The gut microbiota, environment and diseases of modern society

Abstract

The human gut microbiota is a complex community that provides important metabolic functions to the host. Consequently, alterations in the gut microbiota have been associated with the pathogenesis of several human diseases associated with a disturbance in metabolism, particularly those that have been increasing in incidence over the last several decades including obesity, diabetes and atherosclerosis. In this review, we explore how advances in deep DNA sequencing technology have provided us a greater understanding of the factors that influence that composition of the gut microbiota and its possible links to the pathogenesis of these diseases.

Introduction

The human gut microbiota is one of the most densely populated bacterial communities on Earth with approximately 10¹¹ organisms per gram of fecal weight composed of over a 1000 species, most of which are obligate anerobes,^1,2 with a collective genome 150-fold greater than that of its human host.¹ The larger view of the mammalian physiology should take into account that we are a biologic “supraorganism” that is dynamic and carries out functions in parallel or cooperatively. Although there are over 50 bacterial phyla on Earth, human-associated bacteria largely belong to one of four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes.^3,4 Mammalian hosts have coevolved to exist with our gut microbiota in a mutualistic relationship where we provide a uniquely suited environment in return for physiological benefits provided to us by our gut microbiota.⁵ Examples of the latter include the fermentation of indigestible carbohydrates to produce short chain fatty acids that are utilized by the host, biotransformation of conjugated bile acids, synthesis of certain vitamins, degradation of dietary oxalates and education of the mucosal immune system.⁵ Indeed, when viewed as a whole, the “supraorganism” of the gut can carry out enzymatic reactions distinct from those of the human genome and harvest energy that would otherwise be lost to the host. The consequences of these enzymatic reactions suggest that over the millennia, mammalian metabolism, physiology and disease have shaped and been shaped by the gut microbiota. Commensal bacteria may also directly inhibit the growth of specific pathogens, such as Clostridium difficile, by competitive inhibition thus providing an adequate niche for expansion.

Despite the importance of the gut microbiota in maintaining the health of the host, growing evidence suggests that it may also be an important factor in the pathogenesis of a variety of diseases, particularly those that have shown a rapid increase in incidence over the past few decades.

Methods to Analyze the Microbiota

Traditional bacterial culture techniques have a limited value in characterizing the composition of the gut microbiota because most community members are obligate anerobes that are fastidious and difficult to grow in vitro. Tremendous advances in deep DNA sequencing technology over the past decade have revolutionized the approach to characterize complex bacterial communities. There are two main methods to characterize the microbiota that are culture-independent. First, small-subunit rRNA (rRNA) studies in which the 16S rRNA gene sequences (for Archaea and Bacteria), or 18S rRNA gene sequences (for Eukaryotes) are used as stable phylogenetic markers to define the lineages that are present in a sample.⁶ Second, shotgun metagenomic sequencing can be performed, in which community DNA is sequenced in totality permitting not only an evaluation of microbial community structure but also allowing an evaluation of the genomic representation of the community. The latter can be used to help understand the functions encoded by the genomes of the gut microbiota.⁷ In addition, there are techniques that evaluate gene expression directly, including metatranscriptomics and metaproteomics. These analyses may provide a deeper understanding of microbiota function.⁷

Initial approaches utilized “fingerprinting” that provide limited information about the microbial communities, but are less expensive and faster to perform than deep sequencing. These methods, which include denaturing gradient gel electrophoresis (DGGE), T-RFLP (terminal restriction length polymorphism), TGGE (temperature gradient gel electrophersis), SSCP (single strand conformation polymorphism) and ARDA (amplified rDNA restriction analysis), rely on amplification of a specific gene, usually16S rRNA genes. The technique can be used in conjunction with oligonucleotide probes in order to increase the specificity of the analysis.⁸ Unfortunately, it is difficult to correlate banding patterns to changes in particular species or lineages.^7,8 In addition, the dynamic range is typically limited and not sensitive enough to detect all the diversity present, therefore only a few of the most abundant members of the community can be observed. These techniques can be most useful for checking for stability in the dominant members of a community and for clustering communities according to changes in the dominant members across large numbers of samples.

