Si Ming Man, Nadeem O. Kaakoush and Hazel M. Mitchell

Transcription

1 The role of bacteria and pattern-recognition receptors in Crohn s disease Si Ming Man, Nadeem O. Kaakoush and Hazel M. Mitchell Abstract Crohn s disease is widely regarded as a multifactorial disease, and evidence from human and animal studies suggests that bacteria have an instrumental role in its pathogenesis. Comparison of the intestinal microbiota of patients with Crohn s disease to that of healthy controls has revealed compositional changes. In most studies these changes are characterized by an increase in the abundance of Bacteroidetes and Proteobacteria and a decrease in that of Firmicutes. In addition, a number of specific mucosa-associated bacteria have been postulated to have a role in Crohn s disease, including Mycobacterium avium subspecies paratuberculosis, adherent and invasive Escherichia coli, Campylobacter and Helicobacter species. The association between mutations in pattern-recognition receptors (Toll-like receptors and Nod-like receptors) and autophagy proteins and Crohn s disease provides further evidence to suggest that defective sensing and killing of bacteria may drive the onset of disease. In this Review, we present recent advances in understanding the role of bacteria and the contribution of pattern-recognition receptors and autophagy in the pathogenesis of Crohn s disease. Man, S. M. et al. Nat. Rev. Gastroenterol. Hepatol. 8, (2011); published online 8 February 2011; corrected online 18 March 2011; doi: /nrgastro Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK (S. M. Man). School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia (N. O. Kaakoush, H. M. Mitchell). Correspondence to: H. M. Mitchell h.mitchell@ unsw.edu.au Introduction Crohn s disease is a form of IBD with unknown etiology. It is currently hypothesized that an initiator (either gastrointestinal microorganisms or their by-products), in association with a disruption of the gastrointestinal epithelium, stimulates and subsequently drives a dysregulated immune response in predisposed individuals. 1 Evidence to support the role of microorganisms in the pathogenesis of Crohn s disease has been demonstrated in both humans and animals. In humans, recurrent Crohn s disease can be prevented by postoperative diversion of the fecal stream. 2 6 In these studies, the majority of individuals who underwent fecal diversion exhibited striking clinical improvement within 3 6 months, and of these patients a substantial proportion achieved remission in the long term (Box 1). Furthermore, the use of antibiotics in the treatment of Crohn s disease has resulted in notable benefits, 7 15 which suggests that eradication of certain populations of bacteria may contribute to remission (Table 1). A metaanalysis of six randomized, placebo-controlled clinical trials, published between 1966 and 2006, showed that broad-spectrum antibiotics (metronidazole, ciprofloxacin or co-trimoxazole) seem to improve clinical outcomes in individuals affected by Crohn s disease, and that patients receiving antibacterial therapy were times more likely to show clinical improvement compared with those receiving placebo. 16 Investigations using mouse models of Crohn s disease complement findings Competing interests The authors declare no competing interests. from human studies and have helped elucidate the role of microorganisms in Crohn s disease, an area that has been expertly reviewed recently. 17 Although it is accepted that microorganisms have an essential role in the initiation of Crohn s disease, the microorganism or group of microorganisms involved remains elusive, despite decades of research and technological advances in molecular biology that facilitate their detection. In this Review, we present recent advances in understanding the role of bacteria in Crohn s disease, including discussion of studies investigating overall changes in the gut microbiota, as well as studies examining the involvement of specific groups of bacteria in the etiology of disease. This Review also explores the contribution of impaired bacterial sensing and killing as a consequence of mutations in pattern-recognition receptors, including Toll-like receptors (TLRs), and Nod-like receptors (NLRs), highlighting the dynamics of bacterial host interactions in the pathogenesis of Crohn s disease. Dysbiosis of the gut microbiota The adult human intestinal microbiota of the ileum and colon are composed of >10 4 and >10 12 organisms per gram of luminal fluid, respectively. 18 These bacteria not only have an important role in host nutrition and gut development but they also contribute to immunological homeostasis within the healthy gastrointestinal tract. 18,19 Given these roles, it has been proposed that changes in the intestinal microbiota may lead to adverse effects on health in the host. Breakdown in the balance between protective and harmful intestinal bacteria has 152 MARCH 2011 VOLUME 8

2 been termed dysbiosis (Figure 1), a condition that may promote chronic intestinal inflammation. 20 Over the past decade, evidence has accrued to suggest that dysbiosis may have a pivotal role in the pathogenesis of IBD. Early studies that employed culture-dependent techniques revealed differences in the diversity and composition of specific bacterial groups within the intestinal microbiota of patients with Crohn s disease and healthy controls. However, the fact that less than 40% of intestinal bacteria can be cultured significantly limits this approach. 21 The introduction of culture-independent techniques, such as metagenomics and the large-scale analyses of the 16S rr genes, has allowed a more in-depth analysis of the composition of the intestinal microbiota in patients with Crohn s disease and healthy controls. 22,23 A consistent finding across these studies is that in patients with Crohn s disease, the abundance of members of the Firmicutes (Gram-positive bacteria, including Clostridium and Bacillus species) is decreased, whereas members of the Proteobacteria (Gram-negative rods, including Escherichia spp.) are increased compared with non-ibd or healthy controls; in some studies E. coli in particular was increased (Tables 2 and 3) Consistent with these clinical observations is the finding of a four-log increase of nonpathogenic E. coli in the colon of mice treated with the colitis-inducing agent dextran sulfate sodium (DSS). 