Cardiovascular disease is an inflammatory disorder characterized by the progressive formation of plaque in coronary arteries, termed atherosclerosis. It is a multifactorial disease that is one of the leading causes of death worldwide. Although a number of risk factors have been associated with disease progression, the underlying inflammatory mechanisms contributing to atherosclerosis remain to be fully delineated. Within the last decade, the potential role for infection in inflammatory plaque progression has received considerable interest. Microbial pathogens associated with periodontal disease have been of particular interest due to the high levels of bacteremia that are observed after routine dental procedures and every day oral activities, such as tooth brushing. Here, we explore the potential mechanisms that may explain how periodontal pathogens either directly or indirectly elicit immune dysregulation and consequently progressive inflammation manifested as atherosclerosis. Periodontal pathogens have been shown to contribute directly to atherosclerosis by disrupting endothelial cell function, one of the earliest indicators of cardiovascular disease. Oral infection is thought to indirectly induce elevated production of inflammatory mediators in the systemic circulation. Recently, a number of studies have been conducted focusing on how disruption of the gut microbiome influences the systemic production of proinflammatory cytokines and consequently exacerbation of inflammatory diseases such as atherosclerosis. It is clear that the immune mechanisms leading to atherosclerotic plaque progression, by oral infection, are complex. Understanding the immune pathways leading to disease progression is essential for the future development of anti‐inflammatory therapies for this chronic disease.

Introduction Periodontal disease is a chronic inflammatory disorder that results from alterations in the oral microbiome and causes immune dysregulation and oral bone loss. Mounting evidence has demonstrated an association in humans between periodontal disease and other systemic diseases including diabetes, cardiovascular disorders and atherosclerosis. These systemic diseases are multifactorial, chronic inflammatory disorders that progress with significant involvement of the innate and adaptive immune systems. Periodontal disease and cardiovascular disease share common risk factors including smoking, obesity and diabetes 1. In the USA, more than 47% (~100 million) of adults have periodontal disease 2 and cardiovascular disease is one of the leading causes of mortality worldwide, accounting for 16.7 million deaths each year 3, 4. Given the high incidence of periodontal and cardiovascular diseases and their economic cost to society, defining their mechanistic link has become increasingly important 4. This is illustrated by the increasing number of reports published on the link between periodontal disease and cardiovascular disease in recent years; in 2007, only 73 articles were cited on this topic; however, by 2014 there were close to 4000 5.

Inflammatory mechanisms contributing to periodontal disease Periodontal disease is a microbial induced inflammatory disorder that leads to the destruction of the tissues supporting the teeth, including the gingiva, and resorption of the underlying alveolar bone 6-8. Although there are over 500 bacterial species in the oral cavity, only a few species are implicated in the progression of periodontal disease 9. Organisms such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia comprise a ‘red complex’ which was traditionally considered to be the root of the destructive inflammation present in the oral cavity 10. However, it has been recently revealed that induction of periodontitis is actually more complex and involves the entire microbial community present in the oral cavity 11. Periodontal inflammation is thought to be largely mediated by ‘pathobionts’ which are commensal organisms that under conditions of disrupted homeostasis (i.e. during P. gingivalis infection) have the potential to deregulate the inflammatory response and cause disease 12, 13. In a mouse model of periodontitis, P. gingivalis was shown to be a low‐abundance species, comprising <0.01% of the total bacterial count 14. This model revealed that at this low colonization level, P. gingivalis induced a shift in the oral microbiota, known as dysbiosis, resulting in alterations in the relative abundances of individual components of the bacterial community. It is now generally accepted that periodontal organisms comprising the red complex, such as P. gingivalis, orchestrate rather than directly cause inflammatory bone loss 11. It is important to note that although the tooth‐associated biofilm is required to induce periodontal disease, it is also the host inflammatory response to this microbial challenge that ultimately causes destruction of the supporting tissues of the teeth and gingiva. Immune recognition of periodontal pathogens results in progressive inflammation consisting of an immune cell infiltrate, such as monocytes and B and T cells, and production of inflammatory mediators 15. As inflammation progresses to a more chronic state, the lesion becomes composed of a cellular infiltrate that is predominantly monocytic 8. Monocytes become activated macrophages, which further accelerate bone resorption through differentiation into osteoclasts and production of tissue‐damaging proinflammatory cytokines 16. It has been suggested that monocyte chemoattractant protein‐1 (MCP‐1) has an important role in the activation and recruitment of inflammatory and immune cells in periodontal disease 17. Furthermore, additional proinflammatory mediators such as IL‐8 and IL‐1β have been detected in the gingiva from patients with periodontal disease 18.