Microarrays are an intermediate approach between fingerprinting and sequencing. They are a convenient and quick approach, however are limited by the data from which they are developed. Bias may result as some species are not included on the microarray and therefore not detected. It provides access to large numbers of samples and phylogenetic resolution, but has limits in both dimensions. ARISA (amplified ribosomal intergenic spacer analysis) technique uses short sequence tags of the intergenic spacer of the rRNA⁹ or sequencing of the V6 hypervariable region of the 16S rRNA to make comparisons between samples and measurements of diversity and assessment of taxonomic distribution. However, this approach is less useful when there are new lineages and no close relatives are dominant.⁷ qPCR (quantitative polymerase chain reaction) and FISH (fluorescent in situ hybridization have been utilized to study the microbiota as well, however they are limited by the fact that they are only as good as the specificity of the primers or probes designed.⁷ Nevertheless, these approaches may be useful as adjuncts to deep sequencing by providing absolute quantification of bacterial taxa and spatial localization within a sample, respectively.

The main advantage of deep sequencing techniques over fingerprinting are that sequences can be classified according to taxonomy and function. Sequences provide greater dynamic range and have the ability to compare complex samples–sequences from different that studies are able to be compared with one another and placed in the same phylogenetic tree.¹⁰ Sequencing is especially useful when asking which specific genes or species contribute to differences among communities. It is also helpful when the discovery of many new genes or species lineages is anticipated in samples, which have been poorly characterized. 16S rRNA gene sequences are especially important for characterizing microbial assemblages at a taxanomic level.⁷ Whereas shot-gun metagenomic sequencing can provide both taxanomic and functional information, the latter through the characterization of bacterial gene relative abundance.

Second generation sequencing, such as 454 Life Sciences (Roche) GS FLX or Titanium when combined with a barcoded pyrosequencing approach, permits the generation of high-throughput 16S rRNA gene sequences simultaneously in multiple samples thus greatly increasing efficiency and reducing costs. The analysis of 454 sequencing data improves the accuracy of building phylogenetic trees and classifying functions of metagenomic reads. It extends the range of rare species reported in a sample and provides deep views into hundreds of microbial communities simultaneously. A small fragment of the 16S rRNA gene is sufficient to make taxanomic assignments by binning sequence reads into “operational taxanomic units” (OTUs) used for many community analyses, including those based on a phylogenetic tree. Due to significant advances, the computational methods for comparing large numbers of microbial communities have greatly improved. This includes UniFrac and network based comparisons, which has allowed for rapid progress to be made. In addition, sequence databases have grown tremendously.⁷ Together with the development of even more sophisticated bioinformatic tools, rapid advances in next generation DNA sequencing technology that increase throughput and accuracy while reducing cost, will ultimately permit whole genome and shot-gun metagenomic sequencing to replace the 16S rRNA gene sequencing approach currently used by many laboratories.

Environmental Impact on the Gut Microbiota

Colonization of the gut begins at birth and, following an initial chaotic community structure during the first year of life, the human gut microbiota becomes more stable and adult-like¹¹ concurrent with the introduction of solid foods into the diet.¹² Several studies have explored the impact of diet on the newborn gut microbiota and have compared breast feeding and formula feeding. A consistent finding has been the higher proportion of Bifidobacteria in breast fed infants as compared with formula fed infants.^13-16 Later on in life, dramatic effects of diet on the gut microbiota of an infant has been observed with the introduction of solid foods.¹² The mode of delivery on the intestinal microbiota has also been examined. Multiple studies utilizing culture dependent techniques have shown that infants born via vaginal delivery are colonized from their mother’s vaginal and intestinal microbiota, in contrast to infants born via caesarean section whose intestinal microbiota are colonized with environmental microbiota.^17-19 Interestingly interindividual differences in the characteristics of the bacterial microbiota are also observed early in life, within months, and persist at 1 y of life.¹¹ Indeed, interindividual differences in the gut microbiota are the largest source of variance among healthy individuals that appear to be relatively stable over time, at least in the short term.³