45 In addition, the same study used interleukin (IL) 10 / mice as a model of intestinal inflammation to elegantly show that orally introduced nonpathogenic E. coli efficiently colonize in high numbers in the colon (~10 8 colony-forming units/g of colon), which concomitantly displaces members of the Firmicutes. In a study examining the colitogenic microbiota in a T bet / /Rag2 / ulcerative colitis (TRUC) mouse model that develops spontaneous colitis, the authors showed that these mice harbor low numbers of bacteria belonging to the orders Clostridiales (phylum Firmicutes) and δ proteobacteria (phylum Proteobacteria), and high numbers of bacteria belonging to the order Bacteroidales (phylum Bacteroidetes). 46 Findings from these mouse models of colitis indicate that in the presence of inflamma tion, prolific coloniza tion by certain commensal bacteria (for example, members of the Enterobacteriaceae) drives the depletion of other groups of bacteria (for example, Firmicutes). It is probable that this microbial succession event is one of the underpinning factors that further aggravates intestinal inflammation. Findings of disease-related changes in the human microbiota for other bacterial groups, such as the Bacteroidetes (Gram-negative rods, including Bacteroides spp. and Faecalibacterium prausnitzii), are more inconsistent. For example, based on 16S rr amplification, cloning and sequencing, Rheman et al. 24 showed Bacteroidetes to be more prevalent in patients with Crohn s disease compared with controls; by contrast, Frank et al. 25 used quantitative-pcr (q-pcr) to demonstrate that Bacteroidetes were significantly depleted in Crohn s disease compared with controls. In addition, Ott et al., 31 using 16S rd based single-strand confirmation polymorphism (SSCP) fingerprinting, cloning and Key points Current evidence suggests that the diversity and abundance of specific groups of bacteria differs between patients with Crohn s disease and healthy controls To date, no specific groups or any single bacterium has been definitively associated with the etiology of Crohn s disease Polymorphisms in pattern-recognition receptors and autophagy proteins are associated with susceptibility to Crohn s disease Defective sensing and killing of bacteria owing to impaired pattern-recognition receptors, autophagy and defensin production may have a role in the etiopathogenesis of Crohn s disease Box 1 The impact of fecal diversion in patients with Crohn s disease Of 31 patients with perianal Crohn s disease who underwent fecal diversion, 81% showed evidence of early remission as defined by objective assessment of the patient s condition, resolution of active fistulas, and resolution of sepsis in those diagnosed with perianal sepsis. The remaining 19% failed to respond. In the early remission group, 32% required no further surgery at a median duration of 81 months after fecal diversion. The remaining 68% relapsed at a median duration of 23 months after fecal diversion 2 Of five patients with Crohn s disease who underwent ileal resection and fecal diversion, the neoterminal ileum of all patients was normal after 6 months as determined by ileocolonoscopy and histology. All five patients developed recurrent inflammation after reanastomosis. In comparison, of 75 patients who underwent a one-step resection with end-to-end ileocolonic anastomosis (no fecal diversion), 71% developed new lesions in the neoterminal ileum 3 Of 75 patients with Crohn s disease who underwent loop ileostomy to establish fecal diversion, 91% (72 patients) showed early clinical improvement after 3 months (assessed by improvement in symptoms and serum markers). A follow up study of the 72 patients showed that 19% required no further surgery within 3 6 years, 39% had undergone elective resection, 33% required further surgery due to relapse, 4% had recurrent disease due to closure of the ileostomy, 4% died 4 Of 13 patients who underwent ileal or colonic resection and fecal diversion, all patients noted improved clinical condition, 46% (six patients) showed objective improvements (determined by barium enema roentgenographic and proctosigmoidoscopic examination) and four of these six achieved complete remission 3 months after fecal diversion 5 In three patients with Crohn s disease who underwent ileocolonic resection and postoperative fecal diversion, re-exposure of fecal content in the normal neoterminal ileum for 8 days resulted in histologic abnormalities, characterized by elevated infiltration of mononuclear cells, eosinophils, and polymorphonuclear cells in the lamina propria, epithelioid transformation, and transendothelial lymphocyte recruitment 6 q PCR of 16S rr genes, showed the microbial diversity in biopsy samples from Crohn s disease patients to be reduced (by 50%) owing to the loss of normal anaerobic bacteria, including Bacteroides species. Swidsinski et al., 30 using fluorescent in situ hybridization (FISH), reported Bacteroides species to predominate in mucosal biopsies from patients with Crohn s disease. One potential reason for the failure to consistently identify similar changes in patients with Crohn s disease compared with controls is likely to relate to the use of different molecular approaches to survey the intestinal microbiota. Each approach not only differs in depth and breadth of coverage, but also in sensitivity and specificity, all factors that clearly impact upon the detection and quantification of the intestinal flora. Other factors contributing to the lack of consistency between studies relate to study design, in particular with respect to the TURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY VOLUME 8 MARCH

3 Table 1 Evidence that antibiotics are effective in inducing remission in active Crohn s disease Therapy used Median duration of therapy Primary end point and outcome Ref. 1) Rifaximin (600 mg daily) (n = 18) 2) Rifaximin (600 mg daily) plus steroids (n = 31) 1) Rifaximin (800 mg twice daily) (n = 27) 2) Rifaximin (800 mg daily) plus placebo (twice daily) (n = 25) 3) Placebo (twice daily) (n = 27) 1) Ciprofloxacin (500 mg twice daily) (n = 28) 2) Placebo (n = 19) 1) Oral ciprofloxacin (500 mg twice daily) plus metronidazole (500 mg twice daily) (n = 64) 2) Placebo (n = 66) All patients also received oral budesonide (9 mg daily) 1) Ciprofloxacin (500 mg twice daily) (n = 18) 2) Mesalazine (2 g twice daily) (n = 22) 1) Ciprofloxacin (500 mg twice daily) plus metronidazole (250 mg four times daily) (n = 22) 2) Methylprednisolone (n = 19) Ciprofloxacin (500 mg twice daily) plus metronidazole (250 mg three times daily) (n = 72) Of the 72 patients, 29 also received prednisone and 9 received aminosalicylic acid preparations 1) Metronidazole (10 mg/kg body weight) (n = 33) 2) Metronidazole (20 