The Gut Microbial Endocrine Organ: Bacterially-Derived Signals Driving Cardiometabolic Diseases

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of death in most developed countries, and despite the widespread use of statin drugs one in six deaths in the United States is still attributable to CVD (1–3). Given this unmet need for effective therapies, there is increasing interest in targeting novel pathways that underlie CVD pathogenesis. Recent genome-wide association studies have provided clear evidence that genetic mutations can predispose to CVD development (4–6), and these studies have identified several new attractive therapeutic targets. However, genetic variation only accounts for less than 20% of CVD risk (7,8). Clearly, environmental factors play a predominant role in the development of CVD, and understanding how environmental cues drive CVD pathogenesis will be central in drug discovery moving forward. Although many environmental factors converge to promote CVD, here we focus on how diverse components within our diet (macronutrients, micronutrients, symbionts, pathogens, etc.) participate in meta-organismal (microbe to host) signaling pathways to promote CVD risk. Until recently, dietary constituents were assumed to be simply absorbed or metabolized by our human cells primarily for energy needs and general cell health. However, we now understand that microbial communities resident in the human gastrointestinal tract play major roles not only in allowing us to efficiently harvest energy from our food (9), but also serve as a key endocrine organ secreting metabolites that act as hormone-like factors that are sensed by dedicated receptor systems in the human host. Gut microbes can also signal to the host to regulate innate immunity through metabolism-independent pathways, where constituents of the microbial cell wall are sensed by host cells through pattern recognition receptors (PRR) to further impact CVD progression. Collectively, through both nutrient metabolism-dependent and metabolism-independent mechanisms, the gut microbiome forms a largely overlooked plastic endocrine organ that integrates input cues from the diet and interfaces with host to play a role in the pathogenesis of CVD and metabolic disorders (Figure 1). Considering the central role the gut microbial endocrine organ plays in human disease, it remains possible that targeting gut microbes may be a provocative new way to move forward in CVD drug discovery.

The concept of the meta-organism was first proposed by an insightful German zoologist Karl Möbius, when he discovered that the health of European oyster populations was dependent on the presence of other species in the surrounding ecosystem (10). This meta-organismal theory can also be applied to human health and disease, where it is increasingly accepted that gut microbial communities act as an underappreciated endocrine organ, bioactivating vitamins and producing other factors from dietary nutrients that can impact normal physiologic processes, educate mucosal immune systems and/or induce pathologic responses (11). Within the last decade, there have been numerous reports linking meta-organismal pathways to human diseases including obesity (9,11), diabetes (12,13), non-alcoholic fatty liver disease (14), osteoporosis (15), cancer (16–18), and innate/adaptive immune responses (19,20). However, the purpose of this review is to highlight recent discoveries linking the gut microbial endocrine organ to CVD risk (21–28). Here we discuss both the current state of knowledge surrounding how meta-organismal pathways involving gut microbial products are linked to CVD risk, and key opportunities and challenges that arise when considering microbe-host interactions as future drug discovery targets or interventions.

THE GUT MICROBIAL ENDOCRINE ORGAN

The human body consists of much more than just human cells. In fact, it has been estimated that greater than 100 trillion (10¹⁴) microbial cells reside in different compartments within the human body, vastly outnumbering human cells (29,30). Amazingly, it has been estimated that less than 10% of DNA found in the human meta-organism derives from Homo sapien origin (30). Fortuitously, the majority of microbes present do not cause harm to the host, and in some cases provide benefit through symbiotic relationships. However, recent evidence suggests gut microbial-driven pathways may actually be causally-linked to several chronic diseases in humans (9–28). Collectively, these realizations have prompted large-scale projects to define microbe-host interaction such as the United States Human Microbiome Project (HMP) and the European Metagenomics of the Human Intestinal Tract (MetaHIT) (31,32). These efforts have provided important clues into the taxonomic diversity of resident microbes in the human gut. It is now well established through such metagenomic sequencing efforts that the human gut possesses a core bacterial microbiome that is predominated by phyla such as Bacteroidetes and Firmicutes (31,32). However lower abundance phyla such as Proteobacteria, Actinobacteria, and Verrucomicrobia are commonly present in the human gut (31,32). It is key to note that the human gut microbiome, while resilient, is also highly dynamic, and can be dramatically altered by antibiotic use, as well as less rapidly impacted by age, host genetics, chronic dietary patterns, and other environmental exposures (33–39). It should be recognized that there also exist virtually unexplored human virome, fungal microbiome (“mycobiome”), and even more expansive “bacteriophagome”, predators of the bacterial microbiome (11,29,30). The types of microbes present and the dynamic nature of these symbionts in the human gut are the topic of several excellent recent reviews (11,39), so this will not be expanded upon here.

