The role of the gut microbiota in health and disease has received considerable scientific interest recently. Especially, the development of new culture-independent techniques has rekindled the interest in intestinal microbial ecology. The gut microbiota has been linked to the risk of gastrointestinal diseases such as inflammatory bowel diseases (IBD) (1–4), irritable bowel syndrome (IBS) (5, 6) and necrotizing enterocolitis (7–9). However the role of the gut microbiota in health and disease may go even beyond the gut as it has also been linked to atopic diseases. This review aims to give a comprehensive overview of observational studies of the association between the gut microbiota composition and atopic disorders, with a special focus on the methods used to characterize this microbiota.
The commensal microbiota of the gastrointestinal tract
The fetal intestine is sterile and bathed in swallowed amniotic fluid. Following delivery, the colonization of the intestines by a variety of microorganisms begins (10). Gastrointestinal colonization involves a succession of bacterial populations waxing and waning as the diet changes and the host develops (11). This assemblage of bacteria inhabiting the gut is usually referred to as the commensal intestinal microbiota. Each human adult harbors approximately 1014 bacteria in the gut, which is about 10 times the number of cells making up the human body (12). There are at least 400–500 different bacterial species and these species can again be divided into different strains, highlighting the enormous complexity of this ecosystem. Furthermore the composition of this microbiota differs depending on their location in the gut. The concentration of bacteria ranges from 103 colony-forming units per millilitre (CFU/ml) in the stomach, where the number of ingested bacteria is dramatically reduced by contact with gastric acid, to 1011–1012 CFU/ml in the colon (Fig. 1) (13). The colonic microbiota is dominated by obligatory anaerobes such as Bacteroides spp., Clostridium spp., bifidobacteria, eubacteria, and fusobacteria. Facultative anaerobes occur in 100- to 1000-fold lower numbers and include lactobacilli, enterococci, streptococci and Enterobacteriaceae (Table 1) (12, 14). In addition to variations in the composition of the microbiota along the axis of the gastrointestinal tract, surface-adherent and luminal microbial populations also differ (15). Bacteria may be free-living in the lumen or attached to the mucus, mucosal surface, food particles or digestive residues. The attached bacteria produce microcolonies, leading to the development of biofilms which initially may be composed of only one bacterial species, but frequently develop into a complex community composed of different bacterial species (16).
|Obligatory anaerobic genera||Facultative anaerobic genera|
Factors influencing the intestinal microbiota composition can be divided into host factors (such as pH, transit time, bile acids, pancreatic enzymes and mucus composition), non-host factors (such as nutrients, medication and environmental factors), and bacterial factors (such as adhesion capacity, enzymes and metabolic capacities) (17). The bacteria in the gut interact with their human host, and although some bacteria are potentially pathogenic and can become a source of infection and sepsis, this host–bacterial interaction is mainly symbiotic. The host provides a nutrient-rich environment and the bacteria can confer important health benefits upon the human host (18). Probably the most important function of the gut microbiota is the so-called colonization resistance. By not only competing for nutrients and adhesion sites, but also by the production of antibacterial substances (bacteriocins), the indigenous gut microbiota makes it difficult for potentially pathogenic bacteria to colonize. Other important functions are the fermentation of non-digestible dietary residues and endogenous mucus, salvage of energy as short-chain fatty acids, production of vitamin K and absorption of ions (18).
Furthermore the gut-associated lymphoid tissue (GALT) is the largest immune organ of the human body, which is exposed to an enormous dietary and bacterial antigenic load. Studies of germ-free animals have shown that the gut microbiota plays an important role in the development of the gastrointestinal immune system. Germ-free animals have among others decreased Peyer’s patch size, decreased number of IgA-producing lymphocytes in the lamina propria, decreased number of intra-epithelial T cells and a delayed immune response after antigenic challenge compared with conventional animals (12, 19–23).
From culture to genotype analysis
Research into the intestinal microbiota composition has relied almost exclusively on the quantitative culture of microbes from fecal samples. Enumeration of particular microbial genera or species relies on the use of selective culture media. The analysis of the composition of the normal microbiota using these media is undoubtedly biased by the inability to culture all of the microbes present in samples (about 40–80% of bacteria as seen by microscopy cannot be cultured), the fact that few selective media are absolutely selective and that these media do not always equally support the growth of different species comprising a population. Even when culturable, the identification to species level using biochemical tests requires experience and is subject to intuitive interpretations (18, 24). Another major disadvantage in using classical culturing techniques in large-scale epidemiological studies is that samples require immediate processing (25). The relative inexpensiveness and wide availability on the other hand make these classical techniques the most applied.