Inflammatory mechanisms contributing to cardiovascular disease Cardiovascular disease encompasses a spectrum of disorders of the heart and associated blood vessels. Traditionally, it was hypothesized that cardiovascular disease involved the passive accumulation of lipids into the arterial wall; however, in the last decade the role of the immune response in disease progression has been increasingly appreciated 3, 19, 20. Cardiovascular disease is characterized by the progressive formation of plaque in coronary arteries, termed atherosclerosis. Atherosclerosis begins with a dysfunctional endothelium, resulting in recruitment of a number of immune cells, such as macrophages and T cells, into the lesion 21 (Fig. 1). Inflammation within the lesion progresses when immune cells become activated by ligands present in the vasculature, resulting in the production of a number of proinflammatory mediators that further propagate inflammation 22, 23. Figure 1 Open in figure viewer PowerPoint 21 40, 42, 43 Cells that participate in the progression and regression of atherosclerotic plaques. Endothelial dysfunction results in recruitment and accumulation of cells, including macrophages, foam cells and T cells, in a progressing atherosclerotic lesion. A hallmark of plaque regression is reverse cholesterol transport, a process driven by cholesterol efflux and accompanied by emigration of macrophages and disappearance of foam cells from the lesion. LDL, low‐density lipoprotein; oxLDL, oxidized low‐density lipoprotein. Within atherosclerotic lesions, cells comprising the vasculature, such as endothelial cells, and cellular infiltrate, such as macrophages, have been shown to upregulate a number of proinflammatory markers. Amongst the most extensively studied markers of inflammation, which have become increasingly associated with inflammatory diseases such as atherosclerosis, are the Toll‐like receptors (TLRs) 24. TLRs were first characterized with regard to their role in detecting invading pathogens, thus serving as a first line of host defence against infection 25, 26. Receptors in the TLR family recognize specific, conserved pathogen‐associated molecular patterns (PAMPs) and, upon ligand recognition, TLRs orchestrate an inflammatory cascade. In addition to recognition of exogenous pathogen‐derived ligands, it is now known that TLRs can also be engaged by endogenous ligands. Further, endogenous ligands, such as oxidized LDL, have been found to engage TLRs expressed on monocytes cells, resulting in lipid‐laden foam cells that characterize the earliest stages of lesion development 3. This finding, amongst others, gave rise to the notion that ligands of microbial origin may also contribute to the detrimental inflammatory reactions within atherosclerotic lesions 27. Pathogens such as Chlamydia pneumoniae, Helicobacter pylori and P. gingivalis have been identified within human atherosclerotic plaques 28-35, suggesting that infectious agents are capable of gaining access to the vasculature. In addition to the progressive accumulation of inflammatory cells in atherosclerotic lesions, plaques also undergo regressive changes, which alter their size, cellular composition and stability 36-39. It is noteworthy that these regressive changes are a distinct set of complex processes leading to clinically measurable decreases in plaque volume as opposed to a simple halting, slowing or reversal of the progressive changes within the vasculature. Francis and Pierce 40 described regression as an interplay between three processes: (i) reduction in or clearance of necrotic and extracellular material from the tunica intima; (ii) endothelial repair, regeneration and return to homeostasis; and (iii) cessation of smooth muscle cell proliferation. Although HDLs have been shown to restore endothelial cell function and decrease smooth muscle cell proliferation and migration, they also play a role in the removal of lipids from the tunica intima 41. An important component of this process is reverse cholesterol transport, which is primarily driven by cholesterol efflux from macrophages but is also promoted by HDL 40, 42. Cholesterol efflux is accompanied by emigration of the macrophage infiltrate and disappearance of foam cells 40, 43 (Fig. 1). Additional steps towards restoration of the plaque environment include reductions in plasma concentrations of apolipoprotein (Apo)B‐lipoproteins and clearance of necrotic debris by healthy phagocytes 42, 43. Plasma cholesterol lowering has been associated with regression, and peroxisome proliferator‐activated receptor (PPAR)‐gamma has recently been identified as a ‘master regulator’ of regression in early lesions 44.