mg/kg body weight) (n = 30) 3) Placebo (n = 36) Median 12.9 weeks Clinical remission defined as CDAI <150 Remission rate in patients treated with rifaximin alone (67%) higher than in those on rifaximin plus steroids (58%) 12 weeks Clinical remission defined as CDAI 150 Remission rate in patients treated with rifaximin 800 mg twice daily (52%) higher than for those receiving rifaximin 800 mg daily plus placebo (32%) or those receiving placebo alone (33%) 6 months Clinical remission defined as CDAI <150 Significant reduction in CDAI score in patients treated with ciprofloxacin (mean CDAI = 112) compared with placebo (mean CDAI = 250, P <0.001) 8 weeks Clinical remission defined as CDAI <150 No significant difference in remission rates in patients treated with antibiotics (33%) compared with placebo (38%, P = 0.55). In patients with colonic disease, 53% in remission after antibiotic treatment compared with 25% who received placebo (P = 0.10) 6 weeks Complete clinical remission CDAI score % of patients with Crohn s disease treated with ciprofloxacin in complete remission versus 54.5% treated with mesalazine 12 weeks Clinical remission defined as a CDAI 150 Clinical remission achieved in 45.5% of antibiotic-treated patients compared with 63% patients treated with steroids 10 weeks Clinical remission defined as a HBI 3 points (equivalent to a CDAI of <150) Clinical remission achieved in 68% of patients with Crohn s disease. Antibiotic treatment particularly beneficial in achieving a clinical response in patients with ileocolonic involvement (84%) compared with those with ileal involvement (63%) >2 16 weeks Clinical remission defined as CDAI <150 Treatment with metronidazole (10 or 20 mg) resulted in significantly reduced CDAI versus placebo (P <0.002). However, no significant difference in percentage of patients entering remission: 36% metronidazole (10 mg), 27% metronidazole (20 mg), and 25% placebo Oral clarithromycin (250 mg twice daily) (n = 25), plus one or more of the following: 5 aminosalicylic acid preparations; prednisolone; or azathioprine Initially for 4 weeks. If partial or complete clinical response observed, treatment continued to 12 weeks (11 of 25 patients) Clinical remission defined as an HBI 4 points At 4 weeks: Remission achieved in 12 of 25 (48%) patients At 12 weeks: Remission achieved in 11 of 25 (44%) patients. 8 of 11 (73%) remained in remission at weeks (median 28 weeks) 15 Abbreviations: CDAI, Crohn s disease activity index; HBI, Harvey-Bradshaw Index. number and age of patients, the stage of disease and its location, the type of samples collected (biopsy or feces), and the control populations used. Although the studies outlined in Tables 2 and 3 provide important information on the prevalence of specific microbial communities in patients with Crohn s disease and controls, to date there are limited data on the transcriptional activity of the observed intestinal microbiota. Interestingly, Rehman et al. 24 have reported that a discrepancy exists between intestinal bacterial richness (measured by rr gene sequences) and bacterial transcriptional activity (measured by rr sequences). This study showed that in Crohn s disease, bacterial richness was significantly lower in clone libraries based on rr as compared with those based on the rr genes (3.10 versus 3.91), while in healthy individuals no difference was observed (3.81 versus 3.85). 24 Specifically, in patients with Crohn s disease, the transcriptional activity of the Firmicutes and Actinobacteria was decreased and that of Bacteroidetes was increased (P <0.01) compared with results obtained from healthy controls. 24 This study adds a new dimension in the investigation of the etiology of Crohn s disease; however, further studies are clearly required. Although the studies outlined above have provided significant insights into alterations underlining the intestinal microbiota of patients with Crohn s disease, these observations are not consistent, and only very general statements mostly on the phylum level can currently be made. Future studies with large cohorts of patients with Crohn s disease matched for disease location, disease activity and stage of disease, and using meta genomic approaches, have the potential to provide a much clearer picture of dysbiosis in Crohn s disease. Mucosa-associated bacteria Mucosa-associated bacteria colonize areas in close proximity to the intestinal epithelium, which places them in a prime position to initiate host microbe interactions. Early studies using electron microscopy and FISH showed that bacteria can be found within intestinal epithelial cells and in the submucosa of patients with Crohn s disease. 30,33, MARCH 2011 VOLUME 8

4 Lumen Predisposing factors: Environment Dietary Genetic Gastroenteritis Mucus layer Intestinal epithelium Submucosa Predisposing factors: Environment Dietary Genetic Gastroenteritis Normal gut microbiota Increased susceptibility to infection Altered microbiota Dysbiosis of the gut microbiota Primary infection Dysregulated immune responses Genetic factors Dysregulated immune responses Genetic factors Crohn s disease Figure 1 Possible pathways in the development of Crohn s disease. The normal human intestinal tract, which is covered by an intact mucus layer, harbors a variety of luminal and mucosa-associated bacteria (top panel). Multiple factors, including environmental, dietary and genetic factors, may induce alterations in the gut microbiota (left panel), which may increase susceptibility to infection (right panel). Conversely, infection of the gut by a specific bacterium may also induce an altered microbiota or damage to the intestinal epithelium. Host factors involving inappropriate immune responses (underpinned by genetic factors) contribute to thinning of the mucus layer, disruption of the intestinal barrier, increased translocation of bacteria to the submucosa and impaired bacterial recognition and killing, which results in the chronically inflamed intestinal milieu that is characteristic of Crohn s disease (bottom panel). This evidence supports the notion that patients with Crohn s disease may be susceptible to colonization and infection by mucosa-associated bacteria (Figure 1). Mycobacterium avium subspecies paratuberculosis Mycobacterium avium subspecies paratuberculosis (MAP) is an obligate intracellular pathogen that causes Johne s disease, a chronic inflammatory condition that primarily affects the small intestine of cattle and other ruminants. Owing to similarities between this bovine disease and Crohn s disease, it has been hypothesized that MAP may have a role in Crohn s disease. A PubMed search of articles containing the terms Crohn s disease and Mycobacterium in the title or abstract yielded over 160 publications in the past 5 years (as of November 2010), indicative of considerable research efforts being invested in clarifying the role of MAP in Crohn s disease. However, there is still no conclusive evidence to indicate that MAP is the etiological agent in Crohn s disease Specific antibodies against MAP proteins that are essential for establishing infection have been identified in patients with Crohn s disease 50,51 and there is evidence to show that anti-map treatments promote disease remission in some patients. 