The definition of an endocrine organ is as follows: “producing secretions that are distributed in the body by way of the bloodstream or lymph.” Although the field of endocrinology has historically focused on host organ systems with endocrine properties (hypothalamus, pituitary gland, pineal gland, thyroid gland, pancreas, adipose, etc.), the gut microbiome also fits this classic definition forming a pseudo-organ with unparalleled endocrine signaling potential. Unlike host endocrine organs, which produce only a few key hormones, the gut microbial endocrine organ has the unique potential to produce hundreds if not thousands of humoral agents generated either through metabolism-dependent or metabolism-independent pathways (Figure 1). Much like hormones derived from human endocrine organs, bacterially-derived hormones are sensed by highly selective host receptor systems that elicit diverse biological responses (Figure 2). Although here we focus on the bacterial-derived products relevant to CVD, a large number of hormone-like compounds have already been described emanating from the gut microbial endocrine organ (21–28,40–56). The current list of bacterially-derived hormones include trimethylamine/trimethylamine-N-oxide (21–28), short chain fatty acids (SCFA) (40–46), secondary bile acids (47–53), polysaccharide A (54), and 4-ethyl phenyl sulfate (55), and catecholamines (56). However, this list only encompasses research within the last decade, and there will likely be an explosion of novel bacterially-derived hormones discovered now that we appreciate the important of this endocrine factory. It is also important to note that gut microbial products have also been shown to functionally interact with the host endocrine system to indirectly alter classic hormonal responses to cortisol (57), ghrelin (58), leptin (59), glucagon-like peptide 1 (60), and peptide YY (61). In fact, there is now a growing appreciation for gut microbiota-driven alteration of neurotransmission in the brain, supporting the idea that hormone-like compounds from the gut can act at distant sites in the central nervous system (62). Collectively, this positions the gut microbial endocrine organ as a central player in both production of its own hormones (21–28,40–56), as well as establishing the signaling tone of host hormones (57–61) It also highlights the importance of the gut microbial filter as a transducer of signals from our major environmental exposure, what we eat. Clearly, gut microbes form a largely neglected endocrine organ with important relevance to human disease.

THE GUT MICROBIAL ENDOCRINE ORGAN AS A BASIS OF CARIOVASCULAR DISEASE: THE TRIMETHYLAMINE-N-OXIDE (TMAO) STORY

With particular relevance to human CVD risk, we have recently described a meta-organismal metabolic pathway that involves multiple interactions between the gut microbial endocrine organ and host metabolic and signaling machinery (21–28). Initially, we used a metabolomic approach to unbiasedly identify small molecule metabolites associated with CVD risk in human plasma (21). Using sequential independent case-control studies as learning, replication, and then large scale validation investigations, we discovered that three metabolites (choline, TMAO, and betaine) of the dietary lipid phosphatidylcholine (PC) were highly predictive of CVD risk (21). In follow up studies, we have shown that feeding atherosclerosis prone mice diets enriched in either choline or TMAO enhances atherosclerosis development and alterations in cholesterol and sterol metabolism (21,22). Importantly, the enhanced atherosclerosis seen with dietary choline supplementation is entirely dependent on gut microbiota, given that antibiotic treatment or germ free conditions abolished dietary choline-driven TMAO generation and atherosclerosis development (21). These original studies uncovered a novel meta-organismal metabolic pathway linking dietary PC intake to CVD risk. This novel pathway involves gut microbiota-dependent metabolism of dietary PC to generate the gas trimethylamine (TMA), which is subsequently metabolized by enzymes of the flavin monooxygenase (FMO) family in the host’s liver to generate the circulating proatherogenic compound TMAO (21,22,25).

