Abstract-Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam 3 CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin ␣ IIb ␤ 3 , generation of reactive oxygen species, and, in human whole blood, formation of platelet-neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/ Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam 3 CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet-neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

SUMMARYA hallmark of infection with the gram-negative pathogen Porphyromonas gingivalis is the induction of a chronic inflammatory response. P. gingivalis induces a local chronic inflammatory response that results in oral inflammatory bone destruction, which manifests as periodontal disease. In addition to chronic inflammation at the initial site of infection, mounting evidence has accumulated supporting a role for P. gingivalis-mediated periodontal disease as a risk factor for several systemic diseases including, diabetes, preterm birth, stroke, and atherosclerotic cardiovascular disease. A growing number of in vitro studies have demonstrated that P. gingivalis infection stimulates cell activation commensurate with expected responses paralleling inflammatory atherosclerotic-type responses. Furthermore, various mouse models have been used to examine the ability of P. gingivalis to stimulate chronic inflammatory plaque accumulation and recent studies have pointed to a pivotal role for innate immune signaling via the Toll-like receptors in the chronic inflammation associated with P. gingivalis infection. In this review we discuss the pathogen and host cell specificity of these responses and discuss possible mechanisms by which this oral pathogen can induce and maintain a chronic state of inflammation at sites distant from oral infection.

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.

Objective-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.Conclusions-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. * Corresponding Author. Caroline A. Genco, 650 Albany St. Boston, MA 02118, USA,, cgenco@bu.edu. 1 These authors contributed equally to this work. Disclosure None.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptAtherosclerosis. Author manuscript; available in PMC 2012 March 1. We previously demonstrated that oral infection with Porphyromonas gingivalis, the etiological agent of human periodontal disease, accelerates plaque accumulation in the aortic sinus in an apolipoprotein E (ApoE −/− ) mouse model [5,8]. While these studies in the ApoE −/− mouse model have described atherosclerotic lesions of the aortic sinus by histological analysis, intra-plaque rupture or signs of plaque disruption at the aortic sinus have not been reported [9]. In contrast, recent studies have documented the presence of ruptured plaques in the innominate artery of ApoE −/− mice [10][11][12][13][14]. The innominate artery has been reported to undergo a high degree of lesion progression, and lesions in this artery reported to express features characteristic of clinical disease in humans [10,11,[15][16][17]. However, the ability of infectious agents t...

Background 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. Conclusions 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.