Human studies The concept of a role of periodontal pathogens in exacerbating atherosclerosis progression is not new. In fact, an association between oral health and cardiovascular disease has been proposed for more than a century 5. The findings of a number of epidemiological studies have indicated an association between periodontal and cardiovascular diseases. Serological studies have shown a link between elevated periodontal bacterial antibody titres and atherosclerotic vascular disease 8, 9, 45-51. The results of case–control studies have confirmed the correlation between periodontal and cardiovascular diseases after adjusting for confounding factors 7, 52. Results from the Oral Infections and Vascular Disease Epidemiology Study revealed an association between periodontal pathogens and atherosclerosis 53, 54. Furthermore, the prevalence and incidence of coronary heart disease are significantly increased in patients with periodontal disease 55; cardiovascular diseases in patients with periodontitis are 25–50% higher than in healthy individuals 5. Specifically, severe periodontitis has been associated with increased intima–media thickening 55. Furthermore, poor self‐reported oral health and tooth loss are positively associated with coronary atherosclerotic burden 5. These reports indicate that periodontal disease can independently predict cardiovascular disease 56. Although these studies have supported a positive association between periodontal and cardiovascular diseases, others have not 57. Numerous studies have evaluated the impact of periodontal treatment, with or without antimicrobial therapy, on systemic inflammation or endothelial dysfunction and have shown mixed results (for extensive review, see 57). Thus, although meta‐analyses of clinical trials support an association between periodontal disease and cardiovascular disorders, including atherosclerosis, large longitudinal studies are needed to strengthen the current evidence supporting periodontal disease as an independent risk factor for cardiovascular disease. During the period between 2003 and 2006, four separate large clinical trials were designed to treat coronary artery disease patients with antibiotics to alleviate disease progression, although these agents were not specific for periodontal pathogens 58-61. The results from these trials, each of which enrolled over 4000 patients with coronary artery disease, showed no significant long‐term benefit of antibiotic treatment in those with established disease. Following the publication of the results of these separate studies, interest in further examining the link between infection and cardiovascular disease considerably declined. Therefore, although significant advancements have been made concerning the association between infection and atherosclerosis, there still remains controversy in the field, especially with regard to whether there is a clinically meaningful association between these two diseases 4. Of note, it has been difficult to establish a causative relationship between periodontal disease and cardiovascular disease in a clinical setting for a number of reasons 4, 62. First, the antibiotics chosen for several of the trials were targeted specifically against C. pneumoniae and not against other infectious agents, including P. gingivalis, that have been linked to an increased risk of cardiovascular disease 27. Secondly, it is difficult to target an individual factor contributing to inflammation, as the inflammatory response in the atherosclerotic lesion is multifactorial 63, 64. Finally, patients enrolled in these trials had advanced, chronic inflammation with ‘end‐stage cardiovascular disease’ 27. The early phases of atherosclerosis involve injury to the endothelium, an event that is likely missed because it is asymptomatic 65. It is plausible that antibiotics would be beneficial in patients with early atherosclerosis; however, clinical trials to examine this possibility would be difficult to design.