Subsequent studies have revealed that other nutrients can feed into this meta-organismal pathway producing TMA and TMAO, broadening its relevance to diet-driven alterations in CVD risk. L-carnitine, a particularly abundant nutrient in red meat, contains a trimethylamine structure very similar to choline. Much like dietary choline (21), the quaternary amine structure of L-carnitine is readily converted to TMA by gut microbiota (22). In fact, bacterial metabolism of L-carnitine can provide an abundant source of TMA that ultimately produces proatherogenic TMAO in both mice and man (22). In agreement, feeding L-carnitine to hyperlipidemic mice alters gut microbe composition, increases blood TMAO levels, and promotes atherosclerosis in a gut microbiota-dependent fashion (22). Importantly, plasma L-carnitine levels predict increased risks for both prevalent CVD and incident major adverse cardiac events (myocardial infarction, stroke, or death) in a large clinical cohort (n=2,595), but only in subjects where TMAO levels were also elevated (22). Moreover, comparisons of omnivores (who have frequent dietary L-carnitine exposure) versus vegans or vegetarians revealed striking differences in the capacity to convert dietary L-carnitine into TMA and TMAO, with vegans/vegetarians showing virtually no TMA/TMAO formation (22). Collectively, our results suggest that chronic exposure to dietary L-carnitine promotes the production of the proatherogenic compound TMAO both in mice and man. Further, they may in part explain why high red meat consumption has been associated with increased CVD and mortality risks (63). These studies also have potentially important implications for not only general dietary recommendations, but the dietary supplement industry, given that supplementation of L-carnitine is so pervasive, particularly in many energy drinks. The safety of chronic dietary exposure to L-carnitine in otherwise healthy subjects ingesting it via red meat, supplements or energy drinks, and whether this is unknowingly fostering the generation of TMAO and advances in long term CVD risk remains an important area of future research.

Although plasma level of several metabolites (choline, betaine, trimethylamine, L-carnitine, TMAO) relevant to this meta-organismal pathway are associated with increased CVD risk, subsequent analyses reveal that the prognostic value is largely confined to TMAO (22–24). Recently, we demonstrated that elevated TMAO levels associate with increased risk of incident major adverse cardiovascular events in a large independent clinical cohort (n=4007) (23). Strikingly, people in the highest quartile of circulating TMAO levels had a 2.5-fold increased risk of having a major adverse cardiac event, when compared to those in the lowest quartile (23). Within the cohort examined, the hazard ratio for TMAO was much higher than traditional risk factors such as LDL-cholesterol. Further, elevated TMAO levels retained strong prognostic value for predicting incident adverse cardiovascular events even after adjustment for traditional risk factors and renal function (24). In addition to the clear link between TMAO and atherosclerotic CVD risk (21–25), TMAO levels have been also been more recently linked to heart failure and adverse prognosis among heart failure subjects (26), as well as high fat diet induced obesity and insulin resistance in a rodent model (64). Given this clear link between circulating TMAO levels and human disease, we have recently developed a quantitative analytical assay for measuring TMAO levels (28), which will be useful for validation studies in additional cohorts. Collectively, these new data suggest that the meta-organismal pathway responsible for the production of TMAO is an important new determinant for increased CVD risk in humans.

Given that blood levels of TMAO are linked to CVD risk in man, the next obvious question is whether circulating TMAO is simply a biomarker of disease or whether TMAO is mechanistically involved in CVD pathogenesis. Current evidence suggests a causative link, given that dietary supplementation of TMAO in hyperlipidemic mice promotes atherosclerosis in a gut microbiota-dependent manner (21,22). However, the mechanism(s) by which circulating TMAO promotes CVD is currently unclear and under active investigation. TMAO has historically been thought of as a key osmolyte, as well as to serve as a small molecule chaperone that stabilizes proteins under denaturing conditions such as high urea or elevated water pressure (65,66). In the broader context of cardiovascular physiology, we have recently shown that TMAO can impact distinct steps in cholesterol and sterol metabolism and macrophage foam cell formation (21,22). Dietary choline or TMAO supplementation results in increased expression of scavenger receptors (CD36 and SR-A1) on macrophages and subsequently promotes foam cell formation (21). In addition, TMAO feeding reduces macrophage reverse cholesterol transport (22), which would be predicted to advance atherosclerosis. Although, TMAO feeding clearly impacts multiple steps of both forward and reverse cholesterol transport (21,22), the underlying molecular mechanisms behind these observations remains unclear. There are many key unresolved questions, such as how circulating TMAO levels are sensed to elicit pathological responses, and additional research is needed to elucidate mechanisms by which TMAO promotes CVD. It is tempting to speculate that circulating TMAO is sensed by a yet to be identified cell surface receptor on target cells (Figure 2). This idea is supported by the fact that structurally similar compound trimethylamine (TMA) signals through a G protein-coupled receptor known as trace amine-associated receptor 5 (Taar5) to both shape reproductive physiology, and as an olfactory receptor (67,68). Identification of a bone fide TMAO receptor system would be a major advance and provide an attractive therapeutic target for atherosclerotic CVD.

