Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as Porphyromonas gingivalis with atherosclerosis and formation of macrophage foam cells. Toll-like receptors and molecules governing their intracellular signaling pathways such as MyD88 play roles in atherosclerosis, as well as host response to P. gingivalis. The aim of this study was to define roles of MyD88 and Lps2 during macrophage foam cell formation in response to P. gingivalis. In the presence of human low-density lipoprotein (LDL) mouse bone marrow derived macrophages (BMØ) cultured with P. gingivalis responded with significant reduction in TNF-α and IL-6. BMØ stained strongly with oil red O, regardless whether bacterial challenge occurred concurrent with or prior to LDL treatment. Heat-killed P. gingivalis stimulated foam cell formation similar to live organisms. BMØ from MyD88-knockout (KO) and Lps2 mice revealed a significant role for MyD88, and a mild role for Lps2 in P. gingivalis-elicited foam cell formation. P. gingivalis-elicited TNF-α and IL-6 was affected by MyD88 ablation and to a lesser extent by Lps2 status. These data indicate that LDL affects TNF-α and IL-6 response of macrophage to P. gingivalis challenge and that MyD88 and Lps2 play important roles in P. gingivalis-elicited foam cell formation.
Innate immunity; Porphyromonas gingivalis; macrophage; foam cell; cytokine
Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease (PD), an infection-driven chronic inflammatory disease that leads to the resorption of tooth supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis-induced alveolar bone loss in vivo and our in vitro work implicated TNF as a key downstream mediator. Here we show that TNF-deficient (Tnf−/−) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental PD. Using bone marrow-derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naïve macrophages is MyD88-dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed HEK cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. While both TLR2 and TLR4 contributed to TNF production in naïve macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis-stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.
Clinical and epidemiological studies have implicated chronic infections in the development of atherosclerosis. It has been proposed that common mechanisms of signaling via toll like receptors (TLRs) link stimulation by multiple pathogens to atherosclerosis. However, how pathogen specific stimulation of TLR4 contributes to atherosclerosis progression remains poorly understood.
Methods and Results
Atherosclerosis-prone apolipoprotein-E null (ApoE−/−) and TLR4 deficient (ApoE−/−TLR4−/−) mice were orally infected with the periodontal pathogen, Porphyromonas gingivalis. ApoE−/−TLR4−/− mice were markedly more susceptible to atherosclerosis following oral infection with P. gingivalis. Using live animal imaging, we demonstrate that enhanced lesion progression occurs progressively and was increasingly evident with advancing age. Immunohistochemical analysis of lesions from ApoE−/−TLR4−/− mice revealed an increased inflammatory cell infiltrate composed primarily of macrophages and IL-17 effector T cells (Th17), a subset linked with chronic inflammation. Furthermore, enhanced atherosclerosis in TLR4-deficient mice was associated with impaired development of T helper type-1 (Th1) immunity and regulatory T cell (Treg) infiltration. In vitro studies suggest that the mechanism of TLR4-mediated protective immunity may be orchestrated by dendritic cell interleukin (IL)-12 and IL-10, prototypic Th1 and Treg polarizing cytokines.
We demonstrate an atheroprotective role for TLR4 in response to infection with the oral pathogen, P. gingivalis. Our results point to a role for pathogen-specific TLR signaling in chronic inflammation and atherosclerosis.
Inflammation; Transgenic/Knockout Mice; Bacterial Infection; Monocytes/Macrophages
Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis–induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf−/−) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow–derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis–stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.
Clinical and epidemiological studies have implicated chronic infections in the development of atherosclerosis. It has been proposed that common mechanisms of signaling via TLRs link stimulation by multiple pathogens to atherosclerosis. However, how pathogen-specific stimulation of TLR4 contributes to atherosclerosis progression remains poorly understood. In this study, atherosclerosis-prone apolipoprotein-E null (ApoE−/−) and TLR4-deficient (ApoE−/−TLR4−/−) mice were orally infected with the periodontal pathogen Porphyromonas gingivalis. ApoE−/−TLR4−/− mice were markedly more susceptible to atherosclerosis after oral infection with P. gingivalis. Using live animal imaging, we demonstrate that enhanced lesion progression occurs progressively and was increasingly evident with advancing age. Immunohistochemical analysis of lesions from ApoE−/−TLR4−/− mice revealed an increased inflammatory cell infiltrate composed primarily of macrophages and IL-17 effector T cells (Th17), a subset linked with chronic inflammation. Furthermore, enhanced atherosclerosis in TLR4-deficient mice was associated with impaired development of Th1 immunity and regulatory T cell infiltration. In vitro studies suggest that the mechanism of TLR4-mediated protective immunity may be orchestrated by dendritic cell IL-12 and IL-10, which are prototypic Th1 and regulatory T cell polarizing cytokines. We demonstrate an atheroprotective role for TLR4 in response to infection with the oral pathogen P. gingivalis. Our results point to a role for pathogen-specific TLR signaling in chronic inflammation and atherosclerosis.