The driving force behind interindividual variation remains to be determined but indirect evidence from a number of studies suggest that early environmental exposures may play a role. Despite individual differences, Palmer et al. showed that there was remarkable similarily in early temporal profiles of the gut microbiota in dizygotic twins that was greater than other relationships that also share 50% genetic identity including mother:baby, father:baby and sibling:baby.¹¹ This observation was also noted in a subsequent study examing the microbiome in obese and lean twins²⁰ where the twin-twin gut microbiota were more similar that twin-mother which were both much more similar than unrelated individuals. Interestingly, the gut microbial communities of monozygotic twin were no more similar than dizytotic twins. In total, these studies suggests that environmental factors early in life may play an important role in shaping interindividual characteristics of the gut microbiota, features that may persist later in life and that are independent of host genetics. The impact of host genetics on the gut microbiota is largely based on studies in model organisms such as rodents and has been reviewed recently.²¹ The impact of genetics on the gut microbiota in healthy human populations, based on current evidence,²⁰ may be relatively modest but further studies are needed.

Many aspects of our environment have been changed dramatically over the past few decades concurrent with the increasing incidence of certain disease processes. Elements of the modern lifestyle that have been postulated to result in changes in the gut microbiota include improved sanitation, vaccinations, increased antibiotic use, decline in parasite infections, caesarean section, decline in H. pylori, smaller family size, refrigeration, less crowded living conditions, sedentary life styles, food processing and diet changes.²¹

Diet and the Gut Microbiota

The development of agriculture and domestication of animals have been major factors in recent human evolution²² with the resultant changes in diet perhaps altering the host-gut microbiota relationship.²³ Over time in industrialized nations, there has been a reduction in fiber consumption with an increase in simple sugars, fats and proteins. It has been hypothesized that this change in diet may have altered the interaction of the host and the microbiota in a manner that has played a role in the increasing incidence of metabolic disorders.²³ Furthermore fluctuations in diet may have consequences for the bacteria and the host, allowing for predisposition to invasion or inflammation.²⁴ At a molecular level, dietary carbohydrates, particularly indigestible carbohydrates by the host, are an important source of energy for the gut microbiota. Intestinal bacteria have highly developed metabolic pathways that regulate carbohydrate metabolism through fermentative processes. These differ in their ability to utilize dietary carbohydrates and host derived carbohydrates in the form of mucin glycoconjugates.^25,26 Bacteroides thetaiotaomicron, for example, metabolizes carbohydrate substrates, due to rich carbohydrate utilization pathways. However in times of dietary carbohydrate restriction of the host, this gut commensal has the capacity to adapt and utilize host produced mucus glycans for energy metabolism.²⁵

Cross sectional studies suggest that there has been a functional evolution of the gut microbiota in relation to diet based on a study examining 16S rRNA surveys of fecal community DNA from herbivores, omnivores and carnivores from 33 mammalian species that represented 10 Orders with varied digestive physiologies (hindgut-fermenters, foregut-fermenters and simple-gut). Microbiota community structure in carnivores and omnivores were distinguished from herbivores in both bacterial 16S rRNA gene and whole community gene data sets implicating the importance of diet in differentiating gut microbial communities.²⁷ Indeed, previous work demonstrated that the fecal microbiome of the same species were significantly more similar than communities of different host species where there may be a core of shared functions implicated by the representation of enzymatic pathways.²⁸ For example, many enzymes that separate carnivore and herbivore microbiomes are involved in amino acid metabolism. By contrast to carnivores, the microbiome from herbivores were enriched in enzymes that map to biosynthetic reactions for 12 amino acids. The microbiome from carnivores had 9 amino acid degradation pathways, while herbivores only contained degradative enzymes involved in the break- down of branched amino acids.²⁸ Based on this evidence, it has been hypothesized that the gut microbiota of carnivores specialize in degradation of proteins as an energy source whereas the gut microbiota of herbivores specialize in the synthesis of amino acid building blocks. More recently, it has also shown that gnotobiotic mice, harboring a 10-member community of sequenced human gut bacteria, can be used as a model to examine the response of the gut microbiota to diet.²⁹