Animal studies Due to the difficulties in establishing the mechanisms linking atherosclerosis to periodontal disease in humans, animal models are frequently used. We have established a model to study this association by infecting atherosclerosis‐prone, ApoE‐deficient mice with the periodontal pathogen P. gingivalis. We have shown that oral infection of ApoE‐deficient mice with P. gingivalis results in chronic inflammation at both local (oral cavity) and systemic (atherosclerotic lesions) sites 66. To understand the effects of a polymicrobial periodontal community on atherosclerosis, Rivera et al. 56 recently infected ApoE‐deficient mice with the pathogens P. gingivalis, T. denticola and T. forsythia (the red complex). The authors observed a positive correlation between the number of species detected by genomic bacterial DNA in the aorta and plaque size in the thoracic aorta, supporting the notion that multiple oral bacterial species are likely associated with progression of atherosclerosis. Therefore, although P. gingivalis is not the only periodontal pathogen that is implicated in cardiovascular disease, its interactions with cells of the cardiovascular system have been extensively studied and thus serve as a model for understanding the impact of periodontal pathogens on atherosclerosis 62. Due to the predominant role of TLRs in atherosclerosis progression, we have investigated the association between TLRs and P. gingivalis‐mediated atherosclerosis. We demonstrated that P. gingivalis‐mediated TLR2 activation contributes to atherosclerosis progression 67. By contrast, TLR4 was shown to play a protective role in P. gingivalis‐mediated atherosclerosis progression 68, 69. Infection with P. gingivalis had a greater effect on plaque accumulation in ApoE‐/TLR4‐deficient mice compared with mice deficient in ApoE alone. 68. These results were initially surprising due to other reports of a detrimental role of TLR4 signalling in atherosclerosis‐prone mice either infected with C. pneumoniae or fed a high‐fat diet 70. It is important to note that the animals were fed a high‐fat diet in the studies with C. pneumoniae, whereas in our studies the animals received a normal chow diet. This makes it difficult to delineate the role of the pathogen versus a high‐fat diet in atherosclerosis. To address this issue, we recently infected ApoE‐deficient mice with either P. gingivalis or C. pneumoniae or fed animals a high‐fat diet (without infection) and assessed the gene expression in the aorta under each condition 71. We found that P. gingivalis infection, C. pneumoniae infection and a high‐fat diet all induced a distinct gene signature in the aorta. For the top 500 differentially expressed genes unique to each group, we observed that P. gingivalis infection resulted in a 76% decrease in expression, including downregulation of the PPAR pathway, which is associated with regression of early atherosclerotic lesions. By contrast, a high‐fat diet increased expression for 96% of the top 500 differentially expressed genes. Notably, C. pneumoniae infection induced an approximately equal increase and decrease in gene expression. These results demonstrate that there is considerable complexity in the ligand‐specific pathways that lead to atherosclerotic disease progression and regression (Fig. 2). Figure 2 Open in figure viewer PowerPoint Porphyromonas gingivalis modifies progression and regression of atherosclerotic plaques. P. gingivalis can stimulate endothelial dysfunction inducing pro‐atherogenic responses within the lesion, resulting in increases in the expression of adhesion molecules (ICAM‐1, VCAM‐1 and E‐Selectin) and the formation of foam cells 88 Porphyromonas gingivalis alters genes responsible for mitochondrial function and downregulates gene expression in the PPAR‐signalling pathway 71 P. gingivalis may prevent the regression of atherosclerotic plaques by interfering with reverse cholesterol transport. PPAR, peroxisome proliferator‐activated receptor; Pg, Porphyromonas gingivalis; LDL, low‐density lipoprotein; oxLDL, oxidized low‐density lipoprotein. Putative mechanisms by which the periodontal pathogenmodifies progression and regression of atherosclerotic plaques.can stimulate endothelial dysfunction inducing pro‐atherogenic responses within the lesion, resulting in increases in the expression of adhesion molecules (ICAM‐1, VCAM‐1 and E‐Selectin) and the formation of foam cells. It has been demonstrated thatalters genes responsible for mitochondrial function and downregulates gene expression in the PPAR‐signalling pathway, which may lead to mitochondrial dysfunction and metabolic imbalance to promote the development of atherosclerosis.may prevent the regression of atherosclerotic plaques by interfering with reverse cholesterol transport. PPAR, peroxisome proliferator‐activated receptor; Pg,; LDL, low‐density lipoprotein; oxLDL, oxidized low‐density lipoprotein.