Studies in humans support a role for the oral pathogen Porphyromonas gingivalis in the development of inflammatory atherosclerosis. The goal of this study was to determine if P. gingivalis infection accelerates inflammation and atherosclerosis in the innominate artery of mice, an artery which has been reported to exhibit many features of human atherosclerotic disease, including plaque rupture.
Methods and Results
Apolipoprotein E-deficient (ApoE−/−) mice were orally infected with P. gingivalis, and Magnetic Resonance Imaging (MRI) was used to monitor the progression of atherosclerosis in live mice. P. gingivalis infected mice exhibited a statistically significant increase in atherosclerotic plaque in the innominate artery as compared to uninfected mice. Polarized light microscopy and immunohistochemistry revealed that the innominate arteries of infected mice had increased lipids, macrophages and T cells as compared to uninfected mice. Increases in plaque, total cholesterol esters and cholesterol monohydrate crystals, macrophages, and T cells were prevented by immunization with heat-killed P. gingivalis prior to pathogen exposure.
These are the first studies to demonstrate progression of inflammatory plaque accumulation in the innominate arteries by in-vivo MRI analysis following pathogen exposure, and to document protection from plaque progression in the innominate artery via immunization.
Atherosclerosis; inflammation; P. gingivalis; infection; innominate artery; MRI
The Human Proteome Organisation’s Proteomics Standards Initiative (HUPO-PSI) has developed the GelML data exchange format for representing gel electrophoresis experiments performed in proteomics investigations. The format closely follows the reporting guidelines for gel electrophoresis, which are part of the Minimum Information About a Proteomics Experiment (MIAPE) set of modules. GelML supports the capture of metadata (such as experimental protocols) and data (such as gel images) resulting from gel electrophoresis so that laboratories can be compliant with the MIAPE Gel Electrophoresis guidelines, while allowing such data sets to be exchanged or downloaded from public repositories. The format is sufficiently flexible to capture data from a broad range of experimental processes, and complements other PSI formats for mass spectrometry data and the results of protein and peptide identifications to capture entire gel-based proteome workflows. GelML has resulted from the open standardisation process of PSI consisting of both public consultation and anonymous review of the specifications.
data standard; gel electrophoresis; database; ontology
Porphyromonas gingivalis is a periodontopathic bacterium closely associated with generalized aggressive periodontal disease. Pattern recognition receptors (PRRs) participate in host response to this organism. It is likely that PRRs not previously recognized as part of the host response to P. gingivalis also participate in host response to this organism.
Methods and Results
Employing qRT-PCR, we observed increased msr1 gene expression at 2, 6, and 24h of culture with P. gingivalis strain 381. Flow cytometry revealed increased surface expression of SR-A protein by the 24h time point. Macrophages cultured with an attachment impaired P. gingivalis fimA- mutant (DPG3) expressed intermediate levels of SR-A expression. Heat-killed P. gingivalis stimulated SR-A expression similar to live bacteria, and purified P. gingivalis capsular polysaccharide stimulated macrophage SR-A expression, indicating that live whole organisms are not necessary for SR-A protein expression in macrophage response. As SR-A is known to play a role in lipid uptake by macrophages, we tested the ability of low-density lipoprotein (LDL) to influence the SR-A response of macrophages to P. gingivalis, and observed no effect of LDL on P. gingivalis-elicited SR-A expression. Lastly, we observed that SR-A knockout (SR-A−/−) mouse macrophages produced significantly more tumor necrosis factor (TNF)-α than wild type mouse macrophages cultured with P. gingivalis.
These data identify that SR-A is expressed by macrophages in response to P. gingivalis, and support that this molecule plays a role in TNF-α production by macrophages to this organism.
Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg.
The Minimum Information for Biological and Biomedical Investigations (MIBBI) project provides a resource for those exploring the range of extant minimum information checklists and fosters coordinated development of such checklists.
Toll-like receptors (TLRs), a group of pathogen-associated microbial pattern recognition receptors, play an important role in innate immune signaling and are differentially regulated in chronic inflammatory diseases such as atherosclerosis. However, the involvement of TLRs in the progression of atherosclerosis is still unclear.