52 Interestingly, when exposed to MAP, peripheral blood mononuclear cells (PBMCs) or mesenteric lymph node cells isolated from patients with Crohn s disease secrete significantly higher levels of the cytokines tumor necrosis factor (TNF), IL 6, IL 8 or IL 10, than cells from patients with ulcerative colitis or healthy controls; the levels of these cytokines produced in response to Listeria monocytogenes or Salmonella enterica serovar Typhimurium (S. Typhimurium) were similar across all groups. 53 Two systematic reviews and meta-analyses argued that there is evidence of a higher prevalence of MAP in Crohn s disease patients than in controls; however, both reports suggest that the role of MAP in Crohn s disease requires further investigation, with an emphasis on evaluating the efficacy of antimycobacterial therapy and the pathogenic potential of MAP. 54,55 Adherent and invasive Escherichia coli Adherent and invasive Escherichia coli (AIEC) are character ized by their ability to invade intestinal TURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY VOLUME 8 MARCH

5 Table 2 Evidence of compositional changes in the mucosal gut microbiota of patients with Crohn s disease Patients Methods Major findings in relation to Crohn s disease Ref. Active Crohn s disease, n = 10 Active ulcerative colitis, n = 10 Healthy controls, n = 10 Amplification of 16S rr gene, T RFLP, cloning and sequencing Firmicutes significantly lower in number in patients with Crohn s disease (52.7%) vs healthy controls 78.6% (P <0.01). Bacteroidetes higher in Crohn s disease (23.1%) vs healthy controls (8.9%). Proteobacteria higher in Crohn s disease (11.2%) vs healthy controls (5.2%); Actinobacteria higher in Crohn s disease (12.95%) vs healthy controls (6.6%). Higher prevalence of E. coli phylotypes in Crohn s disease (7.59%) vs healthy controls (0.37%) 24 Crohn s disease, n = 68 Ulcerative colitis, n = 61 Non-IBD controls (primarily GI cancer patients), n = 61 R sequence analysis, q PCR Members of Bacillus significantly more abundant in the small intestine than in the colon while members of Bacteroidetes and Lachnospiraceae (Firmicutes) less abundant in the small intestine. Sequences representative of Bacteroidetes (P <0.001) and Lachnospiraceae (P <0.001) significantly depleted and Actinobacteria (P <0.001) and Proteobacteria (P <0.001) significantly more abundant in IBD subset (approximately two-thirds of patients with Crohn s disease and three-quarters of patients with ulcerative colitis) vs control subset 25 Ileal Crohn s disease, n = 13 (5 restricted to ileum and 8 ileocolonic) Crohn s disease control group, n = 8 Healthy controls, n = 7 16S rd libraries and sequencing, q PCR, FISH Increased numbers of Enterobacteriaceae sequences (26.4%) (exclusively E. coli) in the ileal mucosal flora of patients with ileal Crohn s disease vs the ileum of Crohn s disease control group (0.5%, P = 0.001) and healthy controls (1.4%, P = ). Lachnospiraceae sequences significantly reduced in ileal Crohn s disease (3.1%) vs healthy controls (15.5%, P <0.0175). Sequences from the Clostridiales (Faecalibacteria and Subdoligranula genera) significantly lower in ileal Crohn s disease (0.4%) vs healthy controls (15%, P = 0.038) and Crohn s disease control group (26%, P = ) 26 Crohn s disease, n = 13 Ulcerative colitis, n = 19 Healthy controls, n = 15 RISA analysis and sequencing, culture Number of Enterobacteriaceae in patients with Crohn s disease 3 4 logs higher than in healthy controls. Number of E. coli (culture) significantly higher in patients with Crohn s disease than controls (P <0.05) 27 Crohn s disease, n = 6 Ulcerative colitis, n = 5 Healthy controls, n = 5 PCR using 16S rr, cloning and sequencing Proteobacteria significantly increased in patients with Crohn s disease (12.9%) vs healthy controls (8.4%, P = ). Bacteroidetes significantly increased in Crohn s disease (74.9%) vs healthy controls (67.4%, P <0.0001). Firmicutes (all Clostridia) significantly decreased in Crohn s disease group (10.0%) vs healthy controls (24.1%, P <0.0001) 28 Crohn s disease, n = 19 Ulcerative colitis, n = 2 Intermediate colitis, n = 1 Healthy controls, n = 15 PCR-DGGE of 16S rr gene Faecalibacterium (Firmicutes) significantly more prevalent in healthy controls (86.7%) vs Crohn s disease group (52.6%, P = 0.035). In particular, F. prausnitzii significantly more prevalent in healthy controls vs Crohn s disease (P <0.05); the Clostridium genus significantly more frequent in Crohn s disease group vs healthy controls (P <0.05). E. coli higher in number in Crohn s disease group (31.6%) vs healthy controls (6.7%, P = 0.074) 29 Crohn s disease, n = 20 Ulcerative colitis, n = 20 Healthy controls, n = 20 FISH Mucosal bacteria at concentrations >10 9 detected in 95% of patients with Crohn s disease vs 35% of controls. Concentration of mucosal bacteria 2 powers higher in Crohn s disease compared with controls (P <0.0001). Mean percentage of Bacteroides-Prevotella within biofilm significantly higher in patients with Crohn s disease (71 ± 21) vs controls (20 ± 11), P <0.001). Biofilm mainly composed of Bacteroides fragilis 30 Active Crohn s disease, n = 26 Active ulcerative colitis, n = 31 Controls, n = 46 (15 with intestinal inflammation and 31 without) 16S rr gene based SSCP fingerprint, and q PCR Mean diversity of microbiota in Crohn s disease (21.7%) reduced compared with noninflammatory controls (50.4%, P <0.0001). Reduction due to loss of anaerobic bacteria, for example, Bacteroides, Eubacterium and Lactobacillus species 31 Active Crohn s disease, n = 16 Crohn s disease surgical samples, n = 15 Non-IBD controls, n = 10 Universal eubacteria 16S rr gene Increased facultative bacteria in colonic biopsies of patients with Crohn s disease vs controls (P <0.001). Significantly increased Ruminococcus gnavus subgroup in small bowel biopsies of patients with Crohn s disease compared with