Role of gut microbiota in Crohns disease

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Role of gut microbiota in Crohn’s disease Expert Rev. Gastroenterol. Hepatol. 3(5), 535–546 (2009)

Phillip I Baker, Donald R Love and Lynnette R Ferguson† Author for correspondence Department of Nutrition, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand [email protected] †

Crohn’s disease (CD), a form of inflammatory bowel disease (IBD), provides a complex model of host–microbe interactions underpinning disease pathogenesis. Although there is not widespread agreement on the etiology of CD, there is evidence that microorganisms lead to the often severe inflammatory response characteristic of the disease. Despite several microbial candidates, no specific microbe has been considered pathogenic. Instead, the concept of the ‘pathogenic community’ has emerged from the evidence, whereby the stability of the microbial ecosystem of the healthy human gut is disrupted in response to host genetics and destabilized immunity, perhaps through changing public health practices leading to altered microbial exposures over time. We discuss the complex microbial ecosystem of the mammalian gut, the underlying genetic factors that predispose to CD, and how these gene variants may alter host–microbe interactions and propagate inflammation. Over the next 5 years, the increased understanding of genes involved in CD and the way in which individuals with variants of these genes respond differently to nutrients and drugs will enable the rational development of personalized therapies, using pharmacogenomic and nutrigenomic approaches. Keywords : Crohn’s disease • genetics • inflammation • inflammatory bowel disease • microbiology

Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn’s disease (CD), which differ in their clinical manifestations, cytokine profiles, genetic pathology and hypothesized etiology. Clinical and experimental evidence suggests that the pathogenesis of CD involves an inflammatory response targeted at the commensal microflora residing in the intestinal tract of a genetically susceptible host. Although the microflora may also be implicated in UC, this review will focus on CD. Four lines of evidence implicate bacteria in CD pathogenesis. First, the sites of highest concentrations of bacteria in the human gut correspond to the most common sites of inflammation in CD patients, and this inflammation may be attenuated by prolonged courses of antibiotics and/or an elemental diet [1,2] . Second, diversion of the luminal stream (which contains a high content of microbiota) away from the inflamed bowel is associated with disease improvement, whereas the exposure of an excluded ileum to fecal matter results in localized inflammation [3,4] . Third, biopsies have provided evidence that tissues from either colonic or small intestinal CD have a reduced ability to kill microbes compared with normal tissues [5] . Last, in www.expert-reviews.com

10.1586/EGH.09.47

animal models of IBD, inflammation occurs in a nonsterile environment but not under germ-free conditions [6,7] . The resident microflora of the healthy human gut are commonly referred to as the commensal microflora, where commensal is defined as a symbiotic relationship between two organisms in which one partner benefits (the microbe) and the other partner neither benefits nor is harmed (the host) [8] . Not all bacteria in the gut fall under this definition, as some confer benefit to the human host, redefining the relationship as mutualistic. However, in IBD these definitions become blurred, as the symbiotic microflora may together become a pathogenic entity or ‘pathobiotic’ [9] . There has been considerable evidence of associations of CD with several specific bacteria and viruses. In particular, increased levels of the bacterium Mycobacterium avium subspecies paratu‑ berculosis (MAP) and of the adherent-invasive Escherichia coli strain LF82, but reduced representation of the phylum Firmicutes, and particularly of the species Faecalibacterium prausnitzii, have been repeatedly observed [8,10–13] . The MAP bacterium has been implicated because it is often found in diseased tissues and is recognized as a chronic enteric pathogen that is able

