The gut microbiota interacts with dietary components74, and data obtained in the past 5 years suggest that microbial metabolites can promote or prevent CVD (Fig. 2).

Atherosclerosis-promoting diet. A large proportion of humans have switched from a healthy diet to a Western diet rich in cholesterols, fats, and sugars. Several components of the Western diet can promote inflammation75, and changes in dietary pattern have been linked to metabolic diseases and atherogenesis54. Moreover, saturated fats in the Western diet increase endotoxin activity in plasma compared with a healthy diet, which could be attributed to a change in microbiota composition and/or increased translocation of microorganisms across the intestinal barrier52,76,77. In Ldlr−/− mice, Western diet induces changes in circulating metabolites and inflammatory markers, indicating an altered cholesterol homeostasis and oxidative stress, which could be partly reversed by switching the mice to a fibre-rich chow diet78. Circulating LPS levels and aortic lesion sizes are also partly reduced through a microbial-dependent mechanism78,79, in agreement with the reduced TLR4-dependent proinflammatory signalling discussed above. By contrast, germ-free Apoe−/− mice fed a low-cholesterol diet had increased aortic lesion sizes compared with conventionally raised mice, whereas no difference was found in mice fed a high-cholesterol diet80. Accordingly, atherosclerosis development in germ-free Apoe−/− mice fed a Western diet was similar to that of conventionally raised mice81. Thus, a diet-dependent microbial mechanism might be involved in the development of atherosclerosis but, on the basis of the data discussed above, the microbiota does not have a causal role in atherosclerosis. However, it cannot be excluded that microbiota induces atherosclerosis and CVD indirectly by causing chronic metabolic dysfunction or, as discussed below, by producing proatherogenic molecules from dietary precursors82,83.

TMAO. Dietary components are also involved in the atherogenic process. One such example is the proatherogenic metabolite trimethylamine N-oxide (TMAO) generated from microbial metabolism of phosphatidylcholine, which is common in red meat, shellfish, and eggs (Fig. 2). Phosphatidylcholine is metabolized by the gut microbiota to generate trimethylamine (TMA), which is further processed in the liver by flavin monooxygenases, leading to the production of TMAO. Plasma levels of phosphatidylcholine-related metabolites and other TMAO-producing molecules, including l-carnitine and γ-butyrobetaine, have been associated with an increased risk of CVD84,85,86,87. Accordingly, increased levels of TMAO have been correlated with an increased risk of cardiovascular events88, prevalent CVD85,89,90, poor prognosis91, and increased risk of death92. Moreover, TMAO levels are higher in patients with chronic heart failure than in healthy controls93.

A study in mice published in 2015 revealed that aortic lesion size positively correlated with plasma TMAO but inversely correlated with choline, and that the combined variations of plasma metabolites accounted for up to 40% of the variation in lesion size94. Apoe−/− mice treated with phosphatidylcholine metabolites resulted in increased aortic lesions, which was dependent on the gut microbiota84. Microbial capacity to produce TMA seems to be important for atherosclerosis development; indeed, the aortic lesions of Apoe−/− mice transplanted with microbiota from high-TMAO-producing mouse strains (C57BL/6J) are increased compared with those of Apoe−/− mice transplanted with microbiota from low-TMAO-producing strains (NZW/LacJ)95. Human intestinal isolates have been screened to identify bacterial strains that can produce large amounts of TMAO96. Nine bacterial strains with TMA-producing properties in vitro led to accumulated plasma TMAO levels when transplanted into germ-free mice96. Taken together, these experimental studies in mice support the potential harmful effect of TMAO on the cardiovascular system.

To date, the mechanisms by which TMAO contributes to atherosclerosis are only partially understood and might involve multiple pathways. In choline-fed Apoe−/− mice, the number of macrophages in aortic lesions as well as foam cell formation are reduced when TMA production is suppressed by antibiotic treatment84. By contrast, different concentrations of TMAO did not affect foam cell formation in mouse macrophages in vitro97. Therefore, the role of TMAO in foam cell formation needs to be clarified.

TMAO can also contribute to atherosclerosis by inhibiting reverse cholesterol transport. However, the mechanisms underlying such an effect are unclear, and might not involve changes in the expression of cholesterol transporters in macrophages85. TMAO was also shown to induce several atherosclerosis-promoting inflammatory proteins in vascular cells, including prostaglandin G/H synthase 2 (also known as cyclooxygenase 2), IL-6, E-selectin, and intracellular adhesion molecule 1 through activation of nuclear factor-κB98. In addition to atherosclerosis, TMAO might also affect thrombosis as increased levels of TMAO are associated with higher rates of thrombotic events and platelet activation99.

