Host–Flora Interactions in Inflammatory Bowel Disease

Advances in complex disorders frequently occur when a research endeavor settles at the interface of seemingly disparate disciplines. The conceptual leaps achieved in hybrid fields, such as neuroimmunology or microbial immunophysiology, are testimony to the importance of thinking beyond the traditional confines of any research field. Modern technology has also expanded the scope of questions that can be asked by curious investigators from different backgrounds. This sharing of minds has been well illustrated in the study of inflammatory bowel disease (IBD), in which there is increasing acceptance of the importance of gene–environment interactions, and particularly host–flora dialogue in the gut. Exploring the cellular and molecular mechanisms underpinning such interactions has already provided a glimpse of exciting future therapeutic strategies. Different aspects of the bacterial flora within the gut have been examined in several comprehensive reviews that include reference to earlier work. 1,–11 Therefore, in this overview of host–flora interactions, emerging concepts, trends, and controversies will be highlighted. In recent years, there has been a sustained resurgence of interest in the commensal bacterial flora within the gastrointestinal tract. This has been driven by several influences, perhaps the most important being increasing recognition of the role of bacteria in the pathogenesis of various complex intestinal disorders, including Crohn's disease, ulcerative colitis, pouchitis, colorectal cancer, and possibly dysfunctional or irritable bowel syndromes. The lesson learned with the discovery of the role of Helicobacter pylori in peptic ulcer disease, gastritis, and gastric cancer has had a sobering effect on many investigators and opened minds to the possibility of a microbial contribution to the etiology or pathogenesis of other disorders. In retrospect, the H. pylori story shows the importance of a holistic investigative approach, spanning traditional research disciplines in the study of complex disorders. It is now evident that the riddle of peptic disease would not have been solved if research remained focused solely on mammalian physiology and host responses to injury. It is only when the host response is examined in the context of the bacterial residents within the gut lumen that the full picture emerges. It seems plausible that prokaryotic–eukaryotic interactions awaiting discovery may underlie or modify other complex syndromes, such as Crohn's disease and ulcerative colitis. In view of the magnitude and complexity of the alimentary ecosystem, it seems surprising that the rediscovery of the importance of the gut flora is only a latter-day occurrence. Thus, it is difficult to escape sensational statistics in describing the enteric flora. This living mass accounts for 1 kg to 2 kg of body weight, has a cellular content that outnumbers eukaryotic cells of the host by ten-fold, represents a combined genetic microbiome exceeding the human genome by one hundred-fold, and exhibits a collective metabolic activity that eclipses that of the liver. The gut flora is tantamount to a hidden organ. 12 The complexity of this virtual organ or ecosystem is greatest at either end of the alimentary tract. In the oral cavity, there are about 200 different bacterial species, whereas the stomach is almost sterile in many cases. In the intestine, bacterial density increases distally with the greatest gradient being across the ileocecal valve, where there are approximately108 bacteria per gram of ileal content versus up to 1012 per gram of colonic content. 4,6 The composition of the flora also varies along the cross-sectional axis of the gut, with the ratios of anaerobes to aerobes being lower at mucosal surfaces compared with the lumen or feces. Comparatively little is known of the metabolic activity of the bacterial residents within the gut; this is probably determined largely by dietary variables and microbe–microbe interactions. There is also some evidence for host genetic influences on the flora. 13 Renewed pursuit of the gut flora is, in large part, also due to the development of modern molecular technology to overcome the limitations of traditional culture-dependent studies. 14,15 About half of the resident bacteria in the gut are non-culturable. In addition, the numerically predominant components of the flora are obligate anaerobes, and this poses methodologic difficulties in sampling, transport, and storage. 16 Rapid molecular profiling of the dominant culturable and non-culturable organisms can be achieved by chemical- or temperature-gradient gel electrophoresis (DGGE or TGGE) using amplified bacterial nucleic acid extracted from the feces or biopsies. 14,15 This can be further refined by using species-specific PCR primers. While the composition of human gut flora was previously thought to be relatively stable after weaning in infancy, this type of molecular fingerprinting has suggested a greater degree of variability depending on host and environmental variables. In addition, the increasing availability of bacterial genomic sequence data has facilitated the development of molecular probes for identification and enumeration of individual species within complex bacterial populations. The methodologic menu includes fluorescent in situ hybridization analysis (FISH), alone or combined with flow cytometry (FISH–FLOW), DNA microarrays, and gene chip technology. Technologic developments to explore specific bacterial involvement in the pathogenesis of inflammatory or other diseases include the use of marker antibodies, generated by hybridoma or phage display, to identify microbial antigens. Anti-neutrophil cytoplasmic antibodies (p-ANCA) associated with ulcerative colitis appear to identify colonic bacteria expressing a p-ANCA-related epitope. 