© 2009 Expert Reviews Ltd

ISSN 1747-4124

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to act as a primary cause of chronic inflammation of the intestine in a range of different species, including nonhuman primates [10] . However, there is not complete agreement as to whether this is just a bystander organism in CD or if it is involved in disease causation, although current evidence favors the latter theory. Although it is not believed that this bacterium is responsible for the gross inflammatory response in CD, it seems possible that it minimizes its own immune recognition. In this respect, it appears that MAP could be involved in the primary immune dysregulation in CD (as in animals) [14] . This might imply that the gross inflammatory changes that occur in CD result from the perturbed neuroimmune response to the secondary penetration into the gut wall by both the normal gut flora and by dysbiotic gut flora. However, the evidence suggests that these microbes are not pathogenic per se. Instead, it appears that host–microbe inter actions are altered at broader taxonomic levels, and that an inflammatory response is initiated against a pathogenic community of microorganisms rather than one species alone. Metagenomic experiments have shown that entire classes of bacteria may be over-represented or even lost during CD [15] . Given that the innate and adaptive immune responses differ significantly between even closely related species of bacteria, these findings suggest that dysbiosis (the condition of having microbial imbalances on or within the body) may play an important role in propagating inflammation [8,16] . Dysbiosis may also influence other important components of gastrointestinal health, including the synthesis of short-chain fatty acids (SCFAs), lipid storage, colonization resistance, vascular proliferation, and cell growth and differentiation [17–20] . The pathogenesis of CD is strongly associated with host genetics. A meta-analysis of genome-wide association studies (GWAS) of over 8000 individuals identified 30 distinct susceptibility loci, clearly indicating the polygenic nature of the disease [21] . Most interest to date has focused on several single nucleotide polymorphisms (SNPs) within the genes nucleotide-binding oligomerization domain containing-2 (NOD2) and autophagy-related 16-like 1 (ATG16L1). These genes are implicated in the cytosolic recognition and processing of intracellular bacteria and are important components of cellular immunity [22–25] . Of the other identified susceptibility genes, several are also associated with host–microbe interactions [21] . The disease may, therefore, result from the complex interplay between the microflora and host genetics. On this basis, this review aims to understand the complex microbial ecosystem of the mammalian gut, the underlying genetic factors that predispose to CD, and how these gene variants may alter host–microbe interactions and drive the inflammatory response.

after which they are exfoliated into the lumen. This provides a rich nutrient source for the resident microflora, which in humans equates to approximately 250 g per day [28] . Four epithelial cell lineages are derived from omnipotent stem cells. Three of these follow the crypt-to-villus migration process and include the enterendocrine cells, the mucus-producing goblet cells and the absorptive enterocytes, the latter comprising 80% of the epithelial cell mass. The fourth lineage are the paneth cells of the innate immune system, which follow a downward migration to the base of the crypt where they are phagocytosed by adjacent cells. These cells play an important part in regulating the microflora composition of the gut through the secretion of antimicrobial lysozymes and defensins [28,29] .

The human gastrointestinal environment

Microbiology

The gastrointestinal epithelium represents the first line of defense against invading pathogens, containing a complex arsenal of innate and adaptive defense mechanisms, which must discriminate between beneficial and pathogenic bacteria [26,27] . Epithelial cell turnover proliferates from omnipotent stem cells located at the base of the mucosal crypts of Lieberkühn. Migration of cells from the crypt to the distal villus is complete within 2–5 days,

Following the transition from a fat (milk)-rich to a carbohydrate-rich diet during infancy, the microflora shifts significantly in composition towards a community dominated by the Bacteroidetes (Gram-negative obligate anaerobes) and Firmicutes (low guanine and cytosine Gram-positive obligate anaerobes) phyla and other obligative anaerobes [34,35] . Densities of bacteria remain low in the proximal and middle small intestine

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Immunity

The immune system is defined as a nonself-recognition system, with ‘self’ defined as the products derived from the individual host genome and the microflora viewed as commensal entities that cumulatively form the bacterial genome or ‘bacteriome’. Evolutionary processes have stabilized innate and adaptive immune recognition against the bacteriome, which may become destabilized in CD and subsequently propagate inflammation [30] . Epithelial cells, dendritic cells, macrophages and neutrophils recognize pathogens via germline-encoded pattern-recognition receptors (PRRs). These are divided into several groups, including mannose receptors, complement receptors, lectins, scavenger receptors, Nod-like receptors and Toll-like receptors (TLRs) [31] . Each group of receptors has evolved to recognize a limited number of distinct and highly conserved products produced by the microflora, termed pathogen-associated molecular patterns (PAMPs). Girardin et al. showed that NOD2 is differentially expressed when exposed to bacteria from different phylogenetic groups and their metabolites isolated from culture supernatants [32] . The same authors demonstrated that even bacteria of the same genus, for example, Bacillus subtilis and Bacillus vulgatus, have substantially different NOD2 stimulatory effects. This indicates that even subtle changes in the microbial composition may influence the innate immune response and inflammatory processes. Importantly, PRRs do not recognise host-derived products and are therefore a key determinant of host recognition between self and nonself. Despite the name, these patterns are also recognized in nonpathogenic bacteria, thereby allowing a targeted adaptive immune response [27,33] . The net immunomodulatory effect of the interaction of PAMPs with PRRs in CD is an excessive effector T‑cell response. This is mediated through a number of proinflammatory pathways, including the NF‑kB pathway.