Sex might be an important factor to consider when studying TMAO synthesis, because expression of dimethylaniline monooxygenase [N-oxide-forming] 3 (also known as flavin monooxygenase 3; FMO3) is increased in human and mouse females compared with males100. In agreement with these data, germ-free female mice colonized with TMA-producing bacterial strains had host-related and sex-related higher levels of plasma TMAO and increased activity of hepatic FMO3 compared with male mice96. Knock down of Fmo3 in different mouse strains results in decreased TMAO plasma levels, altered lipid and cholesterol metabolism, and reduced atherosclerotic lesion areas101,102,103. FMO3 is regulated by FXR, and injection of FXR ligands in mice induces the expression of FMO3 and TMAO100, thus potentially explaining the reduced atherosclerosis in Fxr-deficient Apoe−/− mice65,66,67. Furthermore, TMAO decreases the total pool of bile acid in chow-fed mice85. Taken together, these data indicate that FMO3 is differentially regulated by sex, is a target of FXR, and is important for lipid and cholesterol metabolism.

Pharmacological elimination or reduction of plasma TMAO is a potential therapeutic approach to reduce atherosclerotic cardiovascular events. However, the resulting accumulation of TMA might induce trimethylaminuria, also known as fish odour syndrome104. Thus, it would be preferable to target the microbiota and TMA synthesis directly. Inhibition of choline trimethylamine-lyase, the microbial enzyme that converts choline to TMA, was demonstrated to reduce TMAO plasma levels and aortic lesions in mice105, but data in humans are lacking. Similarly, the antiatherosclerotic molecule resveratrol increases the abundance of bifidobacteria and lactobacilli in Apoe−/− mice. This increase is associated with reduced TMAO plasma levels through inhibition of microbial TMA production, but the mechanisms are unclear106.

Although a number of studies have pointed to an association between plasma levels of choline, TMAO, and CVD88,89,90,91,92,93, available data are still inconsistent. After bariatric surgery, increased plasma levels of TMAO are observed despite an improved metabolic profile, which could be explained by increased intestinal abundance of Pseudomonas107. Furthermore, Apoe−/− mice expressing human cholesteryl ester transfer protein, which is essential for reverse cholesterol transport, were used to investigate the role of l-carnitine, which can also be metabolized to TMAO and promote atherosclerosis85. Mice fed a Western diet supplemented with l-carnitine had increased TMAO plasma levels and decreased aortic lesion sizes, with no changes in plasma lipid profile97, thus suggesting a protective role for l-carnitine and TMAO in atherosclerosis. Why l-carnitine feeding gave rise to opposite phenotypes in two different laboratories is currently unclear, although these studies used different animal models (mice fed a chow diet85 or a Western diet97).

Dietary prevention of atherosclerosis. Diet modulation with specific dietary components might also be used to prevent atherosclerosis. However, most studies have provided only association data, and mechanistic evidence is lacking. Dietary fibre is thought to provide health benefits as fibre is considered to lower the risk of CVD. In humans, this effect could be attributed to a decreased level of plasma LDL108. Moreover, in healthy people, an inverse correlation exists between dietary fibre and CVD mortality109. However, a limited number of human studies have been performed in this field, and the effects of dietary fibre might be indirectly related to CVD through the regulation of glucose metabolism.

Prebiotics are plant-derived fibres that improve the composition or function of the gut microbiota. Indeed, Apoe−/− mice fed inulin, a prebiotic polysaccharide, have reduced cholesterol levels and atherosclerotic lesions compared with control mice110. Probiotics are live bacteria with health benefits. Lactobacilli are common probiotics that have been evaluated in CVD animal models with various outcomes, probably owing to strain-specific effects. For example, consumption of a specific strain of Lactobacillus plantarum had beneficial effects in a small number of patients, including increased intestinal bacterial diversity111, and decreased inflammatory risk factors for atherosclerosis112. Furthermore, although some studies showed that lactobacilli reduce blood cholesterol levels in mice and humans113,114,115, three different strains of Lactobacillus reuteri were shown to have no effect on cholesterol levels or atherosclerotic lesions in Apoe−/− mice116,117.

An example of a bacterium with beneficial effects on atherosclerosis is Akkermansia muciniphila, which is part of the normal microbiota and is important in protecting the intestinal barrier in diet-induced obesity118,119,120. Apoe−/− mice fed a Western diet supplemented with A. muciniphila have reduced atherosclerotic lesions, an effect that could be attributed to an improved intestinal barrier and inflammatory profile121. To summarize, data from mouse studies are conflicting, possibly because of different bacterial strains used. Furthermore, different dietary sources might explain the discrepancies between various studies. Large studies in patients with atherosclerosis are needed to determine any potential beneficial effect of probiotics. The modification of bacterial composition through specific dietary components, or regulation of microbial metabolite formation, might have long-term effects on the development of atherosclerosis, whereas short-term treatments might not be clinically effective.