The development of molecular techniques to investigate ecological microbial communities has provided the microbiologist with a vast array of new techniques to study the human intestinal microbiota. With these techniques, unculturable species are detectable, anaerobic handling and expertise are not required and samples can be kept frozen for later analysis (25). Analysis of bacterial communities using molecular techniques has so far targeted 16S rRNA gene sequences because the small ribosomal sub-unit RNA (16S rRNA in the case of bacteria) contains regions of highly conserved nucleotide base sequences interspersed with hypervariable regions (26). These hypervariable regions contain the signatures of phylogenetic groups, and, sometimes even species.
Fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) combined with denaturing- or temperature-gradient gel electrophoreses (PCR-DGGE/PCR-TGGE) and real-time PCR are molecular techniques which have found application for studying gut microbiota (27). These techniques all have their advantages and limitations. FISH is based on the use of fluorescent oligonucleotide probes targeting 16S ribosomal RNA sequences of intact bacterial cells. Technical difficulties can influence the accuracy of the results. Probes must reach their target sequence, which is inside the bacterial cell, bypassing the cell wall. Gram-positive bacterial cells such as lactobacilli, for example, are more difficult to permeabilize than others (28). Furthermore this method is rather insensitive with detection limits of 106 bacterial cells per gram. This technique is particularly useful to visualize the spatial distribution of microbes within the intestinal ecosystem.
Another molecular method is PCR-TGGE/-DGGE. In this method of analysis, bacterial DNA is extracted from the fecal sample and fragments of the 16S rRNA gene are amplified by PCR; subsequently the 16S molecular species within the resulting mixture are separated by TGGE/DGGE. The double-stranded 16S fragments migrate through the polyacrylamide gel until each kind of fragment is partially denatured by the prevailing temperature or chemical conditions (26, 28). The advantage of PCR-DGGE/-TGGE is that it generates a bacterial fingerprint of the dominant bacteria in a sample. Knowledge about the bacterial composition is unnecessary. The technique is however not quantitative, rather insensitive and very laborious, making it unsuitable for analysis of large numbers of samples.
The quantitative real-time PCR method monitors the amount of PCR products of DNA as they are amplified by the use of fluorescent oligonucleotide probes. The fluorescence intensity emitted during the amplification process reflects the amplicon concentration in real time. From the change of amplicon concentration throughout the amplification cycles, the initial concentration of the target DNA/RNA can be estimated (29, 30). Real-time PCR lends itself well as a tool for the quantification of intestinal populations as it combines the specificity of fluorescent oligonucleotide probes with the sensitivity of PCR (28). Care should however be taken regarding the method used for DNA/RNA extraction, as DNA/RNA may not be extracted with equal efficiency from all bacteria (13).
The microbiota hypothesis in atopy
Very recently it was hypothesized that the gut microbiota may also be involved in the etiology of atopic diseases. Atopic diseases are chronic inflammatory disorders caused by aberrant T-helper 2 (Th2)-type immune responses against common ‘innocuous’ environmental antigens (allergens) in susceptible individuals (31). The worldwide rise in atopic diseases (eczema, food allergy, hay fever and asthma) was most predominant in the westernized countries and occurred in such a pace that this could never be solely explained by changes in the genetic make-up (32, 33). Therefore the causes of the atopic epidemic are generally believed to be of environmental origin. In 1989 Strachan hypothesized that this increase in atopic disease was the result of a lack of infections in early infancy. This hypothesis was based upon Strachan’s observations that infants with higher number of siblings were at decreased risk for developing atopy (34). Although sibship size (35, 36), and other indirect markers of microbial exposure such as rural and farm-living (especially contact with livestock) (37, 38) were consistently shown to be associated with a decreased risk of developing allergies, studies of the association between viral and bacterial infections and allergy were less consistent (38, 39). In 1998 Wold suggested that rather than a decrease in viral or bacterial infections, an altered normal intestinal colonization pattern in infancy, which fails to induce immunological tolerance, could be responsible for the increase in allergies (40). This idea of a potential role of the gut microbiota was based on the observations that (i) it is difficult to achieve oral tolerance in germ-free animals (41); (ii) administration of lipopolysaccharide (LPS; a constituent of the outer membrane of gram-negative bacteria) together with food antigens increases the tolerizing effect of feeding (42); (iii) and bacterial toxins may break oral tolerance (43). Since then, several observational studies of the gut microbiota composition and allergy have been conducted.