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Role of gut microbiota in Crohn’s disease

but increase significantly in the distal small intestine and the colon to approximately 108 bacteria/ml and 1010 –1012 bacteria/ ml of intestinal content, respectively [1] . Owing to the large tissue surface area, low redox potential, relatively neutral pH and slow transit time, the distal colon is the site of the highest bacterial concentration and growth, and is subsequently the most common region of inflammation in CD [36,37] . Approximately 30–40 bacterial species comprise 99% of the microflora population, but determining the true diversity of the human gut microbiota was initially restricted due to limitations in culturing technology [1] . Assays using subtle variations in the highly conserved bacterial 16S rRNA gene are now regularly used to more accurately measure the population diversity of intestinal microbial communities. These studies indicate that at 99% genetic similarity, approximately 500 species of bacteria are thought to reside in the healthy human gut [38,39] . What appears to be lacking in microbial diversity is made up for in abundance, as the same microflora achieves the highest cell density of any ecosystem known to science [18] . Of the eight divisions present, by far the most dominant are the Firmicutes and Bacteroidetes. Lower estimates suggest that each division accounts for 30% of the bacteria present in the mucosa and feces, whereas higher estimates suggest that together these account for over 90% [18,40] . The Proteobacteria (Gram-negative facultative anaerobes and sulfate reducers) also represent a major taxonomic group, although they do not appear to dominate [18] . The only representative member of the Archaea, Methanobrevibacter smithi, is also abundant, although the relative importance of this species is unknown as it is currently the only microbe in the human gut known to produce methane via fermentation [39] . The evidence outlined earlier described a number of factors that shape the gastrointestinal ecosystem and suggest that these may underpin the complex interplay between host genetics, environmental exposures and the gut microflora. Central to our understanding of CD pathogenesis is the importance of understanding the characteristics of the microflora community during CD and how dysbiosis may influence host–microbe interactions. Host–microbe interactions

Hooper, Gordon and coworkers have contributed significant insights into our understanding of bottom-up influences on host biology [19] . They have shown that regardless of host genotypedependent intra- and inter-individual differences in bacterial composition, the microflora retains a functional stability and capacity to undergo a range of biochemical processes that allow it to utilize nutrients that the host can produce. These processes include the fermentation of sugars and the resulting production of SCFAs and gases, the degradation of polysaccharides and proteins, the production of amines from amino acids, and the synthesis of vitamins. The outcome of these processes produce nutrients for the host that would otherwise remain unused [1,18,41–43] . The microflora of the mammalian gut has been shown to influence host biology in a number of other ways, including immunity, vascular proliferation and bowel physiology [17,44–46] . www.expert-reviews.com

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As a result of this, the host has a strong incentive to manipulate the composition of the microflora because the resident gut microflora can extensively influence host nutrition, immunology and physiology. Ley et al. have reported that the microflora composition differences between ob/ob and lean mice of different litters showed a 50% increase in Firmicutes and the same proportional decrease in Bacteroides in obese mice [44] . The authors further suggest that the ob/ob genotype in the mice may favor Firmicutes as a response to promote adiposity, or as an adaptive response to limit energy uptake through limiting microbial fermentation and the uptake of polysaccharides [44] . Evidence of this nature establishes the host genotype as an important driver of microbial selection in the gut. Microbiology of CD

There are two primary hypotheses for the role of microorganisms in the pathogenesis of CD. The first suggests that inflammation is mediated by one or several pathogenic microorganisms. Several papers have indicated MAP, a bacterium common in dairy foods, as more common in fecal and biopsy samples from patients with CD [47] . Similar arguments may be applied to E. coli LF82 [11] and other organisms. However, an infectious origin may be argued against on clinical evidence, and the available data leads to the conclusion that this is the effect, rather than the cause, of the disease [8,48,49] . The second, more accepted, hypothesis is that the microflora and their metabolites, regardless of species, mediate inflammation [50] . Ley et al. define the microflora in some disease states as a ‘pathogenic community’, where no single microbe is pathogenic alone [35] . Instead, they suggest that the community in its entirety is an environmental risk factor and a pathogenic entity. Furthermore, the pathogenic community mediates inflammation relative to other risk factors, such as host behavior (and therefore environmental exposure), diet and genotype [35] . There appears to be more evidence for this hypothesis. Despite some degree of uncertainty about the mechanism, it is evident that the microflora per se is altered in CD pathogenesis, as summarized in Table 1. Therefore, altered host–microbe relationships may be important underlying drivers of inflammation. What is not clear, however, is how the microflora may be altered. Numerous studies provide examples of how host genetics selects for a stabilized microflora during the lifespan of the organism. Drawing on evidence from ecological studies, the characteristics of a microbial population will be influenced not only by host genetics but also the availability and utilization of energy substrates, the abundance of niche habitats, microbial competition and predation, immigration and emigration, and importantly the physical characteristics of the ecosystem, including pH, temperature and redox potential. Models of CD must, therefore, account for environmental as well as host genetic factors. Role of paneth cells in antimicrobial host defense

