Common chronic infections may contribute to up to 40% of newly developed atherosclerotic cases (39
). A role for P. gingivalis
-mediated periodontal disease as a risk factor for atherosclerotic cardiovascular disease is well documented (10
). The observation that innate immune signaling triggered by P. gingivalis
is dysregulated within atherosclerotic lesions has sparked interest in the association between oral infection and induction of innate immune cascades in atherosclerosis progression (40
). Most experimental studies have focused on the proatherogenic consequence of TLR signaling in mouse models of atherogenesis; many involving the influence of high-fat diet (5
). In contrast to studies reporting diminished high-fat diet-induced atherosclerosis in TLR4-deficient mice, we report the unexpected finding that TLR4-deficient mice are markedly more susceptible to atherosclerosis after infection with P. gingivalis
. Live animal imaging demonstrated that enhanced disease severity occurred progressively, long after cessation of the infectious stimulus and at two anatomically relevant sites, in large (aortic sinus) and medium (innominate artery) size vessels. Enhanced atherosclerosis progression in ApoE−/−
mice compared with ApoE−/−
mice is unlikely to be due to differences in plasma cholesterol or triglycerides, which were similar among all groups. Minimal atherosclerotic lesion area in the innominate artery was observed in uninfected ApoE−/−
mice, and this is likely due to the fact that animals were fed a normal chow diet. In our recent study in which atherosclerosis progression was examined using MRA in the innominate artery of uninfected and P. gingivalis
mice, animals were fed a high-fat diet for the duration of the study (35
). High-fat diet enhances atherosclerosis progression in ApoE−/−
mice. In the absence of high-fat diet and infection, plaque accumulation within the aorta and innominate artery progresses more slowly and is minimal at the time point examined in the current study. Effective control of immune-mediated pathology in P. gingivalis
mice coincided with an increase in Tregs within the innominate artery. In contrast, the exacerbated inflammatory pathology in P. gingivalis
mice was associated with increased lesion macrophage numbers and T cell infiltration and enhanced expression of IL-17. Tregs play a critical role in maintaining immunological tolerance and controlling the extent of immune-mediated pathology, especially in cases of chronic infection (41
). Our studies indicate that in the absence of TLR4, mice fail to develop protective Th1 immunity and are unable to regulate adaptive immune responses mediated by Th17 cells after P. gingivalis
infection. We propose that this results in a breakdown of immunological tolerance, owing to impaired Treg function, leading to unrestricted activation of pathogenic T cells that mediate arterial inflammation. The unique TLR4 evasive properties of P. gingivalis
lipid A position this organism to disrupt effector T cell mechanisms at the level of DC activation, the interface of innate and adaptive immunity.
TLR2 expression was increased markedly in aortic lesions by P. gingivalis infection in ApoE−/− mice and further increased in ApoE−/−TLR4−/− mice. It is plausible that enhanced TLR2 expression in ApoE−/−TLR4−/− mice may have contributed to increased vascular inflammation and atherosclerosis in ApoE−/−TLR4−/− mice. Thus, the increase in atherosclerosis in ApoE−/−TLR4−/− mice may be a result of not only TLR4 deficiency but also high TLR2 expression. This increased TLR2 expression in activated macrophages and the endothelium may reflect the development and maintenance of a hyperinflammatory state in the absence of TLR4 expression. This observation was an unexpected finding of this study, as was the finding that plaque development was enhanced in the absence of TLR4.
Our results also showed that after in vitro restimulation with Ag, T cells from P. gingivalis
mice predominantly produced IL-17, whereas IFN-γ was the predominant cytokine produced by T cells from infected ApoE−/−
mice. We also demonstrate that TLR4 deficiency was associated with markedly inhibited production of the Th1 polarizing cytokine IL-12 by P. gingivalis
-infected DCs. Taken together, our findings suggest that an initially impaired Th1 response in TLR4-deficient mice results in Th17 skewing of the adaptive immune response, which may be the mechanism for exacerbated atherosclerosis. Indeed, research supports a complex relationship between the Th1 and Th17 cell lineages, and many T cells expressing IL-17 coexpress IFN-γ. We previously reported that IFN-γ is significantly upregulated at the protein level in atherosclerotic lesions from P. gingivalis
mice relative to uninfected controls (28
). This finding is in agreement with IFN-γ contributing to the development of atherosclerosis as suggested by one study (43
). Several studies demonstrate that both IFN-γ and IL-17 may be proatherogenic in mouse models, and circulating levels of both of these cytokines are increased in patients with coronary atherosclerosis (44
). A large body of literature also supports a proatherogenic role for IL-17 in mouse models of atherosclerosis, and neutralization of IL-17 has been demonstrated to reduce pathogen- and diet-induced atherosclerosis in ApoE−/−
). These studies demonstrate that the development of atherosclerosis is multifactorial and may be influenced by the inciting stimulus (i.e., high-fat diet versus pathogen-mediated).
Notably, humans are specifically impaired in their ability to recognize penta-acylated lipid A, a phenomenon reflecting bacterial adaptation to the human host (46
). Species-specific discrimination of atypical penta-acylated lipid A is mediated by a hypervariable 82-aa sequence in the middle region of TLR4, a region where human polymorphisms are common (31
). Our results provide a mechanistic link regarding the conflicting reports on the association of human TLR4 polymorphisms and atherosclerotic diseases. Thus, although we only see a hyperinflammatory phenotype in TLR4-deficient mice, it is plausible that common human TLR4 polymorphisms (47
) that attenuate receptor signaling may predispose individuals to an increased risk of atherosclerosis associated with bacterial infection, which is in contrast to atherosclerosis risk associated with a Western high-fat diet.
Recent studies reported that ApoE−/−
mice fed a high-fat diet and infected intranasally with C. pneumoniae
exhibited diminished atherosclerosis compared with infected ApoE−/−
). A separate report demonstrated a protective role for TLR4 deficiency in diet-induced atherogenesis (8
). It is important to note that these results could not be recapitulated under germ-free conditions (50
), indicating a potential interaction between hyperlipidemia and indigenous microbes. On the basis of these observations, it was proposed that common mechanisms of signaling via TLR2, TLR4, and MyD88 link stimulation by multiple pathogens and endogenous ligands to atherosclerosis, and that therapeutic TLR4 antagonism could prove beneficial in the treatment of chronic atherosclerosis (8
). Our results clearly point to a critical role for specific TLR signaling, in particular, TLR4, in chronic inflammation and atherosclerosis induced by P. gingivalis
. Our results raise caution for the safety and efficacy of TLR4 antagonists for the treatment of atherosclerosis, especially in patients with comorbid conditions including periodontal disease and other infectious diseases.