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Innate Immune Signaling and Porphyromonas gingivalis-accelerated Atherosclerosis

¹ Department of Medicine, Section of Infectious Diseases, and
⁶ Department of Microbiology, Boston University School of Medicine, Evans Biomedical Research Center, 650 Albany Street, Room 637, Boston, MA 02118, USA;
² Department of Conservative Dentistry, Tokushima University School of Dentistry, Tokushima, Japan;
³ Department of Oral Microbiology, Kanagawa Dental College, 82 Inaokoa-cho, Yokosuka 238-8580, Japan;
⁴ School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; and
⁵ Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University Medical Center, Boston, MA, USA

View larger version (69K): [in a new window]   Figure 1. Models linking mechanisms governing pathogen-accelerated atherosclerosis. Four putative mechanisms by which infection may contribute to accelerated atherosclerosis include the following: (1) direct microbial invasion of vascular endothelium, whereby these infected cells would become immunologically activated in a manner that would set into motion events that would lead to the deposition of atheroma (detailed in Fig. 2); (2) immunological sounding, in which the host response to an extravascular infection leads to seeding of cytokines and chemokines into the circulation, with subsequent activation of vascular endothelium (detailed in Fig. 3); (3) pathogen trafficking, whereby pathogens are shuttled from a site of infection inside inflammatory cells to activated endothelium to gain access to this site (detailed in Fig. 4); and (4) auto-immune reaction, whereby bacterial molecules elicit a specific antibody that is cross-reactive with host molecules (detailed in Fig. 5). These pathways can occur alone or concurrently, and may not necessarily be mutually exclusive.

View larger version (33K): [in a new window]   Figure 2. Direct invasion of vascular endothelium. (A) In this model, invasion of the vascular endothelium by pathogenic bacteria such as P. gingivalis (red circles) results in the induction of a local inflammatory response, defined by the expression of cell adhesion molecules (CAMs; green trapezoid), Toll-like receptor (TLRs; blue triangle), chemokines, and cytokines. These inflammatory molecules have all demonstrated significant roles in the initiation and/or acceleration of atherosclerosis. The ability of P. gingivalis to stimulate host endothelial cell activation, both in vitro and in vivo, is a function of surface-expressed major fimbriae. P. gingivalis that does not possess fimbriae (fimA) fails to enter endothelial cells efficiently, whereas those organisms that possess fimbriae (wild-type, WT) readily enter these cells (Deshpande et al., 1998b; Khlgatian et al., 2002; Nassar et al., 2002). Following uptake, P. gingivalis-infected endothelial cells, possibly via a receptor-mediated signaling event, activate gene transcription and stimulate these cells to produce a variety of innate immune markers, including CAMs (ICAM-1, VCAM-1), TLRs (TLR-2, TLR-4), pro-inflammatory cytokines (TNF-, IL-1ß), and chemokines (MCP-1 and IL-8). These mediators are believed to be involved in the immunological switch of endothelial cells from a normal anti-thrombotic to a pro-thrombotic state. (B) Following P. gingivalis invasion/activation of vascular endothelial cells, these cells recruit monocytes, and, in the presence of elevated circulating lipids such as ox-LDL, atheroma forms.

View larger version (34K): [in a new window]   Figure 3. Infection-induced stimulation of accelerated atherosclerosis by immunological sounding. Persistent local infection, such as oral infections by P. gingivalis, may promote atherosclerosis via chronic up-regulation of inflammatory cascades involving TNF-, IL-1, IFN, IL-8, MCP-1, and CRP. These cytokines, chemokines, and acute phase mediators could be shed into the vasculature from a focus of P. gingivalis infection in the periodontium. Once in the circulation, these mediators may subsequently activate vascular endothelial cells in a manner that shifts them from a normally anti-thrombotic state to one expressing high levels of inflammatory mediators, including CAMs (blue triangles) and TLRs (green trapezoid), that become pro-thrombotic. This activated endothelium would likely be a site for subsequent atheroma formation, independent of direct pathogen involvement at this site. This further immunological activation results in the recruitment of monocytes, as well as the stimulation, migration, and proliferation of smooth-muscle cells that, together with elevated levels of circulating lipids such as ox-LDL, ultimately results in acceleration of the atheroma.

View larger version (35K): [in a new window]   Figure 4. Trafficking of pathogens. Localization of pathogens to the endothelium may occur via host immune cells. Oral infection by pathogens such as P. gingivalis leads to considerable tissue damage and stimulates a complex cellular inflammatory lesion that partly characterizes periodontitis. Phagocytic mononuclear cells such as macrophages are responsible for clearance of non-self antigens via phagocytosis and can ingest P. gingivalis at the site of infection in the oral cavity. Upon phagocytosis, it may be possible for some pathogens to resist phagocytic killing and persist within these cells. Without a chemokine gradient to localize to the site of infection, due either to P. gingivalis cysteine protease (gingipain)-mediated cleavage of inflammatory mediators, or to infection-elicited localized chemokine paralysis, infected phagocytes could leave the site of infection and emigrate back to the circulation. These circulating infected macrophages could then interact with immunologically activated endothelial cells at the site of a developing atheroma, first by localizing via a chemotactic gradient (IL-8 and MCP-1), followed by tight adherence to the endothelium via CAMs (green trapezoid). At this point, bacterial antigens or viable bacteria could be released from these cells, and, ultimately, together with elevated levels of circulating lipids such as ox-LDL, result in acceleration of atherosclerosis.