Unlike other epithelial cell types of the small intestine, the paneth cells are long-lived and reside at the crypt base for approximately 20 days [28] . They prevent the invasion of host tissues by bacteria through the release of defensins and other antimicrobial 537

538

Biopsies from TI, TC, Rec, An

Biopsies from TI, TC 6 CD patients and Rec 5 UC patients 5 controls

TGGE

16S rRNA clone library

Biopsies from TI, RC, LC and Rec

Biopsies from cecum and Rec

TTGE

ARISA and T‑RFLP

Shannon Simpson’s Chao2 ICE MM

Pearson coefficient

[54] [54]

↓ Bacteroidetes ↑ Clostridia

[53]

NSD between inflamed and noninflamed mucosa

SD between CD and control SD between UC and CD NSD inflamed vs noninflamed mucosa

↑ Genus Bacteroides

[50]

[52]

↑ Bacteroidetes ↑ Bacteroides fragilis ↑ Proteobacteria ↓ Firmicutes class Clostridia

↓ Overall diversity ↓ Bacteroidetes ↓ Clostridia NSD inflamed vs noninflamed mucosa SD between CD and UC

[49]

↓ 50% overall diversity NSD ↓ diversity of patients NOD2 SNP positive ↓ Bacteroidetes ↓ Proteobacteria order Enterobacteriaceae ↓ LAB with inflammation

↓ Bacteroidetes ↓ Clostridia ↑ Enterobacteria

↓ Overall diversity

[48]

[51]

↓ Species Clostridium leptum but not Clostridium coccoides

↓ Overall diversity ↓ Clostridia

Ref.

↓ Diversity with time ↓ Bacteroidetes ↓ LAB

Conclusion quantity

Conclusion diversity

An: Anus; ARISA: Automated ribosomal intergenic spacer analysis; CD: Crohn’s disease; DGGE: Denaturing gradient gel electrophoresis; ICE: Incidence-based coverage estimator; LAB: Lactic acid bacteria; LC: Left colon; MM: Michaelis-Menten; NSD: Nonsignificant difference; RC: Right colon; Rec: Rectum; SD: Significant difference; SNP: Single nucleotide polymorphism; SSCP: Single strand conformation polymorphism; TC: Transverse colon; TGGE: Temperature gradient gel electrophoresis; TI: Terminal ileum; T‑RFLP: Terminal-fragment length polymorphism; TTGE: Temporal temperature gradient gel electrophoresis; UC: Ulcerative colitis.

10 CD patients 15 UC patients 16 controls

15 CD patients

Biopsies from TI, 20 CD patients Shannon RC, TC, LC and Rec 14 controls undergoing colon cancer screening

DGGE

Total band number Shannon

26 CD patients 31 UC patients 46 controls

Biopsies

SSCP

Shannon Simpson’s Fishers-a

5 CD patients 11 CD remission patients 18 controls

Modified similarity index

Pearson coefficient

Total species number

Diversity indices

Temporal qualitative DGGE Feces

8 CD patients 9 CD remission 16 controls

6 CD patients 6 controls

Feces

Qualitative 16S rRNA clone library Quantitative FISH

Participants (n)

Sample type

Method

Table 1. A review of the molecular studies analyzing the phylogenetic composition of the microflora relative to the disease states of inflammatory bowel disease.