17,18 Candidate microbes have also emerged from searches for unique bacterial nucleic acid sequences associated with particular locations or lesions in Crohn's disease. In one series of studies, this strategy implicated a bacterial transcription factor from an apparent commensal organism, Pseudomonas fluorescens,19,–21 the significance of which requires further study. The influence of commensal bacteria on intestinal structure and function was shown in comparative studies of germ-free and conventionally colonized animals several decades ago. Life without bacteria is associated with reductions in mucosal cell turnover, digestive enzyme activity, cytokine production, lymphoid tissue, lamina propria cellularity, vascularity, muscle wall thickness, and motility. In contrast, the absence of bacteria is associated with an increase in enterochromaffin cell area. 22 These observations imply that the flora exerts both positive and negative regulatory effects on the development and function of the intestine. Furthermore, these regulatory signals act at the epithelial and sub-epithelial levels. The identity of these signals can now be pursued with modern technology, including laser capture microdissection and gene array analysis. 23 The flora also has important metabolic functions. Germ-free animals need a significantly greater caloric intake to sustain a normal body weight similar to that of colonized animals. Thus, the normal host–flora relationship is not one in which the host is in nutritional deficit because of the presence of bacterial guests within the gut. Important metabolic effects of the flora include the production of short chain fatty acids—a major energy source for colonic epithelia—from dietary fermentable carbohydrates. Other metabolic activities include breakdown dietary carcinogens and synthesis of biotin, folate, and K vitamins. 4,24 Clinicians treating IBD throughout much of the past century have used the azo reductase activity of the colonic flora to metabolize the prodrug, sulfasalazine, to its active aminosalicylate moiety. Knowledge of the metabolic activity of the intestinal flora is still relatively superficial, but given the scale of the activity of the intestinal microbiome, exploration must offer strong potential for therapeutic exploitation in health and disease. The presence or absence of the intestinal flora has pivotal implications for the development and function of the mucosal immune system. Mucosal-associated lymphoid tissue (MALT) is rudimentary in germ-free animals and associated with defective-cell mediated immunity. 25 This is due to defective microbial education of immune responses early in development and, for example, the absence of microbial induction of interleukin-12 (IL-12). The presence of a commensal flora is necessary for fine tuning of T cell repertoires and TH1/TH2 cytokine profiles. 26 Since environmental exposure including dietary and other variables influence the composition of the colonizing flora early in life, it also influences the development of the mucosal immune system and may also account for individual variations in immunity and apparent species differences in mucosal immune responses. In mice, the normal default mucosal immune response to innocuous antigens within the lumen of the gut is generally thought to be biased toward a TH2/TH3 profile of cytokine production, 20 whereas in humans, mucosal T cell responses are TH1 biased. 27 The immune system mediates the sense of danger within the local microenvironment. As with other senses, it has receptors for detecting environmental signals, an afferent limb for uptake of information, an efferent response limb, and it exhibits memory and learning from experience. 28 Host–microbial exchanges occur primarily at mucosal surfaces, the gut mucosa being the largest area (c.400m2), and is structurally adapted to environmental interaction with only a single layer of epithelium separating the lumen from the internal milieu. Immunologic sampling of the lumen takes place across the epithelium at several sites. M cells, which overlie lymphoid follicles, transport particulate and some microbial antigens to subjacent antigen-presenting cells (dendritic cells, B cells and macrophages). 29 In contrast, surface enterocytes transport soluble antigens and also can serve as afferent sensors of danger within the luminal microenvironment by producing chemokines, which alert the host innate and acquired immune responses and direct them to breaches in mucosal barrier with infection. 30 Specialized Paneth cells within the epithelium may also exhibit microbial discriminatory responses in relation to production of defensins. 31 Dendritic cells within the gut are probably critical for mucosal immunosensory function. 32 These antigen-presenting cells have been observed within intestinal epithelium in rodents, 33 and there is evidence from in vitro modeling that subepithelial dendritic cells may extend into the lumen between the surface enterocytes without disrupting tight junctions. 34 Evidence for dendritic cell heterogeneity has been shown in mice with tissue-specific specialization within the gut that may determine local immune responses to antigens from the lumen. 35 Dendritic cells are pivotal contributors to the initiation of the innate and adaptive immune responses. They exhibit remarkable plasticity of sensory responses to diverse pathogens with pathogen-specific responses demonstrable by gene array analysis. 