Methods and Results
TLR2 and apolipoprotein E double knockout (Tlr2−/−Apoe−/−) mice were generated and the progressive formation of atherosclerotic plaque in the aortas was examined in mice fed a normal chow diet. We demonstrate that inactivation of TLR2 resulted in reduced progression of atherosclerosis in both male and female Apoe−/− mice. Likewise, TLR2 deficiency resulted in a reduction in lipid accumulation and decreased macrophage recruitment to the aortic sinus as well as reduced monocyte chemoattractant protein-1 (MCP-1) levels. Furthermore, macrophages isolated from Tlr2−/−Apoe−/− mice demonstrated significantly reduced MCP-1 production upon stimulation with a TLR2 ligand. However, no differences in acetylated-low-density lipoprotein uptake and foam cell formation were observed in macrophages isolated from Tlr2−/−Apoe−/− mice as compared to Apoe−/− mice.
TLR2 plays a critical role in the progression of atherosclerosis in Apoe−/− mice, which is independent of dietary lipids and macrophage lipid uptake.
atherosclerosis; Toll-like receptor; inflammation; macrophages
The receptor activator of NF-κB ligand (RANKL) and the proinflammatory cytokines are believed to play important roles in osteoclastogenesis. We recently reported that the innate immune recognition receptor, Toll-like receptor 2 (TLR2), is crucial for inflammatory bone loss in response to infection by Porphyromonas gingivalis, the primary organism associated with chronic inflammatory periodontal disease. However, the contribution of macrophage-expressed TLRs to osteoclastogenesis has not been defined. In this study, we defined a requirement for TLR2 in tumor necrosis factor-alpha (TNF-α)-elicited osteoclastogenesis in response to exposure to P. gingivalis. Culture supernatant (CS) fluids from P. gingivalis-stimulated macrophages induced bone marrow macrophage-derived osteoclastogenesis. This activity was dependent on TNF-α and occurred independently of RANKL, interleukin-1β (IL-1β), and IL-6. CS fluids from P. gingivalis-stimulated TLR2−/− macrophages failed to express TNF-α, and these fluids induced significantly less osteoclast formation compared with that of the wild-type or the TLR4−/− macrophages. In addition, P. gingivalis exposure induced up-regulation of TLR2 expression on the cell surface of macrophages, which was demonstrated to functionally react to reexposure to P. gingivalis, as measured by a further increase in TNF-α production. These results demonstrate that macrophage-dependent TLR2 signaling is crucial for TNF-α-dependent/RANKL-independent osteoclastogenesis in response to P. gingivalis infection. Furthermore, the ability of P. gingivalis to induce the cell surface expression of TLR2 may contribute to the chronic inflammatory state induced by this pathogen.
Porphyromonas gingivalis is the principal organism associated with aggressive forms of generalized periodontal disease. Previous reports have suggested that encapsulated P. gingivalis strains are more virulent than unencapsulated strains; however, the contribution of capsular polysaccharide (CPS) to the virulence of this organism is poorly understood. Since periodontal disease presents with a complex inflammatory cell lesion comprised of neutrophils and monocytes, we cultured murine peritoneal macrophages with heat-killed P. gingivalis W83, CPS purified from P. gingivalis strain W83, and the seven known serotype-specific P. gingivalis CPS and assessed the ability of supernatant fluids produced by challenged macrophages to attract naïve inflammatory cells. We also defined JE/MCP-1, KC, MIP-2, and RANTES production in response to the P. gingivalis CPS antigens. We observed that supernatant fluids collected from macrophages incubated with P. gingivalis W83 and serotype K1 CPS stimulated the migration of naïve murine bone marrow-derived polymorphonuclear leukocytes in an in vitro cell migration chamber. CPS from W83 and the K1 serotype elicited potent chemokine secretion patterns for macrophages, while those specific to serotypes K2 to K7 were significantly less stimulatory. Reverse transcription-PCR and enzyme-linked immunosorbent assay revealed JE/MCP-1, KC, MIP-2, and RANTES expression from murine macrophages which had been challenged with purified P. gingivalis W83 CPS. Chemokine production appeared to be dependent on both the dose of and time of exposure to P. gingivalis W83 CPS. These data demonstrate that the P. gingivalis serotype K1 CPS elicits chemokine production from phagocytic cells. Furthermore, these data suggest that the host response to this antigen may contribute to the formation of the inflammatory cell lesion observed during P. gingivalis-elicited periodontal disease.
The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase–null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase–null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.