Mechanisms of pathogen‐mediated atherosclerosis progression The immune mechanisms underlying the exacerbation of inflammatory plaque progression by periodontal pathogens are currently unknown. We, and others, have proposed a number of possibilities, which include both direct and indirect mechanisms. Below, we explore the potential role of these pathogen‐mediated direct and indirect mechanisms in exacerbating inflammatory atherosclerosis. Direct mechanisms Investigation of direct mechanisms of pathogen‐induced endothelial dysfunction followed on from the numerous studies in which a variety of oral bacterial species were identified at the DNA, RNA or antigen levels in atheromatous tissue 62. First, in 2000, Haraszthy et al. 31 reported the detection of genomic DNA of Aggregatibacter actinomycetemcomitans, P. gingivalis and other species utilizing bacterial 16S rRNA PCR analysis. Recently, the results of 16 studies investigating the presence of oral bacteria in atheromatous plaque were reviewed, and A. actinomycetemcomitans and P. gingivalis were identified as the most frequently occurring bacteria 5. The presence of bacterial components, either DNA, RNA or antigens, does not distinguish between live and dead bacteria 62. Therefore, obtaining viable microorganisms from human atheromas would be of value, but unfortunately acquiring viable cultures has proven to be difficult 72, 73. To our knowledge, only one study has demonstrated the viability of both A. actinomycetemcomitans and P. gingivalis from human atherosclerotic plaque 34; this was achieved by culturing atheromatous tissue with human coronary artery endothelial cells. More recently, in 2011, Rafferty et al. 74 reported that viable P. gingivalis could be isolated from atheroma samples following in vitro incubation with macrophages. Although numerous studies have demonstrated the presence of DNA from periodontal pathogens in atherosclerotic lesions, it remains unknown how these organisms and/or their ligands reach systemic sites. It is known that periodontal pathogens enter the circulation following physical perturbation of the gingiva, for example after routine dental procedures or everyday oral activities such as tooth brushing 62, 75, 76. It has been proposed that patients with periodontal disease most likely have higher levels of bacteremia from daily oral activities 62. A comprehensive search of the literature provided a list of 275 bacterial species that have been identified in blood cultures following routine daily events or dental procedures 77, 78, supporting the concept that the gingival sulcus is the main source of and portal to the blood stream for oral bacterial species detected in the blood 4. It is this common and recurrent transient bacteremia that has been proposed to produce chronic insult to the vasculature and contribute to the injury and inflammation that initiates the development of atherosclerosis 56 (Fig. 3). Figure 3 Open in figure viewer PowerPoint Local and systemic effects mediated by periodontal pathogens. SRs, scavenger receptors; CAMs, cell adhesion molecules. One recently proposed mechanism to explain how periodontal pathogens remain undetected in the bloodstream in order to reach systemic sites is that they may enter and survive within host immune cells such as dendritic cells and macrophages 62. It is thought that survival and replication within immune cells represents an important step in the early stages of infection in order to evade the immune response and enable bacterial dissemination 79, 80. In the case of P. gingivalis, this pathogen has been shown to gain entry into dendritic cells and to disrupt their phagocytic function 81. Furthermore, we and others have reported P. gingivalis survival within macrophages 69, 82, 83. P. gingivalis has been detected in circulating dendritic cells in patients with chronic periodontal disease and severe cardiovascular disease 84, suggesting a role for blood myeloid dendritic cells in harbouring and disseminating pathogens from the oral mucosa to atherosclerotic plaque (Table 1). Although studies to date point to survival within host cells as a plausible mechanism of dissemination for pathogens to atherosclerotic lesions, this theory remains unproven. Table 1. Recent clinical studies linking periodontitis/periodontal pathogens and cardiovascular disease References Number of participants Sample types Method of analysis Conclusions Mahendra et al. (2015) 51 cardiac subjects/51 non‐cardiac control subjects Subgingival plaque (all subjects)/coronary atherosclerotic plaques (cardiac subjects only) PCR identification of 8 periodontal pathogens Cardiac subjects had a higher number of periodontal bacteria in subgingival plaques, and this number was significantly associated with the number of bacteria in atherosclerotic plaques Ramírez et al. (2014) 22 cases (chronic, moderate to severe periodontitis)/22 controls (gingivitis and incipient periodontitis) Serum Multiplexed immunocytometric assay for detection of eight biomarkers of cardiovascular disease Significantly higher plasma levels of E‐selectin and myeloperoxidase in cases compared to controls Armingohar et al. (2014) 40 subjects with vascular diseases: 30 with chronic