Review Baker, Love & Ferguson



substances [29] . At least three and possibly up to six or more independent genetic alterations in CD involve small intestinal paneth cells (see next section). Those whose involvement is clearly established are NOD2, TCF7L2 (TCF‑4) and ATG16L1, while there are suggestive data for XBP1 [51] , HD5 [52] (Maker et al., Unpublished Data) and b-defensins [53,54] . It is relevant that NOD2, as well as ATG16L1 and TCF7L2, variants are not associated with CD per se, but primarily CD with small intestinal involvement and this is the site at which paneth cells are expressed. However, as argued by Grimm and Pavli, it is important not to overinterpret this evidence in favour of an exclusive paneth cell-based mechanism, since other sites of disease do occur in association with these variants [55] . Much of the control of the numbers and types of microbes present at mucosal surfaces is affected by defensins, natural peptide anti biotics that are categorized into a or b depending upon the distribution of six conserved cysteine residues and intramolecular disulfide bond connections [56] . At least in a mouse model, two enteric paneth cell a-defensins are secreted following intestinal stimulation by Gram-positive or Gram-negative bacteria (or antigens derived from these), but not by fungal species [57] . Matrilysin is the enzyme responsible for activation of mouse paneth cell a-defensins to the mature intestinal form. A mouse with targeted disruption of the gene encoding matrilysin showed reduced or no a-defensin maturation in the small intestine and was highly susceptible to pathogenesis by enteric bacteria [58] . Such observations add strong support for a role of these a-defensins in mucosal host defense. Although many of the early publications on a-defensins emphasized their antimicrobial properties, it appears that mobilizing and activating phagocytes, macrophages, T lymphocytes and mast cells provides an important immunomodulatory function common to all members of the human a-defensin family that is tightly regulated by b-defensins. These processes lead to the induction of numerous proinflammatory and regulatory cytokines [59] . Wehkamp et al. showed that the expression of paneth cell a-defensins is reduced in ileal CD and these appeared to be the main group of paneth cell products that was decreased in these patients [29] . They also demonstrated that human defensin HD5 regulates the bacterial flora in transgenic mice. Salzman and coworkers reviewed human data implicating this reduced paneth cell defensin expression as a key pathogenic factor in ileal CD and suggested that this mechanism is mediated through changes in the colonizing microbiota [60] . There also seems to be evidence for a role of the Wntsignaling pathway transcription factor TCF‑4 as an independent mechanism for this paneth cell defensin deficiency, at least in ileal CD [61] . The authors reported that the reduced expression of human a-defensins HD5 and HD6 mRNA correlated with low expression of TCF‑4 mRNA. This was true for patients with ileal CD, whether or not they were also experiencing high levels of inflammation, but not true for either colonic CD or UC. A mechanistic association was validated in a murine TCF‑4-knockout model. Nutritional manipulation of the microflora in CD

It is known that various nutrients can lead to changes in the gut microflora [62,63] . There had been a degree of optimism that the use of nutrients to alter microbial ecology, in the form of prebiotics, www.expert-reviews.com

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probiotics or synbiotics, might prove beneficial. However, three recent reviews emphasize the variability of results [8,64,65] . It is possible that this variability may reflect another example of gene–nutrient interactions in CD [66] . Nevertheless, it does seem that certain probiotics have the ability to stimulate the expression of b-defensins and may modulate the microbial flora by this mechanism [67] . SNPs in CD that influence the response to the microflora

Concordance of CD is approximately 35% in monozygotic twins and 4% in dizygotic twins, suggesting a strong underlying genetic basis of the disease, which may partially associate with the microbiota [68,69] . GWAS have identified a number of CD-associated gene variants that may alter host–microbe interactions. A recent comprehensive, case-controlled, meta-analysis confirmed 11 previously known susceptibility loci, and strong evidence for a further 21 [21] . The focus of this section lies on several genetic variants that may be implicated in altered host–microbe interactions. A basic overview of the cellular mechanisms associated with these gene variants is outlined in Figure 1. Variants associated with membrane permeability

The cell walls of endothelial cells and their junctions are the first line of defense against luminal pathogens, and thus comprise an important component of innate immunity. The epithelia have adapted a range of mechanisms that regulate the passage of nutrients across this barrier while concurrently maintaining defensive integrity. Impairment of the molecular mechanisms that regulate this process has been suggested in numerous studies of CD [70] . The SNP rs2274910 in the gene Intelectin‑1 (ITLN1) strongly associates with risk of CD. The gene is expressed in paneth and goblet cells of the mammalian small intestine and recognizes galactofuranosyl residues in microbial cell walls. Immunofluorescence studies suggest it is distributed in highest concentrations at the brush border membrane of enterocyte cells, specifically within glycolipid and cholesterol-rich raft micro domains, which regulate the permeability of the membrane and mediate phagocytosis of luminal bacteria and viruses. Intelectin may, therefore, play an important role in innate immunity at the brush border membrane [71] . Several luminal pathogens have adapted glycolipid sensory systems to exploit these rafts and thus gain access to the underlying organism. This involves the exploitation of raft components to prevent the binding of phagosomes with degradative compartments of the cell such as lysozymes. By utilizing extracellular surface receptors some pathogens may survive within host phagocytes by manipulating phagosomeassociated signaling pathways. Intelectin may help regulate the stability of these rafts in response to pathogens, and impediment of this process may therefore be implicated in CD [71] . Variants associated with autophagy

“Autophagy is a cellular homeostasis mechanism whereby portions of the cytosol and damaged organelles or intracellular pathogens and their products are sequestered into an auto phagosome for degradation in autolysosomes” [72] . This mechanism forms an integral component of cellular innate immunity 539

