Frank C. Gibson

Innate Immune Recognition of Invasive Bacteria Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice

Background—Infectious diseases have emerged as potential risk factors for cardiovascular disease (CVD). Epidemiological studies support a connection between periodontal disease, a chronic inflammatory disease of the supporting tissues of the teeth, and CVD. Methods and Results—To directly test the connection between periodontal disease and atherosclerosis, apoE−/− mice were orally challenged with the periodontal disease pathogen Porphyromonas gingivalis or an invasion-impaired P gingivalis fimbriae-deficient mutant (FimA−). Both wild-type P gingivalis and the FimA− mutant were detected in blood and aortic arch tissue of apoE−/− mice by PCR after challenge. ApoE−/− mice challenged with wild-type P gingivalis presented with increased atherosclerotic plaque and expressed the innate immune response markers Toll-like receptor (TLR)-2 and TLR-4 in aortic tissue. Despite detection of the FimA− mutant in the blood and in aortic arch tissue, apoE−/− mice challenged with the FimA− mutant did not present with periodontal disease, upregulation of TLRs, or accelerated atherosclerosis. Furthermore, we demonstrate that immunization to control P gingivalis–elicited periodontal disease concomitantly prevents P gingivalis–accelerated atherosclerosis. Conclusions—We conclude that invasive P gingivalis accelerates atherosclerosis.

Innate Immune Signaling and Porphyromonas gingivalis-accelerated Atherosclerosis

Periodontal diseases are a group of diseases that lead to erosion of the hard and soft tissues of the periodontium, which, in severe cases, can result in tooth loss. Anecdotal clinical observations have suggested that poor oral health may be associated with poor systemic health; however, only recently have appropriate epidemiological studies been initiated, with defined clinical endpoints of periodontal disease, to address the association of periodontal disease with increased risk for cardiovascular and cerebrovascular disease. Although conflicting reports exist, these epidemiological studies support this connection. Paralleling these epidemiological studies, emerging basic scientific studies also support that infection may represent a risk factor for atherosclerosis. With P. gingivalis as a model pathogen, in vitro studies support that this organism can activate host innate immune responses associated with atherosclerosis, and in vivo studies demonstrate that this organism can accelerate atheroma deposition in animal models. In this review, we focus primarily on the basic scientific studies performed to date which support that infection with bacteria, most notably P. gingivalis, accelerates atherosclerosis. Furthermore, we attempt to bring together these studies to provide an up-to-date framework of emerging theories into the mechanisms underlying periodontal disease and increased risk for atherosclerosis, as well as identify intervention strategies to reduce the incidence of periodontal disease in humans, in an attempt to decrease risk for systemic complications of periodontal disease such as atherosclerotic cardiovascular disease.

Distinct Proinflammatory Host Responses to Neisseria gonorrhoeae Infection in Immortalized Human Cervical and Vaginal Epithelial Cells

ABSTRACT 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.

Fimbria-dependent activation of cell adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells

ABSTRACT 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.

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection.

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.

Prevention of Porphyromonas gingivalis-Induced Oral Bone Loss following Immunization with Gingipain R1

ABSTRACT 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.

Role for fimbriae and lysine-specific cysteine proteinase gingipain K in expression of interleukin-8 and monocyte chemoattractant protein in Porphyromonas gingivalis-infected endothelial cells.

ABSTRACT 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.

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE–/– mice, while an isogenic fimbria‐deficient (FimA‐) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme‐linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro‐inflammatory molecules interleukin (IL)‐1β, IL‐8, monocyte chemoattractant protein (MCP)‐1, intracellular adhesion molecule (ICAM)‐1, vascular cellular adhesion molecule (VCAM)‐1 and E‐selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL‐1β production. In addition, the major and minor fimbria‐mediated production of MCP‐1 and IL‐8 was inhibited in a dose‐dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat‐killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro‐inflammatory MCP‐1 and IL‐8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL‐1β. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL‐1β appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.

Macrophage-Specific TLR2 Signaling Mediates Pathogen-Induced TNF-Dependent Inflammatory Oral Bone Loss

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.

Pathogen-Mediated Inflammatory Atherosclerosis Is Mediated in Part via Toll-Like Receptor 2-Induced Inflammatory Responses

Studies in humans have established that polymorphisms in genes encoding the innate immune Toll-like receptors (TLRs) are associated with inflammatory atherosclerosis. In hyperlipidemic mice, TLR2 and TLR4 have been reported to contribute to atherosclerosis progression. Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclerosis via innate immune system activation is not known. Using a genetically defined apolipoprotien E-deficient (ApoE–/–) mouse model we demonstrate that pathogen-mediated inflammatory atherosclerosis occurs via both TLR2-dependent and TLR2-independent mechanisms. P. gingivalis infection in mice possessing functional TLR2 induced the accumulation of macrophages as well as inflammatory mediators including CD40, IFN-γ and the pro-inflammatory cytokines IL-1β, IL-6 and tumor necrosis factor-α in atherosclerotic lesions. The expression of these inflammatory mediators was reduced in atherosclerotic lesions from P. gingivalis-infected TLR2-deficient (TLR2–/–) mice. These studies provide a mechanistic link between an innate immune receptor and pathogen-accelerated atherosclerosis by a clinically and biologically relevant bacterial pathogen.