controls (P <0.001). Decrease in Clostridium leptum and Prevotella nigrescens subgroups in small bowel biopsies of Crohn s disease vs controls 32 Crohn s disease, n = 54 Ulcerative colitis, n = 119 Intermediate colitis, n = 104 Self-limiting colitis, n = 28 Asymptomatic controls, n = 40 Culture (anaerobic or aerobic conditions), FISH, PCR sequencing and electron microscopy While colonic biopsies from non-ibd controls were almost free of bacteria after being washed of fecal contents, bacterial concentrations in IBD patients remained high. Number of bacteria adherent to mucosal surface in patients with Crohn s disease was 2 powers higher than in controls. All bacteria identified were of fecal origin. No difference in composition between Crohn s disease and controls 33 Active Crohn s disease, n = 12 Active ulcerative colitis, n = 12 Non-IBD, n = 14 FISH Bacteroides-Prevotella cluster reduced in ileum of patients with Crohn s disease (mean 5.1%) vs controls (mean 31.7%). Clostridium coccoides and Eubacterium rectale group decreased in colon of patients with Crohn s disease (mean 10.6%) vs controls (mean 31.7%) 34 Active Crohn s disease, n = 12 Active ulcerative colitis, n = 7 Intermediate colitis, n = 6 Lymphonodular hyperplasia, n = 10 Healthy controls, n = 7 Culture on selective and nonselective agar (anaerobic or aerobic conditions), PCR and q PCR Culture: significantly higher total number of aerobes and facultative anaerobes in ileum, cecum and rectum of patients with Crohn s disease (P = 0.005, P = 0.02, P <0.005, respectively), and Gram-negative bacteria (P = 0.005, P = 0.02, P = 0.00, respectively) vs controls. Reduction in percentage of Bacteroides vulgatus in patients with Crohn s disease vs controls. Increased detection of E. coli in children with Crohn s disease (75%) vs controls (25%) 35 Crohn s disease, n = 20 Ulcerative colitis, n = 15 Healthy controls, n = 14 PCR-DGGE, 16S rr gene clone libraries, and qualitative PCR Bacteria associated with inflamed and noninflamed tissues did not differ. Members of the phylum Bacteroidetes more prevalent in Crohn s disease vs healthy controls but not significant (P >0.05). Increase in unclassified Bacteroidetes in patients with Crohn s disease (10.1%) vs healthy controls (1.5%; P <0.01) 36 Abbreviations: FISH, fluorescent in situ hybridization; PCR-DGGE, PCR-denaturing gradient gel electrophoresis; q PCR, quantitative-pcr; RISA, ribosomal intergenic spacer analysis; SSCP, single strand confirmation polymorphism; T RFLP, terminal-restriction fragment length polymorphism. 156 MARCH 2011 VOLUME 8

6 Table 3 Evidence of compositional changes in the fecal microbiota of patients with Crohn s disease Patients Methods Major findings in relation to Crohn s disease Ref. Crohn s disease, n = 6 Healthy controls, n = 6 Microarray and q PCR Clostridial Cluster XIVa (including Eubacterium rectale) and Clostridial Cluster IV (C. leptum subgroup, F. prausnitzii) 5 10-fold more abundant in healthy controls vs patients with Crohn s disease. Similarly, Firmicutes (Ruminococcus albus, R. callidus, R. bromii) and Bacteroidetes (Bacteroides fragilis and Bacteroides vulgatus) more abundant in healthy controls than in patients with Crohn s disease. Enterococcus spp., Lactobacillus fermentum, Clostridium difficile (Firmicutes), Shigella flexneri, and Listeria spp. more abundant in Crohn s disease compared with healthy controls 37 Crohn s disease, n = 8 Healthy controls, n = 14 Phylogenetic species validation: Crohn s disease control group, n = 16 Healthy control group, n = 16 16S rr gene libraries, PCR, q PCR Validation: microarray Subdoligranutum spp. (Firmicutes) in significantly greater abundance in healthy control group vs Crohn s disease control group (P <0.001). Detection of Oscillibacter valericigenes (Firmicutes) significantly higher in healthy control group (93.75%) vs Crohn s disease control group (12.5%) (P <0.001). F. prausnitzii less abundant in Crohn s disease than in healthy controls and E. coli more abundant in ileal Crohn s disease than healthy controls. Proteus vulgaris and Enterobacter cowanii in greater abundance in Crohn s disease vs healthy controls 38 Crohn s disease, n = 4 Ulcerative colitis, n = 21 Healthy controls, n = 14 Illumina-based metagenomic sequencing Principal component analysis based on the same 155 species clearly separated patients with Crohn s disease from healthy individuals 39 Monozygotic twin pairs with Crohn s disease, n = 10 (discordant [n = 6] and concordant [n = 4]) Healthy twin pairs, n = 8 PCR of 16S rr genes, T RFLP fingerprints using general bacterial and Bacteroides groupspecific primers Bacterial diversity higher in healthy twins (median 0.91) compared with twins with Crohn s disease (median 0.87; P = 0.029). Significantly lower abundance of B. uniformis in Crohn s disease twins with ileal involvement (P <0.0005) compared with both healthy twins (P = 0.006) and twins with colonic disease (P = ). Higher abundance of B. ovatus and B. vulgatus in Crohn s disease twins with ileal involvement than in healthy twins (P = 0.08 and P = 0.12, respectively) 40 Crohn s disease, n = 23 Ulcerative colitis, n = 73 Healthy controls, n = 65 q-pcr, FISH and culture Bacteroidaceae, Bifidobacterium and Veillonella lower in number in patients with Crohn s disease compared with healthy controls (P <0.05, P <0.05 and P <0.01, respectively). Bacteroides fragilis group and B. vulgatus significantly decreased in number in patients with Crohn s disease vs healthy controls (P <0.01) 41 Crohn s disease in remission, n = 6 Healthy controls, n = 6 Metagenomic libraries, macroarray (pooled samples), FISH (individual samples) Significantly fewer Firmicutes ribotypes (ribotypes, n = 13) identified in patients with Crohn s disease vs healthy controls (ribotypes, n = 43) (P <0.025). In particular Clostridium leptum group significantly reduced (P <0.02) in patients with Crohn s disease compared with controls 42 Active Crohn s disease, n = 13 Active ulcerative colitis, n = 13 Infectious colitis, n = 5 Healthy controls, n = 13 FISH adapted to flow cytometry Significantly higher percentage of total bacteria in healthy controls (86.6% ± 12.7) vs patients with Crohn s disease (70.9% ± 15.0). C. leptum group reduced in Crohn s disease (13.1% ± 11.9 vs 25.2% ± 14.2 in healthy controls; P = 0.002). No significant difference in abundance of Bacteroides group in Crohn s disease (13.8% ± 11.8) vs healthy controls (12.1% ± 7.0) 43 Colonic Crohn s disease in remission, n = 9 Active colonic Crohn s disease, n = 8 Healthy controls, n = 16 Quantitative dot blot hybridization and TTGE Relative proportion of Bacteroides group and Bifidobacteria decreased in both active Crohn s disease and quiescent Crohn s disease versus healthy controls; however, only significant for quiescent Crohn s disease (Bacteroides; P = and Bifidobacteria; P = 0.019). Enterobacteria increased in active Crohn s disease (11%) and quiescent Crohn s disease (6%) vs healthy controls (0%) 44 Abbreviations: FISH, fluorescent in situ hybridization; q PCR, quantitative-pcr; T RFLP, terminal-restriction fragment length polymorphism; TTGE, temporal temperature gradient gel electrophoresis. epithelial cells, survive intracellularly in macrophages and induce high levels of TNF. 56,57 AIEC has been associated with ileal Crohn s disease, with one study reporting the isolation of AIEC in 22% of neoterminal ileal biopsy samples from 40 patients with Crohn s disease, compared with 6.2% of 16 non-ibd controls. 