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Lumen Mucosa

MUC19 ITLN1

+ Cytosolic + proteins

TLRs

Defensins

MDP

NOD2

ATG16L1 LRRK2 IRGM

+ +

PGN

Increased intracellular loading + + Intracellular immune response

+ +

Transcription factors

TCF-4 Nucleus

Phagosome -

MHCII

Phagolysosome Presentation to CD4 + T cells

Expert Rev. Gastroenterol. Hepatol. © Future Science Group (2009)

Cytoplasm IL-2 IL-5 IFN-γ TGF-β

Th1

IL-12

IL-1B

Figure 1. Cellular mechanisms associated with Crohn’s disease-associated gene variants. MDP: Muramyl dipeptide; PGN: Peptidoglycan; TLR: Toll-like receptors.

against bacteria and viruses that enter the cytoplasm either pathogenically or accidentally via endocystosis [73] . A GWAS identified the SNP rs2241880 in the ATG16L1 gene as conferring susceptibility to CD [74] . Since then, this SNP has been confirmed as a CD-susceptibility allele by a number of independent studies, although not in a Japanese cohort [75–77] . The autophagy pathway is a key process in the capture and degradation of intra cellular bacteria and therefore innate immunity [24,78] . An in vitro model using human HeLa cells showed that RNAi knockdown of ATG16L1 inhibits autophagy of Salmonella typhimurium [74] . This suggests that gene variants may confer risk of CD by impeding the ability of cells to target and remove intracellular bacteria, thereby propagating bacterial loading within the cell and the subsequent immune response [79] . Autophagy also acts as an adaptation to cellular starvation, which involves the processing of intracellular organelles and proteins, and the generation of amino acids during nutrient deficiency in the cell [80,81] . The autophagy pathway is activated in response to a number of cellular stressors, such as nutrient deprivation, bacterial infection and oxidative damage to 540

cellular organelles, and is directly dependent on the nutritional status of the cell. This suggests that accumulative intracellular bacterial concentrations coupled with poor nutritional status may together confer risk of CD. Clinical evidence to support this stems from the efficacy of elemental diets in attenuating inflammation in the active disease state [82] . A study using a combination of real-time PCR and immunohistochemistry showed that this gene is expressed in a variety of human tissues and cells, including the ileum, colon, spleen, leukocytes and intestinal epithelial cells. However, no difference in expression was found in intestinal epithelial cells between CD patients and controls [52,74] . Loss of function in any of the inter-related pathways of autophagy, phagocytosis or endocytosis would impair the innate immune response. This is especially true for endocytosis, as endosomes can shuttle pathogen surface receptors such as TLR4 to autophagosomes, allowing them to respond to intracellular bacteria [83,84] . Impediment of these pathways would allow invading bacteria to proliferate and necessitate increased inflammation by the subsequent adaptive immune response. Expert Rev. Gastroenterol. Hepatol. 3(5), (2009)


Genome-wide association studies have identified two strongly associated nonfunctional SNPs, rs4958847 and rs13361189, in the immunity-related GTPase – M (IRGM) gene with ileal CD [85,86] . Expression of IRGM has been reported in tissues of the colon and ileum and in peripheral blood leukocytes and monocytes [86] . A study of Mycobacterium tuberculosis infection showed that IRGM is required for the early stage initiation of IFN-g-induced autophagy and the facilitation of phagosome maturation [72] . Mice deficient in the homologous LRG-47 show significantly increased susceptibility to infection by Listeria mono‑ cytogenes and Toxoplasma gondii, further supporting the critical role of IRGM in innate immunity [87] . However, the functional consequences of these human variants that are associated with disease remains unknown, as the amino acid sequence of the gene remains unchanged. This suggests that altered regulatory mechanisms, such as gene splicing and expression, or interactions with other SNPs in linkage disequilibrium, may be responsible for the functional consequences, as opposed to altered protein function per se. A possible mode of action may be the inhibition, or at least the reduction, of response to IFN-g-induced autophagy, resulting in high bacterial loading of enterocytes and peripheral immune cells [72] . Barret et al. identified the SNP rs11175593 on chromosome 12q12 as strongly associated with CD [21] . This locus contains the leucine-rich repeat kinase-2 (LRRK2) and Mucin-19 (MUC19) genes. With relevance to the susceptibility genes ATG16L1 and IRGM, mutant LRRK2 has recently been associated with induction of autophagy in an in vitro neuronal cell transfection model of a common variant associated with Parkinson’s disease. Interestingly, this variant resulted in cellular remodeling, which was attenuated via siRNA knockdown of essential downstream components of the autophagy pathway [88] . Mutant LRRK2 is most highly associated with genetic and late-onset Parkinson’s disease. Initial in vitro studies suggest that pathogenic variants of this gene result in a gain-of-function associated with increased kinase activity [89,90] ; however, functional consequences of CD-associated variants have not yet been explored and may or may not result in cellular remodeling. The MUC19 gene encodes a protein containing multiple threorine- and serine-rich repeats and is expressed in the intestinal mucus layer. Mucin proteins are important structural components of the intestinal mucus layer, which is positioned strategically over the underlying endothelia, thus providing a physical defense mechanism against the luminal microflora. The absence of this layer, induced experimentally in a MUC2-/- mouse model, is associated with intestinal inflammation [91] . Impairment of the mucosal defense is supported by clinical evidence showing significantly higher bacterial loading within the mucus layer of IBD patients compared with controls [92] . Variants associated with microbial sensing & innate immune response

