99th Dahlem Conference on Infection, Inflammation and Chronic Inflammatory Disorders: The normal gut microbiota in health and disease

Abstract

Mammals are metagenomic, in that they are composed not only of their own genome but also those of all of their associated microbes (microbiome). Individual variations in the microbiome influence host health and may be implicated in disease aetiology. Therefore, it is not surprising that decreased microbial diversity is associated with both obesity and inflammatory bowel disease. Studies in germ-free mice have demonstrated that the gut microbiota is required for development of diet-induced obesity as well as inflammatory diseases. However, the underlying molecular mechanism(s) for how the gut microbiota causes metabolic diseases is only beginning to be clarified. Furthermore, emerging data suggest that the gut microbiota may predispose or protect against other important diseases such as cardiovascular disease and diabetes.

Introduction

The adult human intestine is home to an almost inconceivable number of microorganisms. The size of the population – up to 100 trillion – far exceeds that of all other microbial communities associated with the body's surfaces and is ∼10 times greater than the total number of our somatic and germ cells [1]. Accordingly, our gut microbiota can be viewed as a microbial organ placed within a host organ and is composed of different cell lineages with specific metabolic functions and with the capacity to communicate with one another [2].

The normal gut microbiota in obesity

Increased sequencing capacity and bioinformatic capabilities have allowed a dramatic increase in sequencing of the gut microbial metagenomes, in particular the 16 S rDNA gene [3–5]. These studies have significantly improved the understanding of the gut microbial ecology in humans and mice. A recent study [6] has characterized the gut microbial communities in twin pairs and their mothers, an attractive model for assessing the impact of genotype and shared early environmental exposures on the gut microbiome. The results revealed that the human gut microbiome is shared among family members, but that each person's gut microbial community varies in the specific bacterial lineages present, with a comparable degree of co-variation between twin pairs. However, there was a wide array of shared microbial genes among sampled individuals, comprising an extensive, identifiable ‘core microbiome’ at the gene, rather than at the organismal lineage level. Obese twins were found to have phylum-level changes in the microbiota, reduced bacterial diversity and altered representation of bacterial genes and metabolic pathways compared to their lean twin. These results demonstrate that a diversity of organismal assemblages can, none the less, yield a core microbiome at a functional level, and that deviations from this core are associated with different physiological states. Other studies [7] have corroborated these findings in part, whereas another did not [8]. This may be due to different methodologies in each study and highlights the need for standardized enumeration methods of microbial ecosystems. Similar to the decreased levels of Bacteroidetes in obese humans [6,9], obese mice also have decreased Bacteroidetes levels and decreased microbial diversity [5,10]. Metagenomic studies of the obese microbiome have indicated that the distal gut microbiome in obese mice is enriched with genes involved in energy harvest [11]. Importantly, the obese phenotype appears to be a transmissible trait: transplantation of an obese microbiota to germ-free mice results in increased adiposity compared with transplantation of a lean microbiota [11].

While the body is well suited to absorb dietary glucose and to metabolize starch, it does not digest complex polysaccharides readily, which will reach the distal gut and provide energy to the gut microbiota. The gut microbiota is well equipped to cleave most identified glycosidic linkages present in carbohydrates. In fact, the collected genomes of all gut microbes, the microbiome, contains ∼100-fold more genes than our own genome, and one prominent member of the human gut microbiota, Bacteroides thetaiotaomicron, contains 226 glycosidases/lyases alone [12]. It is thus not surprising that germ-free rats cannot metabolize complex polysaccharides such as cellulose [13]. These results indicate how well equipped the microbiota is to extract energy from otherwise indigestible polysaccharides. However, it is inappropriate to assume that the host entirely absorbs this energy. Almost 20% of the fermented carbohydrates are requested to sustain bacterial growth. Moreover, short-chain fatty acids release energy by less efficient pathways than glucose and anaerobic fermentation has only a ∼50% energy conversion rate [13], but may still contribute ∼10% of the daily energy intake in man [14].

