IL-8 degradation by Porphyromonas gingivalis proteases

Gingival epithelial cells are the primary barrier to infection by periodontal pathogens. The production of chemotactic cytokines, including interleukin-8 (IL-8), is one mechanism the host uses in response to infection. In this study it was found that both gingival and oral epithelial cells produced IL-8, but after infection with the periodontal pathogen Porphyromonas gingivalis, accumulation of the cytokine could not be detected. Northern analysis established that infected epithelial cells still expressed IL-8 mRNA, suggesting that the cytokine was destroyed after secretion. Incubation of IL-8 with P. gingivalis cells resulted in rapid loss of the cytokine; however, in the presence of a protease inhibitor the loss was significantly retarded, indicating that IL-8 was degraded by P. gingivalis proteases.     

Effect of Porphyromonas gingivalis and Lactobacillus acidophilus on Secretion of IL1B, IL6, and IL8 by Gingival Epithelial Cells

Porphyromonas gingivalis alters cytokine expression in gingival epithelial cells, stimulating inflammatory responses that may lead to periodontal disease. This study explored the effect of Lactobacillus acidophilus on the specific expressions of the interleukins (ILs) IL1B, IL6, and IL8 induced by the pathogen. Human gingival epithelial cells were co-cultured with P. gingivalis, L. acidophilus, or L. acidophilus + P. gingivalis; the control group consisted of the cells alone. Protein and gene expression levels of the ILs were detected using ELISA and qRT-PCR, respectively. The supernatant from the P. gingivalis group held significantly higher protein and mRNA levels of IL1B, IL6, and IL8, compared to the control group. In the mixed bacterial group (L. acidophilus + P. gingivalis), the levels of all three ILs decreased with increasing concentrations of L. acidophilus and were significantly different from the P. gingivalis group. This suggests that in gingival cells, L. acidophilus offsets the P. gingivalis-induced secretion of these ILs in a dose-dependent manner.     

Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.     

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.     

Interactions of Porphyromonas gingivalis with epithelial cells.

The invasion of gingival epithelial cells by certain pathogenic periodontal bacteria may account for their presence within diseased gingival tissue. To dissect the initial steps of a potential invasion pathway for the periodontal pathogen Porphyromonas gingivalis, laboratory and clinical bacterial isolates were tested for their interactions with a human oral epithelial cell line (KB). Several P. gingivalis strains immobilized on filters could bind oral epithelial cells. Quantitative adherence assays supported these results. The invasion of epithelial cells by P. gingivalis 33277 was measured by assay and confirmed by transmission electron microscopy. These preliminary results demonstrate that certain P. gingivalis strains are capable of internalization by human oral epithelial cells in vitro.     

Role of the Amino-Terminal Region of Porphyromonas gingivalis Fimbriae in Adherence to Epithelial Cells

Porphyromonas gingivalis fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of P. gingivalis FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated P. gingivalis strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of P. gingivalis to oral epithelial cells. To determine the specific region(s) of P. gingivalis FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of P. gingivalis cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of P. gingivalis cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with P. gingivalis fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of P. gingivalis fimbriae.     

Differential activation of human gingival epithelial cells and monocytes by Porphyromonas gingivalis fimbriae

Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.     

Calprotectin expression in vitro by oral epithelial cells confers resistance to infection by Porphyromonas gingivalis

Calprotectin, an S100 calcium-binding protein with broad-spectrum antimicrobial activity in vitro, is expressed in neutrophils, monocytes, and gingival keratinocytes. In periodontitis, calprotectin appears upregulated and is detected at higher levels in gingival crevicular fluid and tissue specimens. How calprotectin contributes to the pathogenesis of periodontal diseases is unknown. To isolate the effects of calprotectin, a calprotectin-negative oral epithelial cell line was transfected with calprotectin genes to enable expression. Porphyromonas gingivalis was permitted to bind and invade transfected cells expressing calprotectin and sham transfectants. Rates of invasion into both cell lines were compared using the antibiotic protection assay. Transfected cells expressing calprotectin showed 40 to 50% fewer internalized P. gingivalis than sham transfectants. Similarly, binding to calprotectin expressing cells was reduced approximately twofold at all time points (15, 30, 45, and 60 min) as estimated by immunofluorescence analysis. Independent of invasion, however, prolonged exposure to P. gingivalis induced epithelial cell rounding and detachment from the substratum. These morphological changes were delayed, however, in cells expressing calprotectin. Using P. gingivalis protease-deficient mutants, we found that Arg-gingipain and Lys-gingipain contributed to epithelial cell rounding and detachment. In conclusion, expression of calprotectin appears to protect epithelial cells in culture against binding and invasion by P. gingivalis. In addition, cells expressing calprotectin are more resistant to detachment mediated by Arg-gingipain and Lys-gingipain. In periodontal disease, calprotectin may augment both the barrier protection and innate immune functions of the gingival epithelium to promote resistance to P. gingivalis infection.     

Porphyromonas gingivalis infection of oral epithelium inhibits neutrophil transepithelial migration.