58 Another study reported the isolation of AIEC in 29% of patients with Crohn s disease compared with 9% of controls. 59 The abundance and richness of AIEC seems to be higher in patients with Crohn s disease compared with healthy controls. 60 In vitro characterization of the Crohn sdisease-associated AIEC strain LF82 revealed that it is able to induce the formation of multinucleated giant cells, resulting in structures resembling that of granulomas. 61 Attachment, invasion, intracellular survival or replication of AIEC in host cells are facilitated by a repertoire of virulence factors, including outer membrane proteins OmpA and OmpC, Type I pili, and periplasmic oxidoreductase DsbA Interestingly, the patho genicity of AIEC is mediated or enhanced by host factors, such as the host adhesion receptor carcinoembryonic antigenrelated cell adhesion molecule 6 (CEACAM6) 65 and an endoplasmic- reticulum-localized stress response chaperone Gp96 64 the levels of which are increased in the ileal epithelium of patients who are in the acute or quiescent phase of Crohn s disease. Mice with DSS-induced colitis challenged with LF82 experience weight loss and substantially worse clinical symptoms compared with those infected with nonpathogenic strains. 66 These studies show that the adherent and invasive properties of AIEC are augmented in inflamed conditions of the gut and the arsenal of virulence factors identified in AIEC argues for its role in potentiating damage to the intestinal barrier. Taken together, there is increasing evidence to indicate that AIEC is a likely trigger of ileal Crohn s disease in susceptible individuals. TURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY VOLUME 8 MARCH

7 Campylobacter concisus In the past 2 years, Campylobacter species other than Campylobacter jejuni, such as Campylobacter concisus, have been implicated in Crohn s disease. C. concisus has been detected and isolated from children with newly diagnosed Crohn s disease. 67,68 Detection of C. concisus D showed that 65% of children with Crohn s disease had a positive PCR result for this bacteria (35 of 54), which is significantly higher than that of healthy (33%, 11 of 33) and non-ibd controls (37%, 10 of 27). 68 A strain of C. concisus isolated from a child with Crohn s disease has been shown to invade and induce a membrane-ruffling phenomenon in Caco 2 intestinal epithelial cells and disrupt barrier function by inducing the movement of tight junction proteins from the membrane to the cytosol in these cells. 69 A range of virulence factors have been identified in clinical strains of C. concisus, including cellbound and secreted hemolysins, zonula occludens toxin, an RTX toxin and a toxin similar to the cytolethal distending toxin capable of inducing cytopathic effects In a mouse model, C. concisus has the ability to colonize the ileum and liver of immunocompetent BALB/cA mice and cause weight loss, intestinal inflammation and occasional formation of liver microabscesses. 74 Given that there are two to four genetically diverse groups (genomospecies) of C. concisus, some of which readily colonize the gut of healthy individuals, it is likely that only a subset of C. concisus strains is pathogenic and clinically relevant in Crohn s disease. Helicobacter species Interest in the possible involvement of Helicobacter species in the initiation of Crohn s disease arose when a number of enterohepatic Helicobacter species (EHS) were found to induce an IBD-like condition in a range of immunocompromised mice. 75 Unlike gastric Helicobacter pylori, EHS colonize the mucus layer that lines the lower bowel mucosa and intestinal crypts of a range of mammals and birds. 76 In humans, the role of Helicobacter species in Crohn s disease remains unclear, as studies have generated inconsistent results. In detection studies, Bohr et al. found EHS D in 12% of patients with Crohn s disease (3 of 25) compared with 4% of controls (1 of 23). 77 Zhang et al. used FISH to show that members of Helicobacteraceae present in the intestinal mucus of children with IBD were viable. 78 In related studies, a significantly higher prevalence of EHS and H. pylori D was found to be present in fecal samples from children with Crohn s disease (17 of 29; 59%) compared with asymptomatic healthy children (1 of 11; 9%; P = 0.01) and symptomatic children with non-ibd pathology (0 of 26; 0%; P <0.0001). 79 A larger follow-up study by Kaakoush et al. also found that the presence of EHS and H. pylori D was significantly higher in patients with Crohn s disease (41.5%) than in controls (22.5%). 80 Of particular interest in both of these studies is that H. pylori D was detected in patients who were negative for gastric H. pylori, suggesting the possibility that some H. pylori strains or H. pylori-like organisms may colonize the small or large intestinal tract. Another study has reported a possible association between the EHS Helicobacter pullorum and Helicobacter canadensis and Crohn s disease. 81 H. pullorum strains have been shown to stimulate IL 8 secretion by human gastric and intestinal epithelial cell lines. This requires bacterial adherence, lipopolysaccharides (LPS), and nuclear factor κb (NFκB) signaling. 