A recent study focusing on the effect of diet on the gut microbiota in healthy human subjects using dietary questionnaires as well as a controlled feeding experiment revealed differences in the impact of long-term vs. short-term diets.³⁰ Long-term diets high in animal protein and fats and low in carbohydrates, similar to a “Westernized” diet, were associated with high levels of Bacteroides and low levels of Prevotella genera. By contrast, diets high in carbohydrates but low in animal protein and fat were associated with the inverse pattern, higher levels of the Prevotella and lower levels of Bacteroides. This relationship was observed with a dietary questionnaire that probed long-term diet but not with a 24-h dietary recall instrument measuring short-term diet. Although a change in dietary consumption leads to an alteration in the human gut microbiota within 24 h, consistent with studies in mice,^31,32 alterations in the relationship between Bacteroides and Prevotella were not observed. These results provide an explanation for previously described clustering of individuals into “enterotypes” dominated by Bacteroides and Prevotella based on the composition of the gut microbiota.³³ These observations are also consistent with a study comparing the gut microbiota of children from a village in the West African country of Burkina Faso to those in Europe,³⁴ as well as a more recent study comparing the gut microbiome of residents in the agrarian Malawi and Ameridian societies to residents in the US,³⁵ where the inverse relationship between Bacteroides and Prevotella genera were also noted. Although the abundance of Prevotella could be considered a discriminative taxon associated with the general agrarian environment, that fact that these associations were also observed in residents of the US³⁰ supports the notion that the observed inversely related proportions of Prevotella and Bacteroides are diet driven. In total, these four studies suggest that long-term diet helps to distinguish a gut microbiota community, or enterotype that is associated with a “Westernized” diet rich in Bacteroides from an enterotype associated with an agrarian diet where the bacteria of the Prevotella genus predominates. Future studies will be needed to determine if enterotypes are distinct clusters or more continuous gradients and whether or not they are associated with human disease.

The ability to distinguish a gut microbiota based upon a “Westernized” vs. a more agrarian diet may be of importance in the interpretation of studies associating various disease states that are more prevalent in industrialized nations with the gut microbiota. The impact of diet on the gut microbiota is still in an early stage of characterization. Clearly it is important that additional studies be performed to address numerous critical issues. Among these include studies to determine whether or not long-term diet can lead to enterotype switching, studies to determine whether or not the Bacteroides vs. Prevotella enterotypes exist as distinct entities or rather represent a continuum (or “enterogradient”) where the observed dietary associations occur at the extremes, and studies to determine if enterotypes association consistently exist at deeper taxonomic classifications. Ultimately, it will be important to determine whether individuals with the Bacteroides enterotype have a higher incidence of diseases associated with a Western diet, and whether long-term dietary interventions can stably switch individuals to the Prevotella enterotype. If an enterotype is ultimately shown to be causally related to disease, then long-term dietary interventions may allow modulation of an individual’s enterotype to improve health. Alternatively, if causal associations are not established, enterotypes may still have utility as a prognostic biomarker of disease.

The Impact of Environment vs. Host Genotype on the Gut Microbiota Associated with Disease

Advances genomic technology, principally DNA sequencing and SNP mapping used for genome-wide association studies (GWAS) combined with biocomputational algorithms, have revealed the genetic underpinnings of various complex disease processes such as inflammatory bowel disease (IBD), type-1 and type-2 diabetes mellitus (T1DM and T2DM), atherosclerosis, asthma, obesity and colon cancer, to name a few. In most circumstances, the contribution of host genetics to the risk of disease development is significantly less that 50% implicating the importance of environmental influences.³⁶ The observation that these diseases have shown a steadily increasing incidence over that past several decades, the geographic distribution of disease clustering in industrialized nations, and immigration studies revealing the adoption of disease risk of the host country within 1 or 2 generations, all emphasize further the importance of environment in the pathogenesis of these diseases. There is no doubt that the consumption of a high calorie “Westernized” diet together with a sedentary life style are environmental/behavioral factors that contribute to the pathogenesis of some of these diseases.