The physiological role of neutrophils is to seek out and destroy invading microbes. Professional phagocytes engulf (phagocytose) these organisms and kill them using bactericidal peptides, enzymes, toxic reactive oxygen species, and reactive nitrogen species produced by neutrophils and macrophages. Unfortunately, the reactive oxygen species unleashed in an oxidative burst response can cause considerable collateral damage and are directly responsible for infection-associated tissues injuries, especially if the invaders are protected against killing by neutrophils. The authors investigated the pathogenesis of Porphyromonas gingivalis, an anaerobic bacterium that is responsible for human periodontal disease and is protected against oxidative stress by the cytoplasmic protein rubrerythrin. We show that P. gingivalis is not only resistant to reactive oxygen species, but that in mice, rubrerythrin shields the bacterium against reactive nitrogen species. These features allow P. gingivalis to proliferate in animals that possess a fully functional oxidative burst response. Furthermore, we demonstrate that the neutrophil oxidative burst response, rather than eliminating the bacteria, exacerbates disease by damaging host tissues and facilitating growth and systemic dissemination of the pathogen. Collectively, this study provides important information on how oxygen-dependent killing mechanisms operate during anaerobic infection and on the role of rubrerythrin in protecting against a pathogenic anaerobic organism, while emphasizing the importance of limiting host-mediated tissue injury in inflammatory diseases caused by bacteria.
Here we report on early inflammatory events associated with Porphyromonas gingivalis-accelerated atherosclerosis in apolipoprotein E knockout (ApoE−/−) mice. Animals challenged with P. gingivalis presented with increased macrophage infiltration, innate immune marker expression, and atheroma without elevated systemic inflammatory mediators. This early local inflammatory response was prevented in mice immunized with P. gingivalis. We conclude that localized up-regulation of innate immune markers early after infection, rather than systemic inflammation, contributes to pathogen-accelerated atherosclerosis.
Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis. Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E−/− mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-type P. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-type P. gingivalis by fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purified P. gingivalis antigens. Following a wild-type P. gingivalis challenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonists Staphylococcus aureus lipoteichoic acid (SLTA; TLR2 ligand) and Escherichia coli lipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-type P. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA and E. coli LPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited by E. coli LPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasive P. gingivalis stimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.
Epidemiological and pathological studies have suggested that infection with the oral pathogen Porphyromonas gingivalis can potentiate atherosclerosis and human coronary heart disease. Furthermore, infection with invasive, but not noninvasive P. gingivalis has been demonstrated to accelerate atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice and to accelerate local inflammatory responses in aortic tissue. In the present study, using high-density oligonucleotide microarrays, we have defined the gene expression profile of human aortic endothelial cells (HAEC) after infection with invasive and noninvasive P. gingivalis. After infection of HAEC with invasive P. gingivalis strain 381, we observed the upregulation of 68 genes. Genes coding for the cytokines Gro2 and Gro3; the adhesion molecules intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and ELAM-1 (E-selectin); the chemokine interleukin-8 (IL-8); and the proinflammatory molecules IL-6 and cyclooxygenase-2 were among the most highly upregulated genes in P. gingivalis 381-infected HAEC compared to uninfected HAEC control. Increased mRNA levels for signaling molecules, transcriptional regulators, and cell surface receptors were also observed. Of note, only 4 of these 68 genes were also upregulated in HAEC infected with the noninvasive P. gingivalis fimA mutant. Reverse transcription-PCR, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting analysis confirmed the expression of ICAM-1, VCAM-1, E-/P-selectins, IL-6, and IL-8 in HAEC infected with invasive P. gingivalis. We also demonstrated that increased expression of ICAM-1 and VCAM-1 in aortic tissue of ApoE−/− mice orally challenged with invasive P. gingivalis but not with the noninvasive P. gingivalis fimA mutant by immunohistochemical analysis. Taken together, these results demonstrate that P. gingivalis fimbria-mediated invasion upregulates inflammatory gene expression in HAEC and in aortic tissue and indicates that invasive P. gingivalis infection accelerates inflammatory responses directly in the aorta.
Active immunization with Porphyromonas gingivalis whole-cell preparations has been shown to prevent P. gingivalis infection and oral bone loss. Employing passive antibody transfer and opsonization, we demonstrate with this study that immunization-elicited P. gingivalis-specific immunoglobulin G facilitates clearance of P. gingivalis in a subcutaneous chamber model and prevents P. gingivalis-elicited oral bone loss.