In addition to modulating energy absorption, the gut communicates with controllers of energy balance in the brain by means of neural and endocrine pathways [15]. Signalling molecules, e.g. hormones, are produced by specialized enteroendocrine cells in the gut epithelium and are produced at low basal levels. Serum levels of most gut hormones increase transiently within minutes of nutrient intake and activate neural circuits that, in turn, communicate with peripheral organs [15]. The gut microbiota influences the release of a number of these hormones and participates in the regulation of gastrointestinal (GI) endocrine cells and the epithelial structure [16]. The total volumes of gastrin- and serotonin-immunoreactive cells are enlarged in the gastric mucosa of germ-free rats, as well as the total volumes of serotonin-producing cells in the ileum and the colonic mucosa [16]. Moreover, the distal small intestines of germ-free rats contain elevated concentrations of somatostatin, and plasma glucagon levels are increased significantly in germ-free rats [16]. For a more detailed discussion about these pathways see [17,18].

Obesity as an inflammatory disease

Obesity is associated with a low-grade chronic inflammation (also known as metabolic inflammation), which has been implicated in the development of the metabolic syndrome and insulin resistance [19]. Obese subjects have elevated levels of serum free fatty acids (FFAs), which activate proinflammatory pathways [20,21]. In addition, obese mice and humans have elevated circulating endotoxin levels, originating most probably from the gut microbial community [22–24]. However, it should be noted that these levels are in the physiological range and are 10–50 times lower than values that seen in septicaemia or other infections [25]. Chronic administration of low levels of endotoxin to mice increased whole-body, liver and adipose tissue weight, fasting glycaemia and insulinaemia [24].

Metabolic endotoxaemia may, at least in part, be responsible for macrophage infiltration into adipose tissue and increased levels of circulating proinflammatory cytokines and triglycerides observed in obese mice and humans [24]. Interestingly, CD14-deficient mice were protected from metabolic disease both after endotoxin infusion and high-fat feeding. These findings demonstrate that signalling through the endotoxin receptor complex [CD14/Toll-like receptor (TLR)-4] regulates insulin sensitivity and the onset of diabetes and obesity and suggests further that factors that altered intestinal barrier function may be important in regulating the development of the metabolic syndrome. These results are corroborated further by experiments performed in Tlr4-deficient mice [26,27].

High-fat feeding alters the gut microbial composition significantly, in part by reducing numbers of bifidobacteria, which have many physiologically positive effects, including improved mucosal barrier function. Mice fed a high-fat diet supplemented with oligofructose restored bifidobacteria levels and decreased endotoxaemia [28]. These observations suggest that increased levels of bifidobacteria may decrease intestinal permeability and lower the circulating levels of endotoxin. Furthermore, the increase in bifidobacteria correlates with improved glucose tolerance, glucose-induced insulin secretion, lower body weight gain and decreased production of inflammatory mediators [28]. Although the underlying mechanism for endotoxin absorption from the gut is unclear, obese mice have significantly increased paracellular permeability within the gastrointestinal tract, which is accompanied by a dramatic redistribution of tight junction proteins [22,29]. In a recent study, Cani and co-workers demonstrated that bifidobacteria increase gut permeability and tight junction morphology in a glucagon-like peptide 2 (GLP-2)-dependent mechanism [30]. However, how endotoxin micelles penetrate the colonic mucus layer and the epithelial layer still remains to be demonstrated directly. Another, perhaps complementary, mechanism could be that endotoxin is absorbed more proximally in the GI system together with chylomicrons [31].

The normal gut microbiota in type 2 diabetes (T2D)

Insulin resistance is a major contributor to morbidity and mortality worldwide, and both epidemiological and animal studies indicate genetic and environmental factors in the aetiology of this disease [32]. As expected, the decreased adiposity in germ-free mice is associated with improved insulin sensitivity and glucose tolerance [33,34]. However, the gut microbiota may have direct effects on glucose sensitivity, as germ-free mice have improved glucose tolerance [33]. Furthermore, Dumas et al. demonstrated that the gut microbiota may affect insulin resistance and steatosis by regulating cholin metabolism [35]. The metabolism of choline to methylamine is performed entirely by the gut microbiota, and trimethylamine (TMA) is absorbed from the gut and transported to the liver, where it is cleared by hepatic first-pass metabolism. The putative mechanism for the increased insulin resistance and steatosis could be elevated microbial metabolism of choline to TMA, which reduces the bioavailable levels of cholin, which is a precursor for phosphatidylcholine and could cause hepatic triglyceride accumulation [36]. Metabolic inflammation caused by elevated endotoxin levels, or microbial regulation of hormones that regulate glucose homeostasis, may be additional pathways by which the gut microbiota affect host glucose metabolism.