Interestingly, inflammation has been strongly associated with many of these “Westernized” disease processes. In addition to IBD and asthma, which are principally diseases due to unrestrained immune processes, T1DM has been associated with type 1 interferon production and altered T cell signaling suggesting an autoimmune response and insulin resistance, the hallmark of T2DM, is associated with an inflammatory reponse in adipose tissue.³⁷ Even obesity and atherosclerosis have been associated with chronic inflammation with elevations of serologic markers such as C-reactive protein (CRP).³⁸

Using our current understanding of disease pathogenesis in IBD as a paradigm, functional genomics has revealed a complex interaction between host innate and adaptive immunity that provide protection against microbial invasion yet demonstrates tolerance to colonization with the microbiota at mucosal surfaces (recently reviewed in³⁹). In the case of IBD, the loss of mucosal tolerance, together with a defect in protective host innate immunity to a dysbiotic microbiota, leads to an unrestrained mucosal immune response, the hallmark of this disease process. As discussed in the following sections, growing evidence demonstrates that the gut microbiota is associated with a number of disease processes associated with a “Westernized” diet and lifestyle. Although a causal relationship has been demonstrated primarily in animal models and a functional effect in human disease is currently lacking, the role that the gut microbiota plays in the establishment of host immunity together with its effects on the inflammatory response suggest that continued investigation may lead to direct evidence for the role of the microbiota in at least some of these disease processes.

Obesity and the Gut Microbiota

Over the last several decades, obesity has become a worldwide epidemic. This has prompted numerous investigations into possible environmental factors associated with the rise in incidence of this disease. There have been several studies, both in humans and murine models, evaluating the relationship between the microbiota and obesity. These studies have found an association between the gut microbiota, host genetics, diet and this disease.

Several experiments have used the gnotobiotic mouse model to elucidate the relationship between gut microbiota and alterations in host metabolism.^40-43 Initial work comparing germ-free mice, conventionally-raised mice and conventionalized mice demonstrated that, in comparison to germ-free mice, the latter two had increased body fat, epididymal fat weight and energy expenditure despite decreased food intake.⁴² Conventionalized animals showed increase in monosaccharide uptake and liver triglyceride content in addition to upregulation of lipogenic enzymes in the liver.⁴² Furthermore, using a reductionist model system, germ-free mice colonized with only two organisms, Bacteroides thetaiotaomicron and Methanobrevibacter smithii (an Archaeon) showed similar changes to conventionalized animals. This suggests that the gut microbiota alters metabolism and physiology of the host in a manner that favors energy storage.

Conversely, 16S rRNA sequencing of obese phenotype (ob/ob) mice has shown that obesity was correlated with a significant reduction in Bacteroidetes compared with lean mice and a similarly greater proportion of Firmicutes,⁴⁴ suggesting that there is a particular gut microbiota that is associated with the obese phenotype. The functionality of this alteration was demonstrated using the gnotobiotic mice whereby cecal contents of an obesity-associated phenotype (ob/ob mouse) were transplanted into germ-free mice. When compared with mice transplanted with a lean-associated gut microbiota, these mice gained significantly more weight despite similar food consumption.⁴¹ Gas Chromatography/Mass Spectrometry and bomb calorimetry methods confirmed increased short chain fatty acid availability in the cecum and decreased energy in the feces of mice conventionalized with the obese-associated microbiota.⁴¹ From a mechanistic standpoint, it appears that short chain fatty acids are not only an additional source of calories for the host but they are also ligands for a G-protein coupled receptor known as Gpr41. Mice that are null for the Gpr41 gene behave similarly to germ-free mice after conventionalization, gaining less weight and adiposity in comparison to germ-free wild-type mice that have been conventionalized.⁴⁵ Germ-free animals are protected against the obesity that occurs after consumption of a Western-style, high fat, sugar-rich diet.⁴³ Fasting-induced adipose factor (Fiaf) is a circulating lipoprotein lipase inhibitor that is inhibited by the presence of gut microbiota. Germ-free mice that lack the gene for Fiaf are not similarly protected from diet-induced obesity, demonstrating increased weight gain and intra-abdominal adiposity despite similar quantities of food intake.⁴³ It should be noted that, unlike mice, humans likely obtain a smaller proportion of caloric requirements from the absorption of colonic short chain fatty acids. Thus the degree to which the human gut microbiota leads to the development of obesity, relative to high caloric consumption and a sedentary lifestyle, may be modest.