Although several of the aforementioned gene variants are integral components of innate cellular immunity, the following gene variants are directly implicated in regulating host–microbe www.expert-reviews.com

Review

interactions across the gastrointestinal epithelia. The year 2001 saw the concurrent discovery by two separate research groups that variants in the NOD2 gene predispose to CD [22,23] . NOD2 codes for a cytoplasmic bacterial sensor protein expressed in monocytes and intestinal epithelial cells, with highest expression in the paneth cells of the ileum [93,94] . Evidence for a role of the NOD protein family in bacterial recognition stems from homology of the LRR region at the C-terminal of the protein with the TLRs, which are involved in the recognition of a range of conserved bacterial components [95,96] . Muramyl dipeptide (MDP), an intracellular derivative of peptidoglycan (PGN) found in the cell wall of both Gram-negative and Gram-positive bacteria, interacts directly with the LRR region of the protein [97] . This activates the two adjoining NOD effector domains and proinflammatory gene transcription via the NF-kB transcription factor and the MAPK signaling pathways [98] . Although three SNPs in this gene are implicated in susceptibility, it is now widely recognized that the 3020insC mutation confers the greatest risk [99] . Peripheral blood mononuclear cells carrying human CD variants have reduced or loss of response to MDP, suggesting a loss-of-function model. This model is also supported by the recessive nature of inheritance [23,99] . NOD2-knockout mice only show increased susceptibility to disease after infection, highlighting the important role of epithelial defense [100] . Loss of NOD2 activity may alter TLRmediated responses. Compared with wild-type cells, monocytes from NOD2 -/- mice appear to lack responsiveness to MDP, but show an elevated NF-kB response to the TLR2 ligand PGN, resulting in elevated levels of the NF-kB target cytokine IL‑12. This suggests that wild-type NOD2 acts as an intracellular sensor of bacterial MDP and may inhibit the extracellular PGN-mediated stimulation of the TLR2 pathway [101] . It is now evident that NOD2 is critical for bacterial recognition and immune response after exposure to TLR ligands in general [102] . The altered immune response described earlier may result in increased intracellular bacteria survival and a subsequent inflammatory response. This concept has become increasingly relevant in light of other susceptibility gene variants that may impede autophagy-mediated clearance of intracellular bacteria and metabolites, and recent findings that suggest NOD2 is a critical component of TLR-mediated bacterial recognition and the subsequent immune response [102] . There is weaker evidence that SNPs in other PRRs, including TLR4 [103] and NOD1 [104] , may play some role in disease etiology, either acting alone or in combination [66] . Koslowski et al. provide evidence associating genetic variants of the Wnt signaling pathway transcription factor TCF‑4 promoter region with small intestinal CD [105] . They reasoned that, since the level of expression of paneth cell antimicrobial a-defensins appears to characterize the disease, and since TCF‑4 is known to regulate the expression of HD-5 and -6, a causal relationship between the events was likely. They sequenced 2.1 kb of the 5´ flanking region of TCF‑4 in small groups (n = 10 each) of ileal CD patients and controls. The results led to the identification of eight SNPs, of which three were in linkage disequilibrium 541