Periodontal diseases are inflammatory disorders caused by microorganisms of dental plaque that colonize the gingival sulcus and, subsequently, the periodontal pocket. As in other mucosal infections, the host response to plaque bacteria is characterized by an influx of polymorphonuclear leukocytes (PMNs) to the gingival crevice. Neutrophil migration through the epithelial lining of the gingival pocket is thought to be the first line of defense against plaque bacteria. In order to model this phenomenon in vitro, we used the oral epithelial cell line KB and human PMNs in the Transwell system and examined the impact of Porphyromonas gingivalis-epithelial cell interactions on subsequent PMN transepithelial migration. We demonstrate here that P. gingivalis infection of oral epithelial cells failed to trigger transmigration of PMNs. Furthermore, it significantly inhibited neutrophil transmigration actively induced by stimuli such as N-formylmethionyl leucyl phenylalanine, interleukin-8 (IL-8), and the intestinal pathogen enterotoxigenic Escherichia coli. The ability of P. gingivalis to block PMN transmigration was strongly positively correlated with the ability to adhere to and invade epithelial cells. In addition, P. gingivalis attenuated the production of IL-8 and the expression of intercellular adhesion molecule 1 by epithelial cells. The ability of P. gingivalis to block neutrophil migration across an intact epithelial barrier may critically impair the potential of the host to confront the bacterial challenge and thus may play an important role in the pathogenesis of periodontal disease.     

Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed.     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.     

Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis

The oral pathogen Porphyromonas gingivalis secretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed to P. gingivalis cell-free culture supernatant or purified RgpB. hBD-2 mRNA in GECs was induced in response to supernatant and purified RgpB from P. gingivalis (P = 0.02 and P = 0.016, respectively). This effect was abrogated by the protease inhibitor tosyl-l-lysine chloromethyl ketone (TLCK) (P < 0.05). In response to P. gingivalis supernatant and to purified RgpB, the hBD-2 mRNA expression was significantly decreased in PAR-2 gene knockdown cells, whereas no change was detected in PAR-1 gene knockdown cells. CCL20 mRNA expression also increased in response to the supernatant of P. gingivalis, and this effect was blocked by the protease inhibitor, TLCK (P = 0.05 and P = 0.024, respectively), and was blocked in PAR-2 gene knockdown cells. Our data indicate that hBD-2 and CCL20 mRNA up-regulation by P. gingivalis supernatant and purified RgpB was mediated via PAR-2, but not via PAR-1, and that proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity.     

Gingipain adhesin domains mediate Porphyromonas gingivalis adherence to epithelial cells

Porphyromonas gingivalis, a Gram-negative oral anaerobe, interacts with epithelium lining the gingival sulcus. Continuing our studies on the role of gingipain cysteine proteinases in P. gingivalis adherence to epithelial cells, we showed that antibody raised to the recombinant adhesin domain of arg-gingipain A blocked bacterial attachment, providing new additional evidence that P. gingivalis adherence to epithelial cells is mediated by gingipain adhesin peptides.     

Bacterial fimbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2.

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), significantly induced IL-8 production and NF-kappaB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2.     

Cytokine profiling of macrophages exposed to Porphyromonas gingivalis, its lipopolysaccharide, or its FimA protein

To characterize the roles of Porphyromonas gingivalis and its components in the disease processes, we investigated the cytokine profile induced by live P. gingivalis, its lipopolysaccharides (LPS), and its major fimbrial protein, fimbrillin (FimA). Using cytokine antibody arrays, we found that P. gingivalis LPS and FimA induced a similar profile of cytokine expression when exposed to mouse peritoneal macrophages but that this profile differed significantly in response to live P. gingivalis. In vitro, mouse peritoneal macrophages were stimulated to produce interleukin-6 (IL-6), granulocyte colony-stimulating factor, and lymphotactin by live P. gingivalis, but not by P. gingivalis LPS or FimA, while RANTES, gamma interferon, IL-17, vascular cell adhesion molecule 1 (VCAM-1), and vascular endothelial growth factor were induced by P. gingivalis LPS or FimA, but not by live P. gingivalis. In vivo, IL-6 mRNA was strongly induced only by live P. gingivalis while monocyte chemoattractant protein 1 mRNA was strongly induced only by P. gingivalis LPS and FimA in mouse calvarial scalp, further confirming the differences of cytokine profile induced in vitro. Cytokine antibody arrays using toll-like receptor 2 (TLR2)- and TLR4-deficient macrophages revealed that most of the cytokines induced by P. gingivalis LPS or FimA signal through TLR2, while most of cytokines induced by live P. gingivalis signal through both TLR2 and TLR4. Interestingly, the activation of TLR2 by live P. gingivalis inhibited the release of RANTES, VCAM-1, and IL-1alpha from mouse peritoneal macrophages. A tumor necrosis factor alpha enzyme-linked immunosorbent assay further confirmed that P. gingivalis LPS and FimA activate mouse peritoneal macrophages via TLR2. These results indicate that host immune cells sense live P. gingivalis and its components differently, which translates into the expression of different inflammatory cytokine profiles.