Porphyromonas gingivalis is an oral pathogen that has recently been associated with chronic inflammatory diseases such as atherosclerosis. The strength of the epidemiological associations of P. gingivalis with atherosclerosis can be increased by the demonstration that P. gingivalis can initiate and sustain growth in human vascular cells. We previously established that P. gingivalis can invade aortic, heart, and human umbilical vein endothelial cells (HUVEC), that fimbriae are required for invasion of endothelial cells, and that fimbrillin peptides can induce the expression of the chemokines interleukin 8 and monocyte chemotactic protein. In this study, we examined the expression of surface-associated cell adhesion molecules on endothelial cells in response to P. gingivalis infection by fluorescence-activated cell sorting FACS analysis and confocal microscopy. Coculture of HUVEC with P. gingivalis strain 381 or A7436 resulted in the induction in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P- and E-selectins, which was maximal at 48 h postinfection. In contrast, we did not observe induction of ICAM-1, VCAM-1, or P- or E-selectin expression in HUVEC cultured with the noninvasive P. gingivalis fimA mutant DPG3 or when P. gingivalis was incubated with fimbrillin peptide-specific anti-sera prior to the addition to HUVEC. Furthermore, the addition of a peptide corresponding to the N-terminal domain of fimbrillin to HUVEC resulted in an increase in ICAM-1, VCAM-1, and P- and E-selectins, which was maximal at 48 h and similar to that observed for live P. gingivalis. Treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also resulted in the inhibition of ICAM-1, VCAM-1, or P- and E-selectin expression. Taken together, these results indicate that active P. gingivalis invasion of HUVEC mediated via the major fimbriae stimulates surface-associated cell adhesion molecule expression. Stimulation of adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by P. gingivalis may play a role in the pathogenesis of systemic inflammatory diseases associated with this microorganism, including atherosclerosis.
Recent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease. Previously, we have established that the periodontal pathogen Porphyromonas gingivalis is capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC). Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions of P. gingivalis with endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis. In this study we examined the consequences of P. gingivalis infection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1). HUVEC were found to constitutively produce low levels of IL-8 and MCP-1. The addition of P. gingivalis fimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses. In contrast, coculture of HUVEC with live P. gingivalis strain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses. Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherent P. gingivalis fimA mutant DPG3 or when P. gingivalis was preincubated with fimbrillin peptide antisera prior to the addition to HUVEC. Furthermore, treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also abolished the constitutive IL-8 and MCP-1 responses. Treatment of HUVEC with E. coli LPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with live P. gingivalis. Analysis of P. gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC. Pretreatment of P. gingivalis with protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production in P. gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated. Coculture of HUVEC with a P. gingivalis mutant deficient in lysine-specific cysteine proteinase (gingipain K [Kgp]) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with the P. gingivalis wild-type strain. These results indicate that P. gingivalis can temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.
The arginine gingipains RgpA and RgpB of Porphyromonas gingivalis are well-documented virulence factors of this organism. Structurally, RgpA and RgpB have nearly identical catalytic domains, while RgpA possesses an additional hemagglutinin domain. In this study, we examined the abilities of these proteins to elicit protection against P. gingivalis-mediated oral bone loss in a murine oral challenge model. Mice immunized subcutaneously with heat-killed P. gingivalis or purified RgpA or RgpB possessed elevated levels of P. gingivalis-specific immunoglobulin G; however, only the animals immunized with P. gingivalis whole cells or RgpA were protected from maxillary bone loss. These data suggest that immunization with RgpA stimulates the production of hemagglutinin domain-specific antibodies, which contribute to the prevention of P. gingivalis-mediated periodontal disease.
In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated, N. gonorrhoeae strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by N. gonorrhoeae.
The unc operon of Escherichia coli was split into two fragments by the restriction endonuclease HindIII. The operator-proximal portion was cloned into plasmid pACYC184, forming plasmid pAN51, which included the genes uncB, uncE, and uncA. When plasmid pAN51 was used as template in an in vitro transcription/translation system, the α subunit (from the uncA gene) and δ subunit of the F1 adenosine triphosphatase (ATPase) were formed. In addition, three polypeptides of molecular weights 18,000, 17,000, and 14,000 were formed, and the significance of these polypeptides is discussed. The operator-distal portion of the unc operon was also cloned into plasmid pACYC184, forming plasmid pAN36, which included the uncD and uncC genes. When this plasmid was used as template in an in vitro transcription/translation system, the β subunit (from the uncD gene) and the ε subunit (from the uncC gene) of the F1 ATPase were formed. A polypeptide of a molecular weight similar to the ε subunit but of different net charge was also formed. Plasmid pAN45, carrying the complete unc operon, was isolated after digestion of a mixture of plasmids pAN51 and pAN36 with the restriction endonuclease HindIII and then religation with T4 deoxyribonucleic acid ligase. It was concluded that a HindIII restriction site occurred within the newly described uncG gene, which was shown, by complementation studies with Mu-induced mutants, to be located between the uncA and uncD genes to give the gene order uncBEAGDC. The uncG gene appears to code for the γ subunit of the F1 ATPase.