An observational study of the gut composition of 12 obese humans placed upon restricted diets over one year showed a relative increase in the abundance of Bacteroidetes and a relative decrease in Firmicutes.⁴⁶ Characterization of the gut microbiota of obese and lean twins (31 monozygotic twin pairs, 23 dizygotic twin pairs and 46 mothers) revealed no significant difference in the degree of similarity in gut microbiota of adult monozygotic compared with dizygotic twin pairs.²⁰ Similar to ob/ob mice, obese individuals were found to have a relative decreased proportion of Bacteroidetes species, although there was no significant difference in Firmicutes and an increased proportion of Actinobacteria.²⁰ Similarly, other studies in human subjects have not observed the obesity associated alterations in the relative proportions of Firmicutes and Bacteroidetes observed in mice.^30,47

The primary cause of obesity in the ob/ob mouse model is increased food consumption due to leptin deficiency, yet the bacteria in the obese mice were “assisting” their host in energy extraction. Many questions remain regarding the regulation of the composition of the gut microbiota and subsequent relationship to phenotype. It is counterintuitive to conclude that the gut bacteria are signaled by obese phenotype individuals to become more energy efficient, while the gut bacteria associated with loss of weight shift to a less energy-efficient population.⁴⁸ Indeed, a number of studies demonstrate that similar alterations in the gut microbiota are observed in mice, with endogenous or humanized gut microbiota, fed high fat, high calorie chow reminiscent of “Westernized” diet.^32,49-51 More recently, it has been suggested that the association between the gut microbiota and obesity in humans may be due, in part, to the consumption of saturated fatty acids.³⁰ It currently appears that the relationship between obesity and the gut microbiota is complex where both the phenotype of the host and the environment provided to the gut microbiota, in the form of a “Westernized” diet, can have an impact on the composition of the microbiota in the gut.

Diabetes and the Gut Microbiota

Similar to obesity, the pathogensis of both type 1 and type 2 diabetes mellitis (T1DM and T2DM) is complex due to the interaction of both host genetic and environmental factors. T1DM is an autoimmune disease that results from T-cell mediated destruction of insulin-producing B cells. It is defined clinically by high blood glucose and absolute insulin dependence, a consequence of destruction of the insulin secreting β cells of the pancreatic islets. There is significant heterogeneity in the presentation of this disease. Patients who are diagnosed under 10 y of age frequently demonstrate islet inflammation or insulinitis, as compared with patients over 10 who show these findings less often.⁵² As seen in obesity, the rapid increased incidence over recent decades has prompted investigators to study environmental contributions to the disease. While controversial, enterovirus has been most associated with T1DM. The link was first reported in 1969, and there has since been higher rates of enterovirus seen in patients with T1DM at diagnosis as compared with controls.^52-54 In addition, prospective studies have demonstrated increased enterovirus infections in children who developed islet autoantibodies or subsequent diabetes or both. There has been a temporal association between infection and autoimmunity.^55,56 The major sensor for enterovirus RNA is the T1DM susceptibility gene IFIH1, encoding MDA5.⁵⁷

The involvement of the gut microbiota with the development of T1DM has been implicated based on the regulatory relationship of the intestinal microbiota on the innate immune system in a murine model. Wen et al. demonstrated that the incidence of spontaneous T1D in non-obese diabetic (NOD) mice is affected by microbial environment and exposure.⁵⁸ MyD88 is an adaptor protein utilized by multiple Toll-Like receptors (TLRs) and other innate immune receptors which recognize microbial stimuli. NOD mice lacking MyD88 protein were examined for variations in gut microbial communities and subsequent differences in the development of T1DM. Germ-free and antibiotic treated MyD88^KO NOD mice developed T1DM at higher rates than the same MyD88^KO mice colonized with specific pathogen free bacteria, suggesting that the presence of normal gut microbiota protects against the development of diabetes.⁵⁹

As mentioned previously, T2DM is characterized by low grade inflammation.⁶⁰ Similar to T1DM, studies have been performed exploring the link between the microbiota and the development of T2DM focusing on toll-like receptors (TLRs). TLRs are a family of type I transmembrane receptors with an extracellular leucine-rich repeat domain and an intracellular Toll/IL-2 receptor (TIR) domain.⁶¹ Activation of these receptors leads to a signaling cascade that triggers the innate immune system and launches a response against pathogenic infection, but the mechanism by which the host differentiates between response to pathogenic gut bacteria and tolerance of commensal nonpathogenic gut microbes remain incompletely characterized.