OTHER EXAMPLES OF MICROBIAL ENDOCRINOLOGY IN CARDIOVASCUALAR DISEASE

TMAO is one of many bacterially-derived products that have hormone-like properties in the host. Alternative gut microbiota-derived metabolites with hormone-like properties are short chain fatty acids (SCFAs), including acetic acid, butyric acid, propionic acid, and valeric acid (37–43) (Figures 1 & 2). It is well established that a subset of anaerobic bacterial found in the cecum and proximal colon produce SCFAs via fermentation of non-digestable carbohydrates (40). Although the human genome does not encode enzymes capable of breaking down many common forms of complex carbohydrates, anaerobic bacteria serve as a dietary filter to ferment several classes of carbohydrates including pectins, gums, hemicelluloses, and galactose-oligosaccharides to produce key metabolites that are then subsequently metabolized by the host or alternatively act as hormones (40). One well documented role for bacterially-derived SCFAs is to provide a key fuel source for the colonic epithelium, as well as regulating intestinal immune homeostasis (40–42). In particular, gut microbial production of butyrate interacts with host receptors on leukocytes and endothelial cells in the intestine to balance Th1 and Th2 immune responses (42). In addition, Gut microbial-derived SCFAs have potent effects on insulin action in peripheral tissues (43,44). Most recently, gut microbe-generated SCFAs have been found in act distally in the central nervous system to regulate integrated metabolic responses (45). Serving as a prime example of meta-organismal endocrinology, gut microbiota-derived SCFAs are sensed by dedicated G protein-coupled receptors (GPR41 and GPR43) that reside on diverse host cell populations in peripheral tissues (43–46) (Figure 2). Collectively, by regulating energy metabolism, insulin sensitivity, and immune cell programs and responses, SCFAs act as key meta-organismal hormones regulating physiology relevant to numerous processes involved in CVD.

Another elegant example of meta-organismal endocrinology with implications in CVD involves a stepwise interaction of host synthesis, bacterial modification, and subsequent host sensing of bacterially-modified bile acids (47–53) (Figures 1 & 2). Bile acids have long been known to be important in solubilizing dietary fat and cholesterol for absorption into the body (53), but within the last decade bile acids have been recognized as hormones, regulating many physiological processes such as energy expenditure, insulin sensitivity, and cholesterol balance (47–53). This meta-organismal pathway is initiated when host enzymes convert cholesterol to primary bile acids, which is a process that is highly regulated by classic feedback regulation (53). Once primary bile acids are synthesized in the host liver, they are secreted into bile, enter into the lumen of small intestine, and traverse through the intestine until they reach the terminal ileum where they are almost completely recovered (>95%) by selective ileal bile acid transporters (53). The small amount of bile acids that are not reabsorbed then encounter a subset of facultative and anaerobic bacteria resident in the large bowel, where they undergo complex deconjugation and hydroxyl group oxidation modifications generating microbe-dependent secondary bile acids (53). Importantly, a small amount of these microbiota-derived secondary bile acids are released into the blood stream, typically in the postprandial state, where they can act as hormones to signal to the host (47–53). There are two major host receptor systems that sense bacterially-derived secondary bile acids (Figure 2). The most well characterized bile acid receptor is a nuclear hormone receptor known as farnesoid X receptor (FXR), which can be activated to elicit transcriptional responses involved in feedback regulation and intestinal bile acid transport (50–52). More recently, a G protein-coupled receptor called TGR5 has been shown to sense plasma bile acid levels to regulate multiple steps in energy balance and insulin sensitivity (47–49). Collectively, microbe-host cross talk in bile acid metabolism and signaling represents an important endocrine axis regulating cardiometabolic pathways relevant to host physiology and CVD (47–53).

