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152 | MARCH 2011 | VOLUME 8 www.nature.com/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

The role of bacteria and pattern-recognition receptors in Crohn’s diseaseSi 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, 152–168 (2011); published online 8 February 2011; corrected online 18 March 2011; doi:10.1038/nrgastro.2011.3

IntroductionCrohn’s disease is a form of IBD with unknown etiol-ogy. 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 dys-regulated 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 bac-teria may contribute to remission (Table 1). A meta- analysis of six randomized, placebo-controlled clinical trials, published between 1966 and 2006, showed that broad-spectrum antibiotics (metronidazole, cipro-floxacin or co-trimoxazole) seem to improve clinical outcomes in individuals affected by Crohn’s disease, and that patients receiving antibacterial therapy were 2.257 times more likely to show clinical improvement com-pared with those receiving placebo.16 Investigations using mouse models of Crohn’s disease complement findings

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 techno-logical 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 examin-ing the involvement of specific groups of bacteria in the etiology of disease. This Review also explores the contri-bution of impaired bacterial sensing and killing as a con-sequence of mutations in pattern-recognition receptors, including Toll-like receptors (TLRs), and Nod-like recep-tors (NLRs), highlighting the dynamics of bacterial–host interactions in the pathogenesis of Crohn’s disease.

Dysbiosis of the gut microbiotaThe adult human intestinal microbiota of the ileum and colon are composed of >104 and >1012 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 immuno-logical 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

Competing interestsThe authors declare no competing interests.

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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 com-position 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 intes-tinal 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 rRNA 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 com-pared with non-IBD or healthy controls; in some studies E. coli in particular was increased (Tables 2 and 3).24–44 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 (~108 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 belong-ing 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 incon-sistent. For example, based on 16S rRNA amplifica-tion, 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 demon-strate that Bacteroidetes were significantly depleted in Crohn’s disease compared with controls. In addition, Ott et al.,31 using 16S rDNA based single-strand confirma-tion 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

q-PCR of 16S rRNA genes, showed the microbial diver-sity 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 speci-ficity, 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

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 diversion2

■ 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 ileum3

■ 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% died4

■ 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 diversion5

■ 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 recruitment6

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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 micro-biota. Interestingly, Rehman et al.24 have reported that a discrepancy exists between intestinal bacterial rich-ness (measured by rRNA gene sequences) and bacterial transcriptional activity (measured by rRNA sequences). This study showed that in Crohn’s disease, bacterial rich-ness was significantly lower in clone libraries based on rRNA as compared with those based on the rRNA genes (3.10 versus 3.91), while in healthy individuals no differ-ence was observed (3.81 versus 3.85).24 Specifically, in patients with Crohn’s disease, the transcriptional activ-ity of the Firmicutes and Actinobacteria was decreased and that of Bacteroidetes was increased (P <0.01) com-pared 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 intes-tinal 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 bacteriaMucosa-associated bacteria colonize areas in close prox-imity 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.30,33,34

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)

Median 12.9 weeks Clinical remission defined as CDAI <150Remission rate in patients treated with rifaximin alone (67%) higher than in those on rifaximin plus steroids (58%)

7

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)

12 weeks Clinical remission defined as CDAI ≤150Remission 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%)

8

1) Ciprofloxacin (500 mg twice daily) (n = 28)2) Placebo (n = 19)

6 months Clinical remission defined as CDAI <150Significant reduction in CDAI score in patients treated with ciprofloxacin (mean CDAI = 112) compared with placebo (mean CDAI = 250, P <0.001)

9

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)

8 weeks Clinical remission defined as CDAI <150No 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)

10

1) Ciprofloxacin (500 mg twice daily) (n = 18)2) Mesalazine (2 g twice daily) (n = 22)

6 weeks Complete clinical remission CDAI score ≤15055.5% of patients with Crohn’s disease treated with ciprofloxacin in complete remission versus 54.5% treated with mesalazine

11

1) Ciprofloxacin (500 mg twice daily) plus metronidazole (250 mg four times daily) (n = 22)2) Methylprednisolone (n = 19)