Potential immunological mechanisms
The innate immune system may be decisive in determining the type of adaptive immune responses elicited against microbial antigens. Of the innate immune cells, dendritic cells (DC) seem to be pivotal in the earliest bacterial recognition and in shaping T-cell responses (44). Innate immune cells recognize microbial antigens through molecules such as Toll-like receptors (TLRs), and nucleotide-binding oligomerization domain (NODs) molecules, which recognize conserved pathogen-associated molecular patterns, including unmethylated CpG motifs characteristic of bacterial DNA, the bacterial LPS and peptidoglycan (45).
The initial immunological explanation for the hygiene hypothesis was a lack of microbial antigen-induced immune deviation from the Th2 cytokine profile to a Th1-type profile, resulting in the development of enhanced Th2-cell responses to allergens (46–48). However, this explanation did not take into account that the prevalence of Th1-associated diseases, such as Crohn’s disease, type 1 diabetes and multiple sclerosis, was also increasing and that chronic parasitic worm (helminth) infections which induce strong Th2 responses and high IgE levels are not associated with an increased risk of allergy (49).
An alternative interpretation conceives anti-inflammatory immune responses to be of fundamental importance in the development of mucosal and systemic tolerance (50). These immunosuppressive mechanisms are orchestrated by regulatory T-cell classes (Treg cells) that control [largely via the production of interleukin (IL)-10 and/or tumor growth factor (TGF)-β] both Th1 and Th2 responses and hence the development of both atopic and autoimmune diseases (50, 51). Indeed the importance of a delicate balance between allergen-specific Treg cells and allergen-specific Th2 cells in healthy and allergic immune responses to common environmental allergens was demonstrated in a study conducted by Akdis et al. (52). Furthermore, a study of duodenal biopsies of healthy infants and infants with multiple food allergy showed that the dominant mucosal abnormality was not Th2 deviation but impaired generation of TGF-β-producing Treg cells (53).
Relatively harmless organisms, including not only bifidobacteria and lactobacilli, but also helminths and saprophytic mycobacteria, may skew immune responses toward immunoregulation by inducing Treg cells, rather than eliciting a proinflammatory immune response. For example, Lactobacillus paracasei has been reported to inhibit the secretion of both Th1 and Th2 cytokines, while inducing the development of a population of CD4(+) T cells producing TGF-β and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis (54).
Lactobacillus reuteri and Lactobacillus casei have been shown to prime monocyte-derived DCs to drive the development of IL-10-producing Treg cells, through binding of the C-type lectin DC-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) (55). The Bifidobacterium genomic DNA has been reported to induce the secretion of IL-10 by peripheral blood mononuclear cells (PBMCs) from healthy donors in vitro (56).
The ‘microbiota hypothesis’ proposes that the loss of exposure to these harmless microorganisms in the westernized environment might explain the increase in immune dysregulatory disorders (57, 58). The epidemiological findings and the experimental evidence available so far suggest that both the reduced immune suppression by Treg cells and the lack of immune deviation from a Th2 to Th1 profile are involved (59). Furthermore, the impact of the gut microbiota on the development of IgA antibody responses, which contribute to pathogen and allergen exclusion in the gut lumen, may also be involved (45).
It has been proposed that the effects of the gut microbiota may not only be related to food antigens, but also to aeroallergens and the manifestation of allergic airway symptoms. Noverr et al. developed a mouse model to demonstrate experimentally that antibiotic therapy, leading to bacterial and fungal microbiota changes, could predispose a host to allergic airway disease (60). Furthermore, oral treatment with live L. reuteri has recently been shown to inhibit the allergic airway response in mice (61). These results support the possibility that afferent events in allergic sensitization may occur outside of the lungs and involve host–microbiota communication.
The mechanisms by which events in the gut can affect the systemic immune system and local inflammation in remote tissues such as the respiratory tract remain to be determined. However, it has been shown that inhaled particles, fluids and microbes are also swallowed. The gastrointestinal tract will, thus, be exposed to any antigens to which the respiratory tract is also exposed. As ingestion of antigens can induce tolerance to that antigen (oral tolerance), the gastrointestinal (GI) tract may act as a ‘sensor’ for the development of tolerance to inhaled antigens (11, 62). Induced regulatory T cells may thereafter home in other tissues throughout the body, in particular in other mucosal tissues such as the respiratory tract (63).