NON-ENDOCRINE PROPERTIES OF GUT MICROBES IMPACTING CARDIOVASCULAR DISEASE RISK

In addition to the ability of bacterially derived metabolites to act as hormones modulating CVD risk (21–28,40–56), components of the bacterial cell wall (lipopolysaccharide and peptidoglycan) can also be recognized by the host’s innate immune system to potentiate CVD pathogenesis (Figure 1). Microbe-associated molecular patterns (MAMPs) such as lipopolysaccharide and peptidoglycan are selectively recognized by host toll-like receptors (TLRs) and nucleotide oligomerization domain-containing receptors (NODs), respectively (69,70) (Figure 2). Although it was long thought that this microbial interaction with the innate immune system was most active in the distal gut (71,72), more recently it has begun to be appreciated that low levels of bacteria can actually make it into the bloodstream to cause chronic low grade inflammation systemically (73). The concept that low levels of gut-derived bacteria can appear in the circulation is commonly referred to as “metabolic endotoxemia” because it has been found to be prevalent in many chronic metabolic diseases such as obesity, type II diabetes, and atherosclerosis (73). Although this type of microbe-host signaling cannot be classified as endocrine in nature, it has clear potential to alter CVD risk. In fact, human mutations and mouse knockout studies demonstrate a key role for Toll-like receptor 4 (TLR4) or nucleotide oligomerization domain 2 (NOD2) activation in atherosclerosis development (69–74). Furthermore, microbial activation of these innate immune receptors promotes inflammation that dampens reverse cholesterol transport, while augmenting insulin resistance, hyperlipidemia, and vascular inflammation (76,77) (Figure 1). Collectively, metabolic endotoxemia and engagement of peripheral pattern recognition receptors plays a major role in the pathogenesis of CVD by reorganizing lipid metabolism and promoting inflammatory responses (76,77).

MOVING FORWARD FROM HERE: A META-ORGANISMAL VIEW OF CARDIOVASCULAR MEDICINE

Although rapidly expanding, the field of meta-organismal endocrinology is still in its infancy. Up to this point, the field has been dominated by metagenomic profiling approaches to correlate the types of bacterial species present with disease phenotypes (29–39). However, simply cataloging the types of microbes in different disease models is quite limited, given that it only allows for correlation, and does not establish causal links. As the field progresses toward new experimental approaches there are several key considerations that will need to be taken into account. First, it is quite clear that gut microbes impact host physiology in a predominant way by generating metabolic intermediates from dietary substrates (21–28,40–56). Therefore, it is imperative that we continue to focus our efforts on identifying bacterially-derived metabolites that have relevance to human disease. Given major advances in the field of metabolomics over the last decade (78), we are now well positioned to identify the entire microbial-generated metabolome by coupling unbiased metabolomic platforms to germ free or antibiotic-treated model systems. In parallel, we must move forward from correlative metagenomics to mechanistic studies linking bacterially-derived metabolites to disease phenotypes in human and animal model systems where gut microbe levels can be experimentally manipulated and determined (21–28). Once key bacterially-derived metabolites are found to be causally linked to human physiology or disease, we must adopt a true endocrine philosophy to subsequently identify the host receptor systems that sense the metabolic hormones produced by gut microbes.

In addition to nutrient-derived metabolites, gut microbes also metabolize a number of xenobiotics to produce modified compounds with broad implications in human health. Although there are many examples of bacterial modification of drugs in humans (79), several elegant examples highlight the importance of our microbial counterparts in shaping the way we respond to drugs. A seminal study by Clayton and colleagues (80) employed a metabolomic profiling approach to identify a microbial metabolite (p-cresol) of the commonly used drug acetaminophen. This work showed that microbial production of p-cresol reduces the ability of the host liver to properly metabolize acetaminophen, likely due to competition with sulfotransferases (80). With relevance to cardiovascular disease, the drug digoxin can be metabolized by a common actinobacteria, functionally inactivating the drug (81). Also, it was shown that the chemotherapeutic drug irinotecan is metabolized in a gut microbe-dependent manner generating a metabolite linked to adverse side effects. Inhibition of the bacterial β-glucuronidase enzyme responsible for this reaction was shown to improve the effectiveness of this chemotherapeutic while decreasing deleterious side effects (82). Most recently, several studies have shown that gut microbial metabolism of chemotherapeutic drugs can actually increase their efficacy, providing novel evidence of “symbiosis” (83,84). These studies highlight the central role that gut microbial metabolism can play in drug metabolism, and also provocatively suggest that dual therapies containing xenobiotics and bacterial modifiers may provide more benefit than either drug alone (82).