periodontitis (CP)/10 without CP Vascular biopsies from subjects with vascular diseases (i.e. abdominal aortic aneurysms, atherosclerotic carotid, and common femoral arteries) V3–V5 region of the 16S rDNA sequencing; species‐specific primers for detection of P. gingivalis Higher bacterial loads and diversity in biopsies from patients with CP P. gingivalis detected in 1 biopsy Koren et al. (2011) 15 subjects with atherosclerosis/15 healthy controls Atherosclerotic plaques (CVD subjects only) oral swab of peridontium; feces (all subjects) 16S rRNA pyrosequencing Plaque, oral, and gut microbiota share many phylotypes, and the abundance of specific bacteria correlated with disease biomarkers Carrion et al. (2012) 40 subjects with acute coronary syndrome (ACS) and moderate to severe chronic periodontitis (CP)/25 healthy controls Blood myeloid dendritic cells (blood mDCs) FACS analysis 16S rDNA sequencing immunofluorescence mDC numbers: healthy controls < CP < ACS/CP Blood mDCs contain P. gingivalis 16S rDNA and intact P. gingivalis within DC‐SIGN+ blood mDCs Figuero et al. (2011) 42 (undergoing carotid artery endarterectomy) Atheromatous plaque from carotid artery endarterectomy PCR identification of 6 periodontitis‐associated bacteria DNA from ≥1 (of 6) bacteria detected in all 42 samples Disruption of endothelial cell function is one of the earliest indicators of cardiovascular disease 62 and there have been numerous studies of the role of infections in endothelial dysfunction 85, 86. Similarly, release into the circulation of bacterial products, such as outer membrane vesicles, gingipains from P. gingivalis or free soluble bacterial components of A. actinomycetemcomitans, can induce pro‐atherogenic responses in endothelial cells 62. Many of the studies of the role of periodontal pathogens in inducing endothelial dysfunction have been performed with P. gingivalis. This pathogen can adhere to and invade various human vascular cells and can stimulate monocyte adhesion to human umbilical vein endothelial cells 55, 87. Co‐culture of human aortic endothelial cells with P. gingivalis increases the expression of adhesion molecules ICAM‐1, VCAM‐1 and E‐Selectin. Stimulation of a murine macrophage cell line with P. gingivalis, in the presence of LDL, resulted in the formation of foam cells 88. Indirect mechanisms Oral infection is thought to indirectly induce elevated production of cytokines or acute‐phase proteins that enter the systemic circulation and reach sites distant from the initial infection 89. Recent studies have focused on how disruption in the microbiome itself could influence systemic production of proinflammatory cytokines and influence systemic inflammatory diseases. For example, dysregulation and low diversity of the gut microbiota have been associated with conditions such as obesity and inflammatory bowel disease 90. It is now becoming evident that dysregulation of the microbiome is associated with other systemic inflammatory diseases including diabetes and atherosclerosis 91 (Fig. 4). Koren et al. 92 reported that bacterial profiles (based on 16S rRNA pyrosequencing) of atherosclerotic plaques contained phylotypes common to oral or gut samples from the same individual with atherosclerosis). In addition, they reported that the amount of bacterial DNA in atherosclerotic plaques correlated with inflammatory markers of atherosclerosis (Table 1). To further elucidate the link between the gut microbiome and atherosclerosis, the same group sequenced the gut metagenomes of age‐ and gender‐matched subjects with and without large vulnerable plaques in the carotid arteries. The genus Collinsella was enriched in subjects with plaques, whereas Eubacterium and Roseburia were enriched in subjects without plaques, further supporting the notion that changes in the gut microbiome are associated with ‘symptomatic’ atherosclerosis. Figure 4 Open in figure viewer PowerPoint 90-92 Porphyromonas gingivalis has been shown to alter the gut microbiota (with concurrent increases in gut permeability and production of proinflammatory cytokines) 99, 100 The oral and gut microbiota in health and disease. Dysregulation and low diversity of the gut microbiota have been associated with conditions such as obesity, inflammatory bowel disease, diabetes and atherosclerosis. In mice, oral administration ofhas been shown to alter the gut microbiota (with concurrent increases in gut permeability and production of proinflammatory cytokines). DC, dendritic cell. Anaerobic bacteria, such as those comprising the gut microbiota, utilize a glycyl radical enzyme to convert choline to trimethylamine (TMA), which is further converted to trimethylamine N‐oxide (TMAO) (in humans) by flavin‐dependent monooxygenase 3 (FMO3) 93, 94. L‐carnitine is also converted to TMAO through a microbe–host metabolism interaction. TMAO is associated with several processes that contribute to the progression of atherosclerosis, such as accumulation of cholesterol in macrophages and accumulation of foam cells in atherosclerotic lesions 95. In a clinical study, levels of TMAO that were readily detectable in both plasma and urine of healthy adults after phosphatidylcholine challenge were almost completely suppressed