82 Although there have been conflicting results in the past with respect to the clinical importance of Helicobacter species in Crohn s disease, 83,84 there are intriguing findings related to this group of mucosa-associated bacteria that warrant further investigation. Other mucosa-associated bacteria in Crohn s disease Several other pathogenic and mucosa-associated bacteria have been associated with Crohn s disease. Investigation of Pseudomonas species in Crohn s disease by Wagner et al. 85 revealed that 58% of children with Crohn s disease were positive for Pseudomonas spp., a prevalence that was significantly higher than that of their non-ibd counterparts (33%; P <0.05). The identified species were closely related to Pseudomonas brenneri, Pseudomonas migulae, Pseudomonas panacis and Pseudomonas proteolytica. In addition, an antigen from Pseudomonas fluorescens, known as I2, has been linked to Crohn s disease severity A number of other studies have also reported that Yersinia enterocolitica and other Yersinia species are associated with Crohn s disease Overall, at least 18 different bacterial species or genera have been implicated in Crohn s disease, and an extensive list of these and the evidence supporting or arguing against their role in disease etiology is shown in Table 4. Bacteria involved in predisposition to Crohn s disease A number of bacterial species have been proposed as possible predisposing factors to Crohn s disease as opposed to being etiological agents. C. jejuni is recog nized primarily for its role in acute gastro enteritis. This pathogen has been reported in some studies to be associated with an increased risk of developing Crohn s disease and ulcerative colitis and with subsequent disease flares By contrast, one study found that exposure to Campylobacter gastroenteritis only increases the risk of developing ulcerative colitis, but not Crohn s disease. 96 To date, studies examining the prevalence of C. jejuni have shown that 3 6% of children with Crohn s disease are positive for C. jejuni. 67,68 Like C. jejuni, Salmonella spp. are known to cause gastro enteritis, as well as typhoid fever in humans. In a population- based cohort study that examined data from 39,364 individuals from Denmark, an increased risk of developing Crohn s disease and ulcerative colitis was demon strated for indivi duals who had previously suffered from Salmonella-associated gastroenteritis. 95 It is possible, therefore, that in recently exposed individuals or in chronic carriers these gastrointestinal pathogens may be predisposing factors for Crohn s disease. In this context, it is clear that recent infection induces dys biosis, an outcome that may eventuate in the onset of Crohn s disease (Figure 1). Whether recent exposure to or chronic carriage of any other gastrointestinal pathogens result in an increased risk of developing Crohn s disease requires further investigation. 158 MARCH 2011 VOLUME 8

8 Table 4 Evidence for the potential role of specific bacterial species in the pathogenesis of Crohn s disease Bacteria* Supporting evidence Contradicting evidence Adherent and invasive Escherichia coli Higher prevalence of adherent and invasive E. coli in patients with ileal Crohn s disease than controls 58,59 Burkholderiales spp. Novel 60-type sequevar associated with perianal Crohn s disease 167 Campylobacter concisus Campylobacter jejuni Chlamydia spp. Coxiella spp. Enterohepatic Helicobacter spp. (EHS) Faecalibacterium prausnitzii Detected and isolated from children with Crohn s disease; 67 detection of C. concisus D showed that 65% of children with Crohn s disease were positive (35 of 54), which was significantly higher than that of healthy (33%, 11 of 33) and non-ibd controls (37%, 10 of 27) 68 C. jejuni gastroenteritis increases the risk of developing Crohn s disease and has been associated with flare ups Associated with exacerbations of gastrointestinal symptoms in patients with Crohn s disease; 169 antibodies against Chlamydia were detected in 69% of Crohn s disease patients, 9.5% of patients with other gastrointestinal disorders, and 2% of healthy controls 170 Associated with exacerbations of gastrointestinal symptoms in patients with Crohn s disease 169 Found to induce a severe IBD-like condition in a range of immunocompromised mice; 75 Helicobacteraceae D was detected in significantly higher numbers of patients with Crohn s disease than in controls; 77,79 81 Helicobacteraceae present in the intestinal mucus were viable 78 Significantly lower levels of F. prausnitzii in patients with Crohn s disease compared with controls; 37,38, F. prausnitzii exhibits anti-inflammatory effects on cellular and colitis models, suggesting a protective effect of this bacterium in Crohn s disease pathogenesis 175 C. concisus has been isolated from healthy and diarrheic individuals 168 Detected in a low number of Crohn s disease patients (3 6%); 67,68 exposure to Campylobacter gastroenteritis only increases the risk of developing ulcerative colitis, but not Crohn s disease 96 Failure to detect any EHS in patients with IBD or no association between the presence of EHS and Crohn s disease 83,84 No correlation between F. prausnitzii abundance and severity of Crohn s disease, and clinical improvement correlated with a significant decrease in the abundance of F. prausnitzii 176 Helicobacter pylori Seems to have a protective role in the development of IBD 177 Reports of similar rates of H. pylori infection between patients with Crohn s disease and controls 177 Klebsiella spp. Listeria monocytogenes Mycobacterium avium subsp. paratuberculosis (MAP) Mycoplasma spp. Pseudomonas spp. Salmonella spp. Staphylococcus aureus Streptococcus spp. Yersinia spp. D identified in patients with Crohn s disease but not in healthy controls. 178 The presence of K. pneumoniae correlated with colitis in a genetically deficient mouse model 46 Reported in more patients with Crohn s disease (75%) compared with either ulcerative colitis (13%) or controls (0%); 179 reported to be more abundant in patients with Crohn s disease than in controls 37 Higher prevalence of MAP from culture, PCR and ELISA methodologies from numerous studies; 47,48,54,55 specific antibodies against MAP proteins that are essential for establishing an infection have been identified in patients with Crohn s disease; 50,51 positive outcomes using anti-map treatments in promoting Crohn s disease remission for some patients 52 Associated with exacerbations of gastrointestinal symptoms in patients with Crohn s disease % of patients with Crohn s disease demonstrated positive test results for this bacteria, which was significantly higher than for non-ibd children (33%); 85 seroreactivity for Pseudomonas fluorescens-associated sequence I2 was observed in 42% of IBD patients and 7.7% of non-ibd cases 88 An increased risk of development of IBD was demonstrated for individuals who had previously suffered from Salmonella-associated gastroenteritis 95 D identified in patients with Crohn s disease but not in healthy controls 178 D identified