The normal gut microbiota in cardiovascular disease

During past decades infectious agents [37] and periodontitis [38] have been associated with increased risk for cardiovascular disease. However, clinical trials to reduce atherosclerosis by antibiotics have been largely unsuccessful [39]. Although the underlying reason remains unclear, one potential explanation could be that these studies have aimed to eradicate only one type of organism (e.g. Chlamydia pneumoniae). A recent study disclosed that bacterial diversity in human atheromas is high and C. pneumonia is present in about 50% of the plaques [40]. Thus, antibiotic treatments that target specific bacteria may be unsuccessful, and more appropriate targets could be signalling pathways that are induced by the bacteria. A recent study from Holmes et al. demonstrated that dietary (alanine) and gut microbial (hippurate)-derived urinary metabolites are correlated with blood pressure in humans [41]. These findings suggest that alterations in the gut microbiota may modulate the development of cardiovascular disease.

Atherosclerosis is now considered an inflammatory disease, and individuals with TLR-4 polymorphism (D299G) have decreased risk for carotid atherosclerosis and reduced levels of acute phase reactants and soluble adhesion molecules [42]. Deletion of Tlr4 and Myd88 in mice on an Apoe^-/- background reduced the development of atherosclerosis by reducing macrophage infiltration and foam cell formation [43,44]. Collectively, these findings suggest that the gut microbiota may contribute to the development of atherosclerosis by activating TLR signalling. In contrast to this hypothesis, Wright et al. reported that mice carrying a point mutation in TLR-4 did not show reduced atherosclerosis, as judged by cholesteryl ester content in whole aortas [45]. Furthermore, they showed that germ-free mice on an Apoe^-/- background were not protected against atherosclerosis. It should be noted that their studies were performed at an advanced state of atherosclerosis and does not exclude that microbes affect the onset of atherosclerosis. Thus, further studies are required to investigate the role of the gut microbiota in the aetiology of atherosclerosis.

The normal gut microbiota in T1D

The incidence of type 1 diabetes (T1D) among children and adolescents has, for reasons unknown, increased markedly in western countries during recent decades. Interestingly, an altered gut microbiota has been associated with T1D progression [46,47]. Decreased levels of Bacteroidetes in the gut microbiota may contribute to the pathogenesis of T1D [46]; interestingly, these changes were detectable long before the clinical onset of the disease [46]. A recent study showed that gut microbiota and its interaction with the innate immune system are important factors affecting predisposition to T1D in the non-obese diabetic (NOD) mouse [47]. Specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple TLRs that recognize microbial stimuli) do not develop T1D. Interestingly, the effect is dependent upon normal gut microbiota, because germ-free MyD88-negative NOD mice have increased incidence of diabetes which is indistinguishable from their wild-type counterparts. The authors demonstrated further that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. These studies suggest strongly that variation in an individual's microbiota may affect the development of T1D.

Conclusions and future challenges

Accumulating data from both patients and animal models suggest that microbial ecosystems associated with the human body, especially the gut microbiota, may be associated with several important diseases, such as inflammatory bowel disease, obesity, diabetes and cardiovascular disease. However, the underlying mechanisms for how bacteria cause disease are only just beginning to be unravelled. One major challenge is to establish uniform methods for determining the microbial composition. Today, each laboratory uses their own methods, which makes cross-laboratory comparisons difficult. An even more difficult challenge will be to identify the means to modulate the gut microbiota (or its signalling) to prevent disease and promote health.

Disclosure

None.