after 1 week of broad‐spectrum antibiotic treatment and restored 1 month after withdrawal of antibiotics 96. Similar results were reported in a parallel study using L‐carnitine challenge 97. Further, 16S rRNA sequencing of faecal samples from the L‐carnitine study participants revealed bacterial taxa associated with (fasting) plasma TMAO levels; specifically, the genus Prevotella was enriched in subjects with plasma TMAO levels that were significantly higher (P < 0.05) than in subjects with profiles characterized by enrichment of the genus Bacteroides 97. In a 3‐year follow‐up study of over 4000 adults undergoing diagnostic cardiac catheterization, elevated (fasting) plasma levels of TMAO were a significant predictor of adverse cardiovascular events after adjustment for risk factors 96. In mouse studies, supplementing a normal chow diet with L‐carnitine was shown to approximately double the plaque burden in the aortic root of ApoE‐deficient mice as compared to mice fed only a normal chow diet, and this increase was inhibited in mice treated with oral antibiotics 97. Gregory, et al. 98 have recently shown that faecal transplantation in mice can transmit atherosclerosis susceptibility. At 20 weeks of age, choline‐fed ApoE‐deficient mice receiving faecal transplantation from an atherosclerosis‐prone donor mouse strain had a significantly higher plaque burden in the aortic root than those receiving faecal transplantation from an atherosclerosis‐resistant donor mouse strain, which showed a similar plaque burden to ApoE‐deficient mice fed a normal chow diet; all ApoE‐deficient mice were treated with antibiotics for 3 weeks prior to the first caecal gavage 98. Thus, it appears that intestinal bacteria interact with the host metabolism to drive disease processes outside the gastrointestinal tract. Recently, oral administration of P. gingivalis has been shown to be associated with alterations of the gut microbiota of C57BL/6 mice. Treatment with P. gingivalis twice a week for 5 weeks resulted in a microbial profile (ileum contents) characterized by an increase in the phylum Bacteroidetes (38.7% vs. 17.0%) and a decrease in the proportion of the phylum Firmicutes (55.4% vs. 72.8%) compared with sham‐infected mice 98. Furthermore, 16S rRNA sequencing of faecal samples revealed the same pattern of dysbiosis after a single oral administration of P. gingivalis 99. These alterations in the microbial profile were not attributed to direct translocation of the administered P. gingivalis. Compared with the sham‐infected group, in mice receiving a single dose of P. gingivalis, mRNA expression of genes related to gut barrier function was decreased and of genes related to proinflammatory cytokines was increased at 48 h postinfection 99, 100. These results indicate that an oral pathogen may contribute to systemic disease progression by causing shifts in the gut microbiota that are followed by increases in intestinal inflammation and gut permeability.

Therapeutic strategies Previous studies have examined the association between periodontal disease and outcomes of coronary heart disease, coronary artery disease, atherosclerotic vascular disease, angina pectoris, carotid calcification, coronary artery calcification, myocardial infarction, acute coronary syndrome and stroke 4. Recent clinical studies have been designed to examine the impact of oral pathogens on cardiovascular disease progression. In a study by Desvarieux et al. 101, changes in periodontal status, assessed both clinically and microbiologically, were associated with progression of carotid atherosclerosis. The authors found that the relative predominance of bacterial species traditionally considered causally related to periodontal disease was most closely linked to atherosclerosis progression. These results provided the first evidence that improvement in periodontal status is related to a decreased progression of carotid atherosclerosis in humans, thereby strengthening the role of pathogens, especially those associated with periodontal disease, in clinical cardiovascular disease. Targeting of inflammatory mediators Overproduction of proinflammatory mediators is known to contribute to inflammatory pathology in a number of chronic diseases 102-104 including atherosclerosis. Although lipid deposition is considered a leading contributor to the inflammation, additional stimuli, such as infectious agents, have been considered as sources for the continuous inflammation 27. We have previously shown that P. gingivalis induces low levels of proinflammatory mediators, particularly IL‐1β, from macrophages in vitro but accelerates chronic inflammatory atherosclerosis in vivo 69. These results would appear paradoxical considering the numerous reports of excessive inflammation leading to atherosclerosis progression. Thus, it remains unclear why a pathogen that induces low levels of inflammatory mediators would also be associated with chronic inflammatory atherosclerosis. IL‐1β is a critical cytokine in host defence against a number of infectious agents 105. Consequently, a number of pathogens have evolved mechanisms to prolong their survival by inhibiting IL‐1β production 106. In agreement with this notion, we observed that low