View larger version (25K): [in a new window]   Figure 5. Infection-induced stimulation of accelerated atherosclerosis by molecular mimicry. Molecular mimicry requires infection by a pathogen that possesses a molecule with significant homology to a host molecule. Following a host response initiated to this molecule, the response then presents as an auto-immune insult against those tissues with cross-reactive epitopes. Depicted here, conserved proteins such as bacterial HSPs (pink diamonds) and host HSPs (pink diamonds with cross) are exposed as a result of tissue damage and the host response to infection. Specific antibody directed toward bacterial HSPs would cross-react with human HSPs, setting in motion a localized auto-immune response. Indeed, cross-reactive antibodies to bacterial and host HSPs have been reported. The resulting inflammatory response could lead to endothelial cell damage, and ultimately, together with monocyte recruitment and the presence of elevated circulating lipids such as ox-LDL, to the acceleration of the atheroma.

View larger version (19K): [in a new window]   Figure 6. MCP-1 expression in HAEC, monocytes, and HAEC/monocyte co-cultures. P. gingivalis 381 (wt) or the fimA mutant DPG3 (fimA) was used to infect HAEC (A) human monocytes or (B) HAEC/human monocyte co-cultures (C) at an MOI of 1:100 (100) or 1:500 (500), and MCP-1 was measured by ELISA after 24 hrs. Uninfected HAEC, monocytes, and HAEC/monocyte co-cultures (control) served as negative controls. Analysis of these data demonstrates that cultures of primary HAEC, human peripheral blood monocytes, and HAEC/monocyte co-cultures actively respond to P. gingivalis challenge. Moreover, the MCP-1 response observed in the co-culture system is not simply additive, but rather, some unknown interaction occurs between endothelial cells and macrophages during P. gingivalis challenge, giving rise to a synergistic response.

View larger version (18K): [in a new window]   Figure 7. IL-8 expression in HAEC, monocytes, and HAEC/monocyte co-cultures. P. gingivalis 381 (wt) or the fimA mutant DPG3 (fimA) was used to infect HAEC (A), human monocytes (B), or HAEC/human monocyte co-cultures (C) at an MOI of 1:100 (100) or 1:500 (500), and IL-8 was measured by ELISA after 24 hrs. Uninfected HAEC, monocytes, and HAEC/monocyte co-cultures (control) served as negative controls. Analysis of these data demonstrates that cultures of primary HAEC, human peripheral blood plastic adherent monocytes, and HAEC/monocyte co-cultures actively respond to P. gingivalis challenge. The IL-8 response observed in these systems parallels that observed for MCP-1 (Fig. 6). The co-culture system supports that some unknown interaction between endothelial cells and macrophages during P. gingivalis challenge is likely occurring, giving rise to a synergistic response.

View larger version (33K): [in a new window]   Figure 8. Model of the interactions of P. gingivalis with host cells. Invasive P. gingivalis can interact with both the aortic endothelium (A) and monocytes (B), and can initiate a potent innate immune response. The combination of the endothelium and the monocytes (C) is more responsive to P. gingivalis than either of the individual cell types alone. In all cases, cells infected with wild-type P. gingivalis (WT; left of dashed line) elicit a more potent response than the major fimbriae-deficient P. gingivalis (FimA-; right of dashed line). Size of arrow indicates relative magnitude of the host response.

View larger version (26K): [in a new window]   Figure 9. Stimulation of mononuclear cells to form foam cells. Infection of mononuclear cells by bacterial pathogens sets in motion a series of events that lead to monocyte activation. Several pathogens epidemiologically linked to the acceleration of atherosclerosis have demonstrated the ability to elicit macrophages to form into foam cells—lipid-laden cells that possess a foamy appearance due to lipid accumulation and are the cells characteristic of an early-stage atheroma called the fatty streak. Recent studies have demonstrated that P. gingivalis stimulates macrophage foam cell formation, and this process appears to be dependent on the attachment of this organism to the macrophage. As part of the characteristic host response of macrophages to infection, these cells express elevated levels of CAMs (green trapezoid) that increase the adhesiveness of these cells to endothelial cells. Additionally, these cells express elevated levels of cytokines and chemokines, as well as TLRs (blue triangles), that could increase macrophage localization to the site of this infected macrophage, as well as increase the sensitivity of these macrophages to specific PAMPs. Ultimately, these mononuclear cells could go on to develop into foam cells as a result of the uptake of serum lipids, including ox-LDL.