12 weeks Clinical remission defined as a CDAI ≤150Clinical remission achieved in 45.5% of antibiotic-treated patients compared with 63% patients treated with steroids

12

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

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%)

13

1) Metronidazole (10 mg/kg body weight) (n = 33)2) Metronidazole (20 mg/kg body weight) (n = 30)3) Placebo (n = 36)

>2–16 weeks Clinical remission defined as CDAI <150Treatment 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

14

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 pointsAt 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 20–60 weeks (median 28 weeks)

15

Abbreviations: CDAI, Crohn’s disease activity index; HBI, Harvey-Bradshaw Index.

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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 paratuberculosisMycobacterium 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.47–49 Specific antibodies against MAP proteins that are essen-tial for establishing infection have been identified in patients with Crohn’s disease50,51 and there is evidence to

show that anti-MAP treatments promote disease remis-sion 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 anti-mycobacterial therapy and the pathogenic potential of MAP.54,55

Adherent and invasive Escherichia coliAdherent and invasive Escherichia coli (AIEC) are character ized by their ability to invade intestinal

Normal gut microbiota

Crohn’s disease

Dysbiosis of the gut microbiota Primary infection

Predisposing factors:■ Environment■ Dietary■ Genetic■ Gastroenteritis

Predisposing factors:■ Environment■ Dietary■ Genetic■ Gastroenteritis

■ Dysregulated immune responses■ Genetic factors

■ Dysregulated immune responses■ Genetic factors

Lumen

Mucus layer

Increased susceptibility to infection

Altered microbiota

Intestinalepithelium

Submucosa

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).

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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 = 10Active ulcerative colitis, n = 10Healthy controls, n = 10

Amplification of 16S rRNA 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 = 68Ulcerative colitis, n = 61Non-IBD controls (primarily GI cancer patients), n = 61

RNA 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 = 8Healthy controls, n = 7

16S rDNA 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 = 0.0006). 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 = 0.0012)

26

Crohn’s disease, n = 13Ulcerative colitis, n = 19Healthy 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)

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PCR using 16S rRNA, cloning and sequencing

Proteobacteria significantly increased in patients with Crohn’s disease (12.9%) vs healthy controls (8.4%, P = 0.0007). 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 = 19Ulcerative colitis, n = 2Intermediate colitis, n = 1Healthy controls, n = 15

PCR-DGGE of 16S rRNA 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


FISH Mucosal bacteria at concentrations >109 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 = 26Active ulcerative colitis, n = 31Controls, n = 46 (15 with intestinal inflammation and 31 without)

16S rRNA 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 = 16Crohn’s disease surgical samples, n = 15 Non-IBD controls, n = 10

Universal eubacteria 16S rRNA 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 = 54Ulcerative colitis, n = 119Intermediate colitis, n = 104Self-limiting colitis, n = 28Asymptomatic 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 = 12Active ulcerative colitis, n = 12Non-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 = 12Active ulcerative colitis, n = 7Intermediate colitis, n = 6Lymphonodular hyperplasia, n = 10Healthy 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%)

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PCR-DGGE, 16S rRNA 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.

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epithelial cells, survive intracellularly in macrophages and induce high levels of TNF.56,57 AIEC has been associ-ated with ileal Crohn’s disease, with one study report-ing 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’s-disease-associated AIEC strain LF82 revealed that it is able to induce the formation of multinucleated giant cells, resulting in structures resembling that of granu-lomas.61 Attachment, invasion, intracellular survival or replication of AIEC in host cells are facilitated by a reper-toire of virulence factors, including outer membrane proteins OmpA and OmpC, Type I pili, and periplasmic

oxidoreductase DsbA.62–64 Interestingly, the patho genicity of AIEC is mediated or enhanced by host factors, such as the host adhesion receptor carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6)65 and an endoplasmic- reticulum-localized stress response chaper-one Gp9664—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 sub-stantially 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.

