivalis LPS-induced production of IL-10, IL-12 p40, and IL-12 p70 by human monocytes was investigated. P. gingivalis LPS selectively activates the PI3K-Akt pathway via Toll-like receptor 2, and inhibition of this pathway results in an abrogation of extracellular signal-regulated kinase 1/2 phosphorylation, whereas the activation of p38 and c-Jun N-terminal kinase 1/2 kinases were unaffected. Analysis of cytokine production following stimulation of monocytes with P. gingivalis LPS revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 synthesis. IL-10 production was suppressed, whereas IL-12 levels were enhanced. Inhibition of P. gingivalis LPS-mediated activation of the PI3K-Akt pathway resulted in a pronounced augmentation of NF-κB p65 that was independent of IκB-α degradation. Furthermore, the ability of the PI3K-Akt pathway to modulate IL-10 and IL-12 production appears to be mediated by the selective suppression of extracellular signal-regulated kinase 1/2 activity, as the MEK1 inhibitor PD98059 closely mimicked the effects of wortmannin and LY294002 to differentially regulate IL-10 and IL-12 production by P. gingivalis LPS-stimulated monocytes. These studies provide new insight into how engagement of the PI3K-Akt pathway by P. gingivalis LPS affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of innate and adaptive immunity.

Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A.

ABSTRACT Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-κB activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-κB p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-α and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties.

Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

Exposure of mononuclear phagocytes to enterobacterial LPS induces a state of transient hyporesponsiveness to subsequent LPS exposure, termed endotoxin tolerance. In the present study, LPS derived from the oral periodontal pathogen, Porphyromonas gingivalis, was compared with that derived from the enterobacterium, Escherichia coli, for the ability to induce endotoxin tolerance. Pretreatment of the human macrophage cell line, THP-1, with E. coli LPS resulted in a severe reduction in the levels of IL-1β, IL-6, and TNF-α upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a mitigation of IL-1β, but not IL-6 and TNF-α production upon subsequent exposure to P. gingivalis LPS: primary or secondary stimulation with ≤100 ng/ml P. gingivalis LPS resulted in comparable levels of IL-6 and TNF-α, while stimulation of THP-1 cells with ≥1 μg/ml P. gingivalis LPS induced a significant enhancement in IL-6 and TNF-α levels upon secondary exposure. To identify possible mechanisms for these differences, changes in the expression of molecules involved in the LPS-signaling pathway were assessed. Pretreatment of THP-1 cells with E. coli LPS resulted in a significant reduction in surface Toll-like receptor 4 (TLR4) expression and an inability to degrade Ι-κB-α or Ι-κB-β proteins upon secondary stimulation. In contrast, pretreatment of THP-1 cells with P. gingivalis LPS resulted in a significant enhancement of both CD14 and TLR2, while maintaining the ability to degrade Ι-κB-β only upon secondary stimulation. Thus, E. coli and P. gingivalis LPS differentially affect CD14 and TLR expression as well as secondary LPS-associated responses.

Characterization of Porphyromonas gingivalis-Induced Degradation of Epithelial Cell Junctional Complexes

ABSTRACT Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (β1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>109 bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and β1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateralP. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and β1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest thatP. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.

Toll-Like Receptor 2 Is Required for Inflammatory Responses to Francisella tularensis LVS

ABSTRACT Francisella tularensis, a gram-negative bacterium, is the etiologic agent of tularemia and has recently been classified as a category A bioterrorism agent. Infections with F. tularensis result in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms mediating this response have not been completely elucidated. In the present study, we determined the role of Toll-like receptors (TLRs) in mediating inflammatory responses to F. tularensis LVS, and the role of NF-κB in regulating these responses. Stimulation of bone marrow-derived dendritic cells from C57BL/6 wild-type (wt) and TLR4−/− but not TLR2−/− mice, with live F. tularensis LVS elicited a dose-dependent increase in the production of tumor necrosis factor alpha. F. tularensis LVS also induced in a dose-dependent manner an up-regulation in the expression of the costimulatory molecules CD80 and CD86 and of CD40 and the major histocompatibility complex class II molecules on dendritic cells from wt and TLR4−/− but not TLR2−/− mice. TLR6, not TLR1, was shown to be involved in mediating the inflammatory response to F. tularensis LVS, indicating that the functional heterodimer is TLR2/TLR6. Stimulation of dendritic cells with F. tularensis resulted in the activation of NF-κB, which resulted in a differential effect on the production of pro- and anti-inflammatory cytokines. Taken together, our results demonstrate the role of TLR2/TLR6 in the hosts inflammatory response to F. tularensis LVS in vitro and the regulatory function of NF-κB in modulating the inflammatory response.