Since the invention of the microscope by game changing scientists such as Robert Hooke and Antonie van Leeuwenhoek, the field of microbiology has matured over almost four centuries of research. However, even with the scientific advances we have today, translating microbiological knowledge into new therapies that impact human physiology and disease presents with many challenges. For instance, although we now have a wealth of knowledge surrounding the taxa of microbes that inhabit the human gut (29–39), the vast majority (<30%) of these species are not amenable to laboratory culture (85). Given, this limitation, the field of microbiology has generated a selective bias in our knowledge base. In other words, our appreciation of the secretory repertoire of distinct microbial taxa has been constrained by our inability to culture and study them in a reduced clonal population system. Therefore, improvements in methods to culture diverse types of human gut-derived microbes are desperately needed. Progress on this front is slowly beginning to happen (86), and this will be central to generating novel probiotic approaches to human health, as is discussed in detail below. Another key consideration for translating microbiology into human health is the concept of horizontal gene transfer (HGT) (87). HGT refers to the transfer of genes between organisms in a manner that does not rely on reproduction. Inter-bacterial gene transfer was first describe by Ochiai and colleagues (88), where they demonstrated that antibiotic resistance could be laterally transferred from one bacterial strain to another. HGT is very common among all bacteria, but interestingly, microbes within the human gut have a 25-fold higher rate of HGT than microbes in other ecosystems (89). The effect of HGT in human physiology is exemplified by the recent study by Hehemann and colleagues, which showed that bacterial transfer of a marine bacterial gene to a human resident symbiont confers that ability to digest seaweed polysaccharides (90). These microbiological concepts, as well as others, will be critical to consider as we move forward to design CVD drugs targeting the microbiome.

THE FUTURE OF CARDIOVASCULAR DRUG DISCOVERY: OPPORTUNITIES TO TARGET THE GUT MICROBIAL ENDOCRINE ORGAN

Historically drug discovery has been dominated by targeting host pathways driving disease pathology, but current and future CVD drug discovery efforts will include approaches targeting the gut microbial endocrine organ. Current strategies for targeting the gut microbial endocrine organ include: 1) dietary modification (altering nutrient metabolite inputs), 2) antibiotics, 3) prebiotics/probiotics, 4) fecal transplantation, 5) bacterial enzyme inhibitors/activators, and 6) host enzyme inhibitors/activators (Figure 3). As a frame of reference, here we discuss therapeutic opportunities within each of these categories relevant to the gut microbial-driven TMAO pathway (21–28). However, as additional meta-organismal pathways are discovered, these same approaches will no doubt also be utilized in a broad array of cardiometabolic drug discovery targets.

The simplest point of intervention is to limit consumption of dietary constituents that serve as substrates for metabolism-dependent generation of proatherogenic hormones such as TMA/TMAO (Figure 3). For instance, we know that both free choline and PC (21), as well as L-carnitine (22), and to a lesser extent, betaine (23), can all serve as dietary substrates for the sequential microbial production of TMA and subsequent host formation of proatherogenic TMAO (21–28). Therefore, limiting the consumption of foods rich in total choline and L-carnitine can be an effective strategy to limit circulating TMAO (21–25). In fact, people suffering from the condition trimethylaminuria, which is caused by mutations in the host enzyme flavin monooxygenase 3 (FMO3), can significantly reduce circulating TMA levels simply by eating a low-fat and low-choline diet (91). Importantly, there are likely other trimethylamines in the food supply that can also enter into microbiota-dependent TMA production that have yet to be identified. Hence, it is important that we continue efforts to identify all major dietary amines that can enter into the TMAO pathway, and apply this knowledge base to other relevant meta-organismal metabolic pathways. This is where recent advances in coupling metabolomic profiling (78) with dietary supplementation studies (21–25) becomes very useful.

Another potential point of therapeutic intervention relies on the use of broad or class-specific antibiotics to eliminate the production of proatherogenic gut microbe-generated hormones. However, this approach is not a likely a long term option due to the fact that many gut microbial products are quite beneficial for the host (29–39), and frequent antibiotic treatment can facilitate the emergence of antibiotic resistant bacterial strains (34,88,89). Even with these potential problems, several clinical trials involving prolonged antibiotic treatment have been conducted to examine effects on CVD endpoints such as myocardial infarction and death (92). Collectively, these trials thus far have shown that while long-term antibiotic treatments were well tolerated, no benefit on cardiovascular endpoints were observed (92). These studies are limited by the fact that despite initial diminution of gut microbes by antibiotic treatment that might generate a specific metabolite, antibiotic resistant strains present in low abundance over time will repopulate and expand their intestinal niche. Further, antibiotic use suppresses both the good and the bad types of commensals alike, making it difficult target specific taxa and favorably impact cardiometabolic phenotypes.