At least 13 TLRs have now been identified, and each has a distinct role in the activation of the innate immune system. Ligands for these receptors number in the dozens, and are extremely diverse in structure and origins. Activation of TLR signaling results in both MyD88 dependent and independent activation of NF-kappa B. Toll-like receptor 4 (TLR 4) was the first characterized TLR in mammals, and is a signal-transducing receptor for bacterial lipopolysaccharide⁶² as well as saturated fatty acids.⁶³ Binding of TLR 4 in conjunction with co-receptors CD14 and MD-2 triggers a downstream signaling cascade that eventually leads to the transcription of genes that encode various proinflammatory molecules such as cytokines, chemokines and other components of the innate immune response.⁶⁴ Alterations in gut microbiota have been implicated in the development of insulin resistance and diet-induced obesity by way of a TLR 4 regulated mechanism.^65-68

Insulin resistance in obesity and diet-induced obesity is associated with elevations in inflammatory markers. In mice, infusion of free fatty acids (FFAs) led to significant increase in TLR4 mRNA expression in adipose tissue.⁶⁹ An increase of NF-kappa B binding to IL-6 increased in these mice as well, a result that was not seen in TLR4−/− mice with the same treatment.⁶⁹ Similarly, in vitro exposure to FFAs increases IL-6 and TNF-α mRNA expression, a result that was prevented by TLR4 knockdown.⁶⁹ Interestingly, when TLR4−/− and wild-type mice are placed on high-fat diets, the knockout mice became more obese relative to controls. Despite increased obesity, however, insulin tolerance tests performed after 39 d on the high fat diet revealed that the knockout mice exhibited decreased insulin resistance and expression of inflammatory markers (IL-6, SOCS3, MCP-1) in both adipose and liver tissue.⁶⁹

In contrast, two additional studies suggested that mice with TLR4 deficiency are protected from the development of obesity as well as insulin resistance on a high saturated fat diet.^65,68 When placed on a high fat diet, C3H/HeJ (loss-of-function mutation in TLR 4) mice showed decrease weight gain, decreased adiposity, increased metabolic rate, improved glucose tolerance and decreased serum TNF-α, IL-6 and adiponectin levels.⁶⁸ 10ScN mice (deletion precluding expression and production of TLR4) were protected from high-fat diet induced obesity, despite the similar caloric intake as control mice.⁶⁵ The protective effect of TLR4 deficiency involves reduced signaling of circulating lipopolysaccharide (LPS) from the gut microbiota, so called “metabolic endotoxemia”⁷⁰. Supportive of this notion is the observation that high fat diet-induced alterations in the gut microbiota are associated with increases intestinal LPS permeability.⁶⁷ An additional study linking TLRs and T2DM demonstrated that mice deficient in TLR5 displayed hyperphagia, became obese, and developed features of the metabolic syndrome. This included the development of hypertension, hypercholesterolemia and insulin resistance. When the gut microbiota of these mice were transplanted into wild-type germ-free mice, they developed similar features of the metabolic syndrome⁷¹ demonstrating the importance of the gut microbiota in the development of the disease phenotype.

Atherosclerosis and the Gut Microbiota

The pathogenesis of cardivascular disease (CVD) includes genetics and environmental factors. A known environmental risk factor is a diet rich in lipids. Atherosclerotic disease is the major cause of severe disease and death among patients with obesity.⁷² The disease is characterized by accumulation of cholesterol and recruitment of macrophages to the arterial wall. Inflammation due to infectious agents has been implicated as a potential factor in the development of CVD though augmentation of proatherosclerotic changes in vascular cells.^73-75 These changes include increased scavanger receptor expression and acitivity, enhanced uptake of cholesterol and modified LDL, increased expression of adhesion molecules and inflammatory cytokines.⁷⁶ However, the link between infection and development of atheroscleosis has not been conclusive. Prospective trials with antibiotic therapy have not demonstrated cardiovascular benefit^77-79 and murine models demonstrate that infectious agents are not necessary for the development of atherosclerotic plaque.⁸⁰