Hydrolysis of Epithelial Junctional Proteins by Porphyromonas gingivalis Gingipains

ABSTRACT Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis.

Enhanced Immunogenicity of a Genetic Chimeric Protein Consisting of Two Virulence Antigens of Streptococcus mutans and Protection against Infection

ABSTRACT The saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutans have been implicated in the initial adherence of S. mutans to saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect against S. mutans infection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P < 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P < 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P < 0.05) in S. mutans colonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization of S. mutans suggests its potential as a vaccine antigen for dental caries.

Bacterial invasion by a paracellular route: divide and conquer

The epithelium of the host plays an important first line of defense against most human pathogens. Microbial factors have been identified that are involved in the destruction of the structures that maintain the integrity of the epithelium. The mechanisms used by several, selected bacteria for the disruption of epithelial cell-cell junctions are discussed.

A Vaccine against Dental Caries

Dental caries continues to be a costly and prevalent oral disease. Research efforts towards developing a well tolerated and effective vaccine against dental caries were initiated following the demonstration of a specific bacterial aetiology for this disease. The cariogenic mutans streptococci are the principal bacteria causing this disease. Specific immune defence against these bacteria is provided mainly by secretory immunoglobulin (Ig) A antibodies present in saliva, which are generated by the common mucosal immune system. Progress in the development of a vaccine against dental caries has increased due to both advancements in molecular biology and our understanding of the mucosal immune system and mucosal vaccines. Advancements in molecular biology have facilitated the cloning and functional characterisation of virulence factors of the mutans streptococci, including the cell-surface fibrillar proteins, which mediate adherence to the tooth surface, and the glucosyltransferase enzymes, which synthesise adhesive glucans and allow microbial accumulation on the teeth.Current strategies for immunisation against dental caries are using these virulence factors as key antigens and incorporating them into novel mucosal vaccine systems and delivering them with or without adjuvants to mucosal IgA inductive sites. The most popular routes of mucosal immunisation are via the oral or nasal route. The mucosal immune system is functional in newborn infants, who develop salivary IgA antibodies as they become colonised by oral micro-organisms. Mucosal immunisation strategies result in the induction of salivary IgA antibody responses and pose fewer problems than parenteral injection of antigen. Therefore, mucosal immunisation of infants prior to the appearance of their first teeth may be a well tolerated and effective way to induce immunity against the colonisation of teeth by mutans streptococci and protection against subsequent dental caries. The purpose of this article is to provide an overview of the recent progress on the development of a vaccine against infection by Streptococcus mutans for the prevention of dental caries, with emphasis on the mucosal immune system and vaccine design.

Role of Mitogen-Activated Protein Kinases and NF-κB in the Regulation of Proinflammatory and Anti-Inflammatory Cytokines by Porphyromonas gingivalis Hemagglutinin B

ABSTRACT Hemagglutinin B (HagB) is a nonfimbrial adhesin expressed on the surface of Porphyromonas gingivalis and has been implicated as a potential virulence factor involved in mediating the attachment of the bacteria to host cells. However, the molecular mechanisms underlying host responses to HagB and their roles in pathogenesis have yet to be elucidated. Mitogen-activated protein kinases (MAPKs) are activated following engagement of a variety of cell surface receptors via dual tyrosine and threonine phosphorylation and are thought to be involved in various cellular responses. The purpose of this study was to determine the role of intracellular signaling pathways including the MAPKs and NF-κB in regulating the production of proinflammatory and anti-inflammatory cytokines following stimulation of murine macrophages with recombinant HagB (rHagB). Stimulation of peritoneal macrophages with rHagB resulted in the production of the proinflammatory cytokines interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), and tumor necrosis factor alpha, as well as the anti-inflammatory cytokine IL-10. We also demonstrated the activation of extracellular signal-related kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 MAPKs by rHagB-stimulated macrophages. Furthermore, blocking of the ERK and p38 signaling pathways by using specific inhibitors revealed differential regulatory roles in the rHagB-mediated production of proinflammatory and anti-inflammatory cytokines. ERK and p38 were important in down-regulation of IL-12p40 and IFN-γ production and up-regulation of IL-10 production. The enhanced levels of IL-12p40 in rHagB-stimulated macrophages by inhibition of ERK or p38 activity were partially attributable to the inhibition of IL-10 production. Moreover, NF-κB was found to be critical for up-regulation of IL-12p40 and down-regulation of IL-10 production in rHagB-stimulated macrophages. Taken together, our results demonstrate a role for the p38 and ERK pathways and the transcription factor NF-κB in modulating key immunoregulatory cytokines involved in the development of immune responses to P. gingivalis HagB.