A more tractable approach at modifying the gut microbial endocrine organ is the use of prebiotics or probiotics (93–95). Prebiotic therapy consists of ingestion of select nutrients or dietary constituents (non-microbial compositions) that provide a growth advantage of “beneficial” bacteria (93). A key example of a prebiotic therapy is the ingestion of non-digestable fiber that can enhance the growth of “beneficial” commensals and alter motility, secondarily impacting gut microbial community structure (93). Probiotic therapy involves the dietary ingestion of one or more live bacterial strain, attempting to take advantage of the mutualism of microbes and potential for horizontal gene transfer to benefit the host (87–89). Although prebiotic and probiotic therapies are still at the early phases of developments, several recent examples highlight the therapeutic potential for these types of approaches. First, the prebiotic ingestion of dietary fructans, which are naturally occurring fructose polymers in many common fruits and vegetables, provides a growth advantage to a beneficial gut microbe family known as Bifidobacteria (93). Importantly, dietary-fructan stimulated Bifidobacteria colonization has been associated with improvements in obesity-induced insulin resistance (93). In addition to prebiotic approaches, several recent examples highlight the potential utility of probiotics in modulating host metabolism and disease (93–95). For instance, administration of the probiotic VSL#3 to mice was shown to alter microbial metabolism of bile acids, with resultant potential anti-atherogenic alterations in host cholesterol and bile acid metabolism (94). Furthermore, probiotic administration of a genetically-modified strain of bacteria designed to generate high levels of a beneficial class of lipids called N-acylphosphatidylethanolamines was recently shown to protect mice from obesity-related disorders (95). These examples highlight the utility of both prebiotic and probiotic approaches for the potential treatment or prevention of CVD. With recent advances describing the effect of diet on microbe-host interactions (35–37) and improvements in microbial culturing conditions (86), we are now poised to take advantage of these types of therapies.

Although prebiotic and probiotic approaches hold great promise, they lack true specificity, given that they impact many types of gut microbial communities due to microbial mutualism and horizontal gene transfer. An alternative therapeutic approach would be to specifically inhibit the bacterial enzyme(s) responsible for the production of TMA or other CVD relevant microbial metabolites. Very recently, Craciun and Balskus identified a bacterial gene cluster responsible for the anaerobic production of TMA from choline (96). Homologues of the choline utilization (cut) gene cluster was identified in 89 bacterial genomes, and the gene products (CutC and CutD) encode a glycyl radical enzyme complex capable of TMA production from choline in vitro (96). Interestingly, a distinct Rieske-type oxygenase/reductase system (CntA and Cntb) has also recently been described as a bacterial enzyme complex capable of converting L-carnitine to TMA (97). It is tempting to speculate that these TMA-producing enzymes, as well as additional as of yet un-identified microbial TMA lyases, may be attractive drug targets within the gut microbial endocrine organ by virtue of their potential to reduce TMA and thus, TMAO levels. Another important consideration regarding TMA and TMAO metabolism by gut microbes is that several common taxa of bacterium including E. coli can reduce TMAO, providing electron acceptors for energy production under anaerobic conditions (98). This additional level of complexity in the meta-organismal TMAO pathway is driven by the bacterial torCAD operon, which encodes a TMAO reductase (TorA), a c-type cytochrome (TorC), and a TorA-specific chaperone (TorD) (98). A recent report has similarly shown that bacteria present in oceanic waters can also degrade TMAO through a novel TMAO demethylase enzyme (Tdm) (99). It remains possible that these bacterial TMAO catabolic pathways can be another potential avenue of therapeutic intervention, given that gut microbes have a high capacity to metabolize TMAO within the human meta-organism. The identification of the bacterial enzymes responsible for TMA production, and TMAO degradation, thus represent important advances, and will form the basis for additional studies to identify means of favorably altering the TMA/TMAO axis through selective microbial TMA lyase inhibitors and/or TMAO degrading enzymes that may be delivered to gut microbes for potential therapeutic benefit.