in patients with Crohn s disease but not in healthy controls; % of patients contained streptococcal antigen of 54 resections (31%) from patients with Crohn s disease contained Yersinia D while all the controls were negative; 89 associated with the development of Crohn s disease in case study Studies have not been able to associate this pathogen with IBD by PCR amplification 180,181 Similar prevalence of MAP using culture, PCR and ELISA methodologies from numerous studies; 49,54 no association between occupational exposure to MAP and Crohn s disease 182 Salmonella-associated gastroenteritis is considered only a possible predisposing factor; thus, the bacterium is unlikely to initiate Crohn s disease *All are invasive pathogens except Faecalibacterium prausnitzii and Helicobacter pylori. Abbreviation:, not available. Bacterial recognition and killing Although there is convincing evidence to argue that bacteria have a role in the pathogenesis of Crohn s disease, host responses are also important. Host recognition of bacteria is part of innate immunity and requires pattern-recognition receptors. These proteins recognize TURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY VOLUME 8 MARCH

9 Box 2 Examples of pattern-recognition receptors Toll-like receptors (TLRs): TLR1 10 NOD-like receptors (NLRs): NLRA/CIITA, NLRB1/IP, NLRC1 5 (including NOD1 and NOD2), NLRP1 14, NLRX1 RIG I-like receptors (RLRs): RIG I, MDA5 C-type lectins receptors (CLRs): Dectin 1, dectin 2 AIM2-like receptors (ALRs): AIM2, IFI16 specific classes of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) and subsequently trigger proinflammatory responses. The theory that bacteria could have a key role in the initiation of Crohn s disease rather than being solely a driving factor was conceptualized in 2001 when the pattern-recognition receptor NOD2 (also known as CARD15) was linked to Crohn s disease. 97,98 Pattern-recognition receptors include TLRs, C type lectin receptors (CLRs), NLRs, RIG I-like receptors (RLRs) and AIM2-like receptors (ALRs) (Box 2). Since 2001, mutations in a growing number of other TLRs, NLRs and related molecules have been identified as risk factors for Crohn s disease, providing further evidence to suggest that defective sensing of bacteria or its constituents may be involved in the etiopathogenesis of Crohn s disease (Figure 2). Bacteria host interaction via TLRs TLRs are vital for the detection of extracellular bacteria or those present in the endosomal compartments of cells. To date, at least 10 members of the TLR family have been identified in humans. 99 Evidence suggests that impaired bacterial sensing as a consequence of mutations in TLRs may be a contributing factor in Crohn s disease suscepti bility (Table 5). TLR4 Examination of biopsy samples from the inflamed intestine of Crohn s disease patients has revealed elevated numbers of TLR4-expressing cells and increased TLR4 mr and protein expression TLR4 recog nizes LPS, which is a major component of the outer membrane of Gram-negative bacteria. Activation of TLR4 by LPS or bacteria leads to the production of pro inflammatory cytokines and/or interferon β in host cells. 104 In 2004, a mutation in TLR4 (D299G) was reported to be associated with Crohn s disease in two predominately white cohorts in Belgium. 105 Other independent studies in Australian, Dutch and Greek cohorts have confirmed that the same polymorphism in TLR4 is associated with Crohn s disease, particularly of the colonic phenotype However, no association of the D299G mutation and Crohn s disease was observed in white populations in Tunisia, Hungary and New Zealand Functional studies have shown that a monocyte cell line transfected with TLR4 carrying the D299G polymorphism abolished NFκB activities following LPS stimulation. 112 Furthermore, the responsiveness to LPS in epithelial cells or macrophages from individuals who have the homozygous D299G mutation is restored following transfection with a wild-type TLR4. Interestingly, individuals who bear the same mutation are hyporesponsive to inhaled LPS but are highly susceptible to infection by Gram-negative bacteria Given that individuals who have suffered from gastroenteritis caused by Gram-negative bacteria have been shown to have an increased risk of developing Crohn s disease, 95 it is reasonable to speculate that TLR4 mutations may modulate susceptibility to Crohn s disease. Other TLRs TLR9 is an endosomal membrane-bound TLR, which detects unmethylated CpG D of bacteria and has been shown to be expressed at similar levels in colonic tissues of healthy individuals and patients with Crohn s disease. 115,116 A German study has found that a polymorphism in the promoter of TLR9 (1237T>C) was linked to susceptibility to Crohn s disease in patients who also carried one or two mutations in NOD The mutation in TLR9 alone did not appear to confer susceptibility to Crohn s disease in the same German cohort nor in a cohort from New Zealand, 111 suggesting that mutations in more than one TLR or NLR may contribute to Crohn s disease susceptibility in a synergistic fashion. A number of studies examining other TLRs have revealed that TLR5, a receptor for extracellular bacterial flagellin, or TLR2, a receptor for lipoproteins, do not appear to be associated with Crohn s disease. 107,111 Intriguingly, a study examining 35 single nucleotide polymorphisms (SNPs) in TLR1 10 in a white Flemish population showed that an SNP in TLR1 (S602I) is negatively associated with Crohn s disease but not ulcerative colitis. 118 A number of studies have shown that the expression profile of other TLRs, including TLR2, TLR3, and TLR8, is different in the ileum or colon between patients with Crohn s disease and non-ibd controls. 101,102,119 Taken together, there is emerging evidence to suggest that defective TLR sensing of bacteria may lead to susceptibility to Crohn s disease. Bacteria host interaction via NLRs NLRs are a large family of receptors that function as sensors of pathogens within the cytosolic compartment of a cell. NLRs consist of an N terminal domain, a central nucleotide-binding domain, and a C terminal domain comprised of leucine-rich repeats. To date, at least 22 members of the NLR family have been identi fied in humans, 120 three of which (NOD1, NOD2, NLRP3) have been implicated in Crohn s disease (Table 6). NOD2 NOD2 recognizes muramyl dipeptide present in the peptido glycan of both Gram-positive and Gram-negative bacteria. NOD2 has been shown to activate NFκB and trigger the release of the proinflammatory cytokines TNF 160 MARCH 2011 VOLUME 8