levels of IL‐1β production were associated with P. gingivalis survival. Therefore, although activation of innate immunity is a necessity for clearing infections, there is a fine balance between activation of innate immunity and unresolved inflammation 20, 107. We propose that future therapies designed to inhibit IL‐1β associated with atherosclerosis should be designed with caution due to the protective role of IL‐1β in immunity. Supporting this concept are results from a recent clinical trial targeting IL‐1β for the treatment of the inflammasome‐mediated disease cryopyrin‐associated periodic syndrome (CAPS) 108. Patients receiving the humanized monoclonal antibody canakinumab, specific for neutralizing IL‐1β, had a 67% increased risk of infection compared with 25% for patients in the placebo group 107. Another clinical trial was recently launched to investigate whether IL‐1β inhibition with canakinumab will reduce major cardiovascular events in patients with pre‐existing coronary artery disease (CAD) 109. Based on our previous in vitro work 69 and the results from the CAPS trial, we suggest that the potential benefit of long‐term use of a neutralizing antibody against IL‐1β in humans at high risk of atherosclerotic vascular disease must be substantial enough to counter the increased risk of infection. It is imperative that the complexity of inflammatory pathways leading to chronic inflammation, particularly those that are accelerated by infection, is considered in the development of future therapies. Antagonizing/neutralizing TLR activation Continuous activation of TLR signalling pathways has been shown to be critical in the progression of chronic inflammation 21. Therefore, a number of therapeutic agents proposed for cardiovascular disease have targeted TLRs 110. Previously, it was proposed that common mechanisms of signalling via TLR2, TLR4 and MyD88 link stimulation by multiple pathogens and endogenous ligands to atherosclerosis progression 111. In support of this proposal, it was demonstrated that activation of TLR4 by Chlamydia infection and lipids exacerbated atherosclerosis 70. Therefore, it would appear that therapeutic antagonism would be beneficial for the treatment of chronic atherosclerosis 112. However, we revealed that antagonism of TLR4 by P. gingivalis exacerbates atherosclerosis 69. These results suggest that therapies that have been designed to specifically antagonize TLR4 for the treatment of atherosclerosis may not be successful. Although TLRs have been commonly associated with detrimental effects in atherosclerosis, we now know the crucial protective role TLR signalling plays in clearing infection 113. We propose that future therapies to block TLR activation should be designed with caution due to the complexities in inflammatory pathways leading to atherosclerosis and in ligand‐specific pathways leading to disease progression.

Future directions Unresolved inflammation is undoubtedly a contributing factor in progressive inflammatory diseases such as atherosclerosis. However, due to the predominant role of inflammation in clearing infections, there is a fine balance between activation of immunity and unresolved inflammation that will need to be considered for the development of future therapies for atherosclerosis. It is clear that the inflammatory pathways that lead to atherosclerosis progression are complex and are likely to be ligand specific. Antagonizing/neutralizing TLR activation has long been considered effective for reducing the inflammation associated with plaque progression. However, due to the role of TLRs in clearing infections, we propose that future therapies to block TLR activation need to be designed with caution. Furthermore, neutralizing IL‐1β has been investigated as a potential therapy for atherosclerosis. However, due to the known role of IL‐1β in clearing infection, we propose that therapies to inhibit IL‐1β should also be carefully designed. It is imperative that these issues are considered when designing future therapeutic agents for the treatment of chronic inflammatory disorders, specifically those exacerbated by infection. Guo et al. 114 recently reported the use of an antimicrobial peptide to selectively kill a specific species of oral bacteria, providing insight into the role of this bacterium within the complex community of the oral microbiota. There is great potential for expanding this approach to other oral bacteria, such as P. gingivalis, to elucidate their roles in modulating the oral microbiome and to identify potential therapeutic targets 115. There is evidence that supports the association between the gut microbiome and systemic diseases. What role the oral microbiome might play in systemic disease progression, however, remains unknown. Recent studies with P. gingivalis in mice have begun to shed light on this issue. For example, P. gingivalis has been shown to alter the gut microbiome, resulting in a concurrent increase in gut permeability and proinflammatory cytokines 99, 100. These results highlight our need for further studies on the impact of oral bacteria on systemic disease through alterations of the gut microbiota.

Conflict of interest statement No conflicts of interest to declare.