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 = 6Healthy 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 = 8Healthy controls, n = 14Phylogenetic species validation:Crohn’s disease control group, n = 16Healthy control group, n = 16

16S rRNA gene libraries, PCR, q-PCRValidation: 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


Illumina-based metagenomic sequencing

Principal component analysis based on the same 155 species clearly separated patients with Crohn’s disease from healthy individuals

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Monozygotic twin pairs with Crohn’s disease, n = 10 (discordant [n = 6] and concordant [n = 4])Healthy twin pairs, n = 8

PCR of 16S rRNA genes, T-RFLP fingerprints using general bacterial and Bacteroides group-specific 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 = 0.0003). 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)

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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 = 6Healthy 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 = 13Active ulcerative colitis, n = 13Infectious colitis, n = 5Healthy 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 = 9Active colonic Crohn’s disease, n = 8Healthy 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 = 0.013 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.

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Campylobacter concisusIn 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 DNA 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 cell-bound and secreted hemolysins, zonula occludens toxin, an RTX toxin and a toxin similar to the cytolethal dis-tending toxin capable of inducing cytopathic effects.70–73 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 occa-sional formation of liver microabscesses.74 Given that there are two to four genetically diverse groups (geno-mospecies) of C. concisus, some of which readily colo-nize 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 speciesInterest 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 immuno-compromised 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 DNA 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 preva-lence of EHS and H. pylori DNA 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 DNA 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 DNA was detected in patients who were negative for gastric H. pylori, suggesting the possibil-ity 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 intesti-nal 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 intrigu-ing findings related to this group of mucosa-associated bacteria that warrant further investigation.

Other mucosa-associated bacteria in Crohn’s diseaseSeveral 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 counter-parts (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 sever-ity.86–88 A number of other studies have also reported that Yersinia enterocolitica and other Yersinia species are associated with Crohn’s disease.89–92 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 diseaseA number of bacterial species have been proposed as pos-sible predisposing factors to Crohn’s disease as opposed to being etiological agents. C. jejuni is recog nized primar-ily 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 ulcer-ative colitis and with subsequent disease flares.93–95 By contrast, one study found that exposure to Campylobacter gastroenteritis only increases the risk of developing ulcer-ative 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.

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Bacterial recognition and killingAlthough there is convincing evidence to argue that bac-teria 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 pat-tern-recognition receptors. These proteins recognize

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 controls58,59

NA

Burkholderiales spp. Novel 60-type sequevar associated with perianal Crohn’s disease167 NA

Campylobacter concisus

Detected and isolated from children with Crohn’s disease;67 detection of C. concisus DNA 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. concisus has been isolated from healthy and diarrheic individuals168

Campylobacter jejuni C. jejuni gastroenteritis increases the risk of developing Crohn’s disease and has been associated with flare ups93–95

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 disease96

Chlamydia spp. 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 controls170

NA

Coxiella spp. Associated with exacerbations of gastrointestinal symptoms in patients with Crohn’s disease169

NA

Enterohepatic Helicobacter spp. (EHS)

Found to induce a severe IBD-like condition in a range of immunocompromised mice;75 Helicobacteraceae DNA 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 viable78

Failure to detect any EHS in patients with IBD or no association between the presence of EHS and Crohn’s disease83,84

Faecalibacterium prausnitzii

Significantly lower levels of F. prausnitzii in patients with Crohn’s disease compared with controls;37,38,171–174 F. prausnitzii exhibits anti-inflammatory effects on cellular and colitis models, suggesting a protective effect of this bacterium in Crohn’s disease pathogenesis175

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. prausnitzii176

Helicobacter pylori Seems to have a protective role in the development of IBD177 Reports of similar rates of H. pylori infection between patients with Crohn’s disease and controls177

Klebsiella spp. DNA 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 model46

NA

Listeria monocytogenes

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 controls37

Studies have not been able to associate this pathogen with IBD by PCR amplification180,181

Mycobacterium avium subsp. paratuberculosis (MAP)