Another potential site of therapeutic intervention lies at the level of the host enzyme machinery necessary for the conversion of TMA to TMAO. The flavin mono-oxygenase (FMO) enzyme family carries out this critical oxidation step, with at least 5 paralogues present in human liver (FMO1, FMO2, FMO3, FMO4, and FMO5) (100). In addition to TMA oxidation, the FMO enzyme family is responsible for the oxygenation of a plethora of nitrogen- and sulfur-containing compounds present in xenobiotics, as well as endogenous substrates (100). FMO3 and FMO1 are the major human FMO enzymes that can efficiently oxidize TMA to form TMAO, with FMO3 having the highest specific activity towards TMA (25). Interestingly, loss-of-function mutations of the FMO3 gene results in the inherited disorder trimethylaminuria, which is also known as fish odor syndrome (100). This autosomal recessive disease arises from the inability of those affected to convert TMA, which smells like rotten fish, to TMAO (100). The FMO3 gene is under complex transcriptional control, where expression is dynamically regulated by inflammation, sex hormones, and the bile acid-activated nuclear receptor FXR (25,100). Although pharmacologic inhibition of FMO3 is expected to provide therapeutic benefit by reducing host TMAO production, it is unlikely that FMO3 inhibitors would be attractive drug targets. This is primarily due to the fact that the accumulation of the FMO3 substrate (TMA) would be expected to result in untoward odorous side effects (100). Also, since FMO3 itself is a xenobiotic metabolizing enzyme (100), there is a strong potential for drug-drug interactions with FMO3 inhibitors, further dampening enthusiasm for FMO3 inhibition as a therapeutic strategy. Regardless of therapeutic utility, it will be important to better understand how hepatic FMO activity is regulated under physiological and pathophysiological conditions given its central role in determining circulating TMAO levels (100). In addition, it will be informative to determine whether patients with extremely low levels of circulating TMAO due to FMO3 mutations have a lower incidence of CVD. Likewise, FMO3 inhibitor or genetic knockout studies in animal models of atherosclerosis are warranted.

Given that circulating levels of TMAO are linked to CVD risk in humans (21–28), and dietary supplementation with TMAO promotes atherosclerotic CVD in mice (21,22), a key therapeutic opportunity lies at the level of blocking the ability of circulating TMAO to elicit a biological response. One potential therapeutic avenue might be development of absorbent agents that can bind and help eliminate TMA and TMAO at the level of the gut. An alternative would be to theoretically intercept TMAO at the level of a molecular receptor (i.e. through a TMAO receptor antagonist). Although a dedicated host TMAO receptor system has not yet been identified, it would not be surprising if such a sensing mechanism exists. In fact, virtually all gut microbial products described to date have dedicated host receptor systems that carefully sense the levels of these biologically active gut microbial products (Figure 2). The concept of a TMAO receptor is bolstered by the fact that gut microbe-generated TMA is sensed by the host G protein-coupled receptor Taar5 (67,68). Identification of a host TMAO receptor system, and subsequent development of TMAO receptor antagonists, would represent an extremely attractive therapeutic approach without the potential drawbacks of dietary manipulation or microbe-modifying drugs. It is quite clear that the interaction between gut microbes and the host they inhabit occurs through delicate host sensing mechanisms (Figure 2), and further characterization of such signaling processes will provide exciting new therapeutic opportunities.

CONCLUSIONS AND PERSPECTIVES

The recent discovery that our gut microbiota plays a central role in diet-dependent CVD susceptibility has broad implications. Although largely overlooked, our gut microbiome functions as a dynamic yet resilient endocrine organ, produces a plethora of metabolism-dependent and metabolism-independent signals that play regulatory roles in CVD development in the host (Figure 1). On one hand, commensals present in the human gut serve as a “metabolic filter”, significantly influencing how dietary inputs are assimilated by the host. Gut microbiota-derived hormones are already identified that alter energy metabolism and cardiometabolic disease relevant phenotypes and CVD pathogenesis. On the other hand, chronic low levels of inflammation driven by metabolic endotoxemia can also apparently reorganize cholesterol balance, insulin resistance, and vascular inflammation as well (Figure 1). Given the central role the gut microbial endocrine organ plays in how environmental factors impact CVD in humans, substantial drug discovery opportunity lies ahead in the area of meta-organismal crosstalk. Although the field has been dominated by correlative metagenomic approaches, simply cataloging microbial communities in disease models is insufficient as we move forward. Instead, functional studies coupling microbial product identification (metabolomics, proteomics, etc.), dietary manipulation, and host receptor discovery in relevant disease model systems are necessary. Furthermore, it will be important to advance the concepts we have learned from microbes resident in the gut lumen to other relevant microbial communities present on our skin and exposed mucosa. Although drug discovery has historically targeted host enzymes, a fertile period in biomedical research lies ahead where instead we target the microorganisms that live within us to either improve human health or prevent disease. The challenge lies in identifying the key points of therapeutic intervention within relevant meta-organismal pathways.