36 The molecular mechanisms underpinning host-flora interactions are now an area of focused investigation. Incoming bacterial signals include secreted chemoattractants, such as the formulated peptide f-met-leu-phe, cellular constituents such as lipopolysaccharide (LPS) and peptidoglycans, and bacterial nucleic acids (CpG DNA). Detection of bacterial stimuli by the host and discrimination of pathogens from commensals is mediated, in part, by pattern recognition receptors (PPRs), which include toll-like receptors (TLRs) 37 and C-type lectins. TLRs are expressed differentially among immune cells which appear to respond to different stimuli. TLRs are also expressed by intestinal epithelial cells which may be differentially altered in ulcerative colitis and Crohn's disease. 38,39 Multiple TLRs are probably used simultaneously by immunocytes to recognize the features of a specific microbe. TLR2 recognizes lipoproteins and peptidoglycans and triggers the host response to gram-positive bacteria and yeast; TLR4 mediates responses to LPS primarily from gram-negative bacteria; TLR1 and TLR6 participate in activation of macrophages by gram-positive bacteria, whereas TLR5 and TLR9 recognize flagellin and bacterial DNA DNA and whereas eukaryotic DNA and for cytokine production in response to pathogens are not known has focused on the transcription factor factor and the factor Thus, is a of epithelial responses to pathogens such as some components of the flora have been shown to responses by of the factor of a of mechanisms by which pathogens can escape or immune in the gut. and bacterial pathogens have been shown to to the internal by the M cell 29 whereas the human immune also the sampling function of cell by bacteria are These include the of and of within of and the of epithelial to increase mucosal surface area. and pathogens may dendritic cell function and escape immune by pattern recognition receptors such as the C-type (dendritic mucosal and in with it is remarkable that bacterial has not of bacteria from the gastrointestinal to including the and other end to occur at a in The have a effect on and bacteria are probably In humans, has been in it is in the particularly in presence of bowel and in disorders While to be in T cell and in is in these In contrast, bacteria are frequently with in with defective bacteria in to from the gastrointestinal tract. and other gram-negative anaerobes at a greater obligate anaerobes and gram-positive Thus, the bacterial species most to are generally the as that in The pathogenesis of IBD a of genetic environmental and immune This to be a of most or In the most view is that the development of disease an immune response to normal flora in a a normal response to flora. the must for heterogeneity within both Crohn's disease and ulcerative colitis, and as a in a of be The complexity of interactions is well illustrated in of of the genetic bacterial is an for full of disease whereas the of disease in a given varies with the into which it is In humans, the of interactions among bacteria, and immunity has been the discovery of the gene and its with Crohn's disease. The of this gene as an for bacterial peptidoglycans and is with the activation of which the of account for to of Crohn's disease, this genetic factor is necessary for development of the disease. It seems that will be that the of the microbial microenvironment the host response to This would be with evidence for defective of bacteria and bacterial in the mucosa of with Crohn's disease, compared with and inflammatory disease Other defective innate immunity to bacteria in with the evidence for a in immune in IBD is While that in immunity can to also that defective immunity is not necessary and that a heterogeneity of may to mucosal T cell activation for any is associated with of tissue and there is evidence for defective of mucosal T cell activation at in with Crohn's disease. responses are by induction of in T of mucosal T cells to to and of in Crohn's disease. The molecular of this is in part, to and of T by of in Crohn's disease. of mucosal T cell in part, for the of the T cell activity is also in by regulatory T cells T cells are defective in human IBD is but the of T cells that has shown therapeutic in animals. in intestinal T activity also some of the effects of in and with activity at the of T seems in of apparent in both and disorders. to the pathogenesis of IBD is shown by in for Crohn's disease and ulcerative colitis influences are to be and at different levels. example, has effects on disease with an effect on the of Crohn's disease in to an apparent effect on ulcerative colitis. most environmental triggers or have not been but are implicated by recent increases in the and of particularly with of the short the of IBD be for by in but it be due to a the lesson of H. pylori and peptic ulcer disease, it would be to an contribution to the pathogenesis of It is that gut mucosa has a of immune to and intestinal pathogens have been shown to be of mucosal cytokine responses similar to in of a within the gut not be in specific IBD because of a to or the has been The most as of IBD is While has been from intestinal of with Crohn's disease or in a and effect relationship is from the lumen has not been the of laser capture microdissection has been used to for DNA in the of tissue, on the that are the most in which be DNA is in some molecular of other of bacteria such as are also This may innate immunity to bacteria in a specific and effect relationship with disease. 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Furthermore, it is not necessary to a traditional to account for the environmental contribution to the of As the environmental influence may be and to in associated with modern The of IBD associated with been by similar in other inflammatory disorders, such as and It is that of these are by different Since some of immune is to it seems plausible that environmental act at the of the immune system at the organ. 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