Review


and were found more frequently in CD patients, while one was located in a predicted regulatory region. They considered the distribution of this SNP in three cohorts of IBD patients and controls, and demonstrated a significant association with ileal CD, but not with either colonic CD or UC. A role for human defensins in CD is further supported by other evidence showing that a lower chromosomal HBD-2 gene copy number predisposes to an inflamed colonic phenotype [54,106] . Of relevance, a mouse model shows this impairment may be localized within the intestine, as Defcr4 is inactive in the duodenum but is most highly expressed in paneth cells [107] . In CD patients carrying NOD2 variants, the expression levels of a-defensins (HD-5 and HD-6) are significantly lower than in control groups, and ileal lesions develop in areas corresponding to areas abundant in paneth cells [106,108,109] . We have also associated SNPs in HD-5 with an increased risk of CD in the New Zealand population [110] . Given the results of the previously mentioned studies, impairment of defensin production could both damage and promote the microbial colonization of differing clades of bacteria in the distal ileum, and therefore alter microflora characteristics in downstream areas of the intestinal tract. This provides evidence for a ‘microflora–dysbiosis’ model of pathogenesis. The nature of the genes described earlier suggests that several potential mechanisms exist for altered host–microbe interactions in CD. Broad qualitative and quantitative changes to microbial composition, for example via reduced luminal defensins, could directly trigger an inflammatory response, while altered cellular permeability at the brush border and impaired microbial processing in the cytoplasm via autophagy may in turn propagate inflammation via normal innate and adaptive immune responses. An increase in the frequency of CD in the population over recent years may have resulted from changing public health practices and their implications for dysbiosis [111] . Expert commentary & five-year view

This review provides an overview of the mammalian gastro intestinal ecosystem, the altered microbial state characteristic of CD, and an outline of the potential genetic polymorphisms

that may alter host–microbe interactions in CD pathogenesis. The previous observations suggest that altered host–microbe interactions, driven in part by host genetics, may be the primary mechanism for differential susceptibility to CD. Future studies may benefit from exploring the presence of pathogenic microorganisms in the intestine of CD patients, including those that are known to exploit the brush border as a means of entry. These may include an array of bacteria and viruses, including, with relevance to CD, Mycobacterium spp. Another critical next step is the functional characterization of the variants reviewed here. Potentially, understanding the variation in these genes may unravel the elusive mechanism by which bacteria drive intestinal inflammation in CD. Transfection cell models using these variants may yield significant insights. The development of symbiosis between humans and microbes over the eons through evolutionary selective pathways has generated a complex biochemical powerhouse, the result of which offers significant nutritional benefit to both microbe and human. As we begin to understand the array of host (and microbial) genes that regulate this relationship, and the interplay of these genes with diet, we may uncover many possible therapeutic targets for the treatment of CD. Although probiotics have not proved to be consistently useful in the treatment of this disease, it is very probable that genetic subsets of patients will have differing underlying mechanisms of altered host–microbe interactions and, therefore, different clinical manifestations. Nutrigenomic and pharmacog enomic approaches may become especially important in the control of this disease over the next 5 years. Financial & competing interests disclosure

Donald Love and Lynnette Ferguson are members of Nutrigenomics New Zealand, which is a collaboration between AgResearch Ltd., Plant & Food Research and The University of Auckland, with funding through the Foundation for Research Science and Technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • Crohn’s disease (CD) is a form of inflammatory bowel disease for which there is evidence of host–microbe interactions underpinning disease pathogenesis. Certain microorganisms may exacerbate the severe inflammatory response characteristic of the disease. • No specific microbe appears to be pathogenic, but rather the stability of the microbial ecosystem of the healthy human gut is disrupted by a combination of host genetics and dysfunctional immunity. • Genome wide association studies help to reveal the genetic basis of CD. • Several of the genes revealed thus far are important for microbial sensing and response. • Single nucleotide polymorphisms or copy number variants in this category fall into one of three main groups: – Variants associated with membrane permeability – Variants associated with autophagy – Variants associated with microbial sensing and the innate immune response • Understanding the way in which CD patients with genetic variants respond to nutrients and drugs will enable the rational development of personalized therapies. • Both pharmacogenomic and nutrigenomic approaches may be relevant.

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Affiliations •

Phillip I Baker, BSc, PGradDipHSc, MHSc(Hons) PhD Candidate, National Centre for Epidemiology and Population Health The Australian National University Canberra, ACT 0200, Australia Tel.: +612 6125 5611 Fax: +612 6125 0740 [email protected]

•

Donald R Love, BSc(Hons), PhD (Adelaide) MIBMS, CSci, MIFST, CBiol, FIBiol, FLS, FRCPath Associate Professor, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand and LabPLUS, PO Box 110031, Auckland City Hospital, Auckland, New Zealand Tel.: +64 9373 7599 ext. 87228 Fax: +64 9373 7417 [email protected]

•

Lynnette R Ferguson, MSc(Hons), DPhil(Oxon.), DSc Professor, Department of Nutrition, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand Tel.: +64 9373 7599 ext. 86372 [email protected]