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 patients52

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 disease182

Mycoplasma spp. Associated with exacerbations of gastrointestinal symptoms in patients with Crohn’s disease169

NA

Pseudomonas spp. 58% 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 cases88

NA

Salmonella spp. An increased risk of development of IBD was demonstrated for individuals who had previously suffered from Salmonella-associated gastroenteritis95

Salmonella-associated gastroenteritis is considered only a possible predisposing factor; thus, the bacterium is unlikely to initiate Crohn’s disease

Staphylococcus aureus

DNA identified in patients with Crohn’s disease but not in healthy controls178 NA

Streptococcus spp. DNA identified in patients with Crohn’s disease but not in healthy controls;178 44% of patients contained streptococcal antigen179

NA

Yersinia spp. 17 of 54 resections (31%) from patients with Crohn’s disease contained Yersinia DNA while all the controls were negative;89 associated with the development of Crohn’s disease in case study90–92

NA

*All are invasive pathogens except Faecalibacterium prausnitzii and Helicobacter pylori. Abbreviation: NA, not available.

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specific classes of pathogen-associated molecular pat-terns (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 constitu-ents may be involved in the etiopathogenesis of Crohn’s disease (Figure 2).

Bacteria–host interaction via TLRsTLRs are vital for the detection of extracellular bacte-ria 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).

TLR4Examination of biopsy samples from the inflamed intes-tine of Crohn’s disease patients has revealed elevated numbers of TLR4-expressing cells and increased TLR4 mRNA and protein expression.100–103 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.106–108 However, no association of the D299G mutation and Crohn’s disease was observed in white populations in Tunisia, Hungary and New Zealand.109–111 Functional studies have shown

that a monocyte cell line transfected with TLR4 carry-ing the D299G polymorphism abolished NFκB activities following LPS stimulation.112 Furthermore, the respon-siveness 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 suscep-tible to infection by Gram-negative bacteria.112–114 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 TLRsTLR9 is an endosomal membrane-bound TLR, which detects unmethylated CpG DNA 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 poly-morphism 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 NOD2.117 The muta-tion 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 bacte-rial 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 ulcer-ative 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 con-trols.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 NLRsNLRs 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).

NOD2NOD2 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

Box 2 | Examples of pattern-recognition receptors

Toll-like receptors (TLRs): ■ TLR1–10

NOD-like receptors (NLRs): ■ NLRA/CIITA, NLRB1/NAIP, 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

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and IL-8.121 Three mutations in NOD2 have been found to be associated with Crohn’s disease susceptibility, including two amino acid substitutions (R702W and G908R) and a frameshift mutation (1007fs).97,98 These mutations associ-ated with Crohn’s disease are located within the leucine-rich repeats domain, which has been hypothesized to be involved in ligand recognition. Convincing evidence demon strates that host cells carrying these mutations generally respond poorly to bacteria and bacterial-derived ligands, suggest-ing that inappropriate detection of intra cellular bacteria may result in the initiation of Crohn’s disease. Monocyte-derived dendritic cells or PBMC from patients with Crohn’s disease exhibiting the NOD2 frameshift mutation have a substantially impaired ability to secrete a range of cyto-kines, including TNF, IL-1β, IL-8, IL-10, IL-12 or granulo-cyte–macrophage colony-stimulating factor (GM-CSF) in

response to M. tuberculosis, S. Typhimurium or muramyl dipeptide.122–126 In addition, HEK293T cells transfected with a NOD2 variant associated with Crohn’s disease fail to activate NFκB, which suggests that these mutations inhibit NOD2 functions.127,128 Following TLR priming and muramyl dipeptide stimulation, dendritic cells from patients carrying the NOD2 frameshift mutation also have a reduced capacity to promote IL-17 production in memory T cells.129 Furthermore, macrophages with wild-type NOD2 that were exposed to muramyl dipeptide did not show elevated proinflammatory responses when they were re-challenged with muramyl dipeptide, lipid A (a TLR4 ligand), or Pam3Cys (a TLR2 ligand), whereas macrophages with the NOD2 frameshift mutation failed to exhibit tolerance (that is, they produced high levels of proinflammatory cytokines).125