Nevertheless, there is some evidence suggesting an association between microbes and the development of atherosclerosis. Several studies that have supported the association between CVD and infection have included periodontal disease.⁸¹ Furthermore, metabolic activity of the gut microbiota has been shown to correlate with blood pressure.⁸² Characterization of bacterial composition by 16S rRNA gene surveys in atherosclerotic plaque, oral and fecal samples obtained from patients with atherosclerosis and healthy control subjects revealed similaries in bacterial taxa between the former and the latter two sources suggesting that the atherosclerotic plaque microbiota may at least in part be derived from the oral cavity and/or the gut. The authors of this study hypothesized that bacteria reached the atherosclerotic plaque through phagocytosis of microbes by macrophages at the epithelial lining of mucosal surface. Once activated, the macropages leave the blood stream and enter the atheroma and form a cholesterol-laden foam cell.⁷²

The gut microbiota may also augment the development of atherosclerosis through the production of certain metabolites of dietary lipid phosphatidylcholine that are associated with the risk for the development of CVD. Using a targeted approach to identify plasma metabolites which predict CVD in patients, Wang et al. identified a novel pathway linking dietary lipid intake, intestinal microbiota and atherosclerosis. Foods rich in phosphatidylcholine are a major source of choline. Catabolism of choline by the intestinal microbiota results in the formation of the gas TMA (trimethylamine) that is metabolized by the liver to form trimethylamine oxide (TMAO) and augments the development of atherosclerosis in animal models thus providing the first link between dietary lipid intake, the intestinal microbiota, and the risk for the development of atherosclerosis.⁸³ Interestingly, consistent with this notion, the consumption of choline is positively correlated with the human gut microbial enterotype rich in Bacteroides that is associated with a “Westernized” diet.³⁰

Summary

Some of the most common disease entities responsible for great morbidity and mortality are associated with both genetic and environmental factors. Many of these, such as obesity, diabetes, atherosclerosis, asthma, inflammatory bowel disease, have shown a dramatic increase in incidence over the past several decades. Together with acquired disease risk by immigration, these observations implicate a powerful environmental effect of societal modernization as a factor in disease pathogenesis. Alterations in diet, sedentary lifestyle, sanitation, antibiotic use, cesarean sections, and vaccinations are among a few of the many recent alterations in lifestyle that have been implicated. It is therefore of great interest that these same factors have been associated with alterations in the gut microbiota, observations made only recently due to dramatic advances in DNA sequencing technology and the development of sophisticated bioinformatic tools. Similarly, alterations in the composition of gut microbial communities have also been associated with some of these disease processes suggesting that the gut microbiota may play a role in the pathogenesis of some diseases. Given the enormity of the microbial biomass in the human intestinal tract, our co-evolution, and the well-established function of the gut microbiota on the regulation of a multitude of normal host physiologic functions, it seems very plausible that the gut microbiota will indeed play an important role.

In this review, we highlighted three diseases for which the gut microbiota has been implicated in disease pathogenesis focusing on those associated with the consumption of a “Westernized” diet. However, there is evidence that similar associations may be found in other disease processes such as inflammatory bowel disease, colon cancer, asthma, necrotizing enterocolitis, and irritable bowel syndrome, to name a few. It must be kept in mind, however, that these associations with human disease do not prove cause-and-effect relationships. Indeed, most data supporting a functional effect of an altered microbiota on host physiology are based primarily on murine models. Although such studies provide fundamentally important information about disease mechanisms demonstrating “proof of principle,” the degree to which they reflect human pathophysiology awaits further investigation. Some additional questions fundament to the field of human gut microbial ecology include the following:

• What are the determinants of the gut microbiota responsible for interpersonal differences?

• What is the long-term stability of the gut microbiota?

• What is the resilience of the gut microbiota to change in immigrant populations?

• Does colonization with microbiota from modern society in early life predispose individuals to an inflammation and autoimmunity-prone immune system?⁵²

• Can the gut microbiota be permanently altered in a way that is beneficial to the host and is the window of opportunity early in life?

The answers to these, and other fundamental questions in the field of gut microbial ecology, await additional studies in human subjects in whom clinical metadata are carefully collected together with continued investigation in animal models in the era of rapidly advancing broad-based technologies such as DNA sequencing, transcriptomics, proteomics, and metabolomics.