TLR5TLR4*TLR2 CD14*

MyD88MAL*

TLR1

Lipoproteins

MDP

LPS Flagellin

FIINDCARD

CARD9*NOD2*

NLRP3*

NLRP3in�ammasome

NLRP3ligands

Pro-caspase-1

Caspase-1

IL-1βIL-18

TNFIL-6, IL-8

Pro-IL-1β/IL-18

CARD8*

ASC

CARDCARD

PYD

CARD

CARD

PYD

NBDMAPKK PYDNBDLRRs

LRRsPYD

Proin�ammatory cytokines

Extracellular

Intracellular

NBDLRRs CARDRIPK2

CARD

MyD88

p65

NFκB

Nucleus

TLR9*

Lysosome

Autophagy

ATG16L1*

CpG DNA

Autophagosome

Enzymaticbreakdown

p50

Figure 2 | Bacterial recognition and signaling via pattern-recognition receptors and autophagy in Crohn’s disease. Extracellular bacteria are recognized by membrane-bound Toll-like receptors (TLRs). TLR1 and TLR2 recognize triacylated lipoprotein. TLR4 detects lipopolysaccharides (LPS), which is amplified by CD14. TLR5 recognizes extracellular flagellin. Activation of these TLRs mediates nuclear factor κB (NFκB) activation, which requires the adaptor protein MyD88. TLR1/2 and 4 also require MyD88 adaptor-like protein (MAL). TLR9 recognizes endosomal CpG DNA and signals through the MyD88 and MAPKK pathways, both of which lead to the production of proinflammatory cytokines. Nod-like receptors (NLRs), including NOD2 and NLRP3, detect intracellular pathogens. The leucine-rich repeats (LRRs) of NOD2 sense muramyl dipeptide (MDP), and NOD2 recruits RIPK2 for signaling via the NFκB pathway, or CARD9 via the MAPKK pathway. Both pathways lead to the production of proinflammatory cytokines. CARD8 functions as an inhibitor of NFκB activation. NOD2 also mediates autophagy by recruiting ATG16L1 to the cell membrane. Autophagy promotes degradation of bacteria via lysosome–phagosome fusion. Disruption of the phagosome releases a range of ligands, some of which are detected by NLRP3. NLRP3 self-oligomerizes via the nucleotide-binding domain (NBD), and the pyrin domain (PYD) then interacts with the PYD of apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC). The CARD of ASC recruits and activates caspase-1 via a CARD–CARD interaction. The formation of the NLRP3 inflammasome results in cleavage of pro-interleukin-1β (pro-IL-1β) and pro-IL-18, generating a matured form of the cytokines. TLR4, TLR9, and NOD2 also mediate the production of Type I interferon. This pathway is not represented in this illustration as the role of Type I interferon in Crohn’s disease is not clear. An asterisk denotes evidence that the presence of one or more polymorphisms confer susceptibility to Crohn’s disease.

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In addition to impaired cytokine production, muta-tions in NOD2 associated with Crohn’s disease also hinder efficient killing of Gram-positive and Gram-negative bacteria, including AIEC, Bacillus subtilis, Staphylococcus aureus, and S. Typhimurium.97,130–132 Furthermore, in an epithelial cell line, primary human macrophages or dendritic cells, killing of S. Typhimurium is enhanced by muramyl-dipeptide-induced NOD2 stimulation.128 Indeed, in epithelial cells, NOD2 has been shown to mediate the production of reactive oxygen species required for the killing of L. mono cytogenes; the NOD2 frameshift mutation was shown to compromise the

efficiency of this killing.133 Results from mouse studies generally agree with those from human studies. Nod2–/– mice injected with muramyl dipeptide fail to rapidly release IL-6, the neutrophil chemoattractant CXC ligand 1 (KC) or monocytic chemotactic protein 1 (MCP1).134 Nod2–/– mice have also been reported to secrete less IL-1β after 72 h post-infection with S. Typhimurium135 and harbor a substantially higher number of E. coli and S. aureus bioparticles in the Peyer’s patches compared with wild-type controls.136 These studies collectively suggest that NOD2 is important for bacterial sensing and killing, potentiating proinflammatory responses,

Table 5 | Toll-like receptors implicated in the pathogenesis of Crohn’s disease

TLR Ligand Known function Relative expression in Crohn’s disease compared with controls

Polymorphism or mutation implicated in Crohn’s disease (region in which the polymorphism is located, if known)

Possible effects of the polymorphism

TLR1 Triacylated lipoprotein (with TLR2)

Production of proinflammatory cytokines

NA S602I (intracellular domain)118 Negatively associated with ileal Crohn’s disease118

TLR2 Triacylated lipoprotein (with TLR1); diacylated lipoprotein (with TLR6)

Production of proinflammatory cytokines

Increased levels of TLR2-positive epithelial cells and TLR2 protein in inflammatory cells of lamina propria; increased TLR2 expression on CD14+ monocytes102,103,183

NA NA

TLR4 LPS Production of proinflammatory cytokines and Type I interferon

Increased TLR4-positive cells, mRNA and protein levels in intestinal tissue100–103

D299G (extracellular domain)105–108,111 LPS hyporesponsiveness112

TLR9 CpG DNA (viruses and bacteria); DNA–immunoglobulin complexes

Production of proinflammatory cytokines and Type I interferon

Similar TLR9 mRNA levels in colonic mucosa of patients with Crohn’s disease and controls116

–1237T>C (promoter region) in combination with Crohn’s-disease-associated NOD2 single nucleotide polymorphisms111,117

Increased TLR9 transcriptional activity (transfection studies)184

Abbreviations: LPS, lipopolysaccharide; NA, not available; TLR, Toll-like receptor.

Table 6 | Nod-like receptors implicated in the pathogenesis of Crohn’s disease

NLR Ligand Known function Relative expression in Crohn’s disease compared with controls



NOD1 (also known as CARD4)

GM-TriDAP (primarily from Gram-positive bacteria)

Production of proinflammatory cytokines; initiation of caspase-9-dependent apoptosis; recruitment of ATG16L1 for autophagy

NA ND1+32656*1 (an intronic region of the leucine-rich repeats domain)138,139

E266K (G796A) (nucleotide-binding domain)185

ND1+32656*1: reduced binding to an unidentified nuclear protein in a lung adenocarcinoma cell line186

NOD2 (also known as CARD15)

Muramyl dipeptide (Gram-positive and Gram-negative bacteria)

Production of proinflammatory cytokines; recruitment of ATG16L1 for autophagy

Expressed in Paneth cells of Crohn’s disease-affected and control tissues187

R702W, G908R, 1007fsinsC (all within the leucine-rich repeats domain)97,98

Decreased proinflammatory cytokine production in immune cells of patients with Crohn’s disease;122–125 impaired bacterial killing and autophagy131,162

NLRP3 (Cryopyrin, NALP3, CIAS1, and Pypaf1)

Nigericin, maitotoxin, ATP, amyloid-β, uric acid crystals, silica, aluminium hydroxide, aluminium phosphate adjuvants

Inflammasome formation and IL-1β and IL-18 production; initiation of caspase-1-dependent pyroptosis; maintenance of intestinal epithelial integrity

Similar NLRP3 mRNA levels in monocytes of patients with Crohn’s disease and controls188

Multiple SNPs downstream of the NLRP3 gene, possibly a regulatory region145

Q705K/2107C>A (nucleotide-binding domain) in combination with C10X of CARD8 and wild-type NOD2147

Reduced NLRP3 mRNA levels in peripheral blood cells and monocytes of Crohn’s disease patients with SNP rs4353135 (upstream of NLRP3)145

Decreased IL-1β production in LPS-stimulated monocytes of Crohn’s disease patients with SNP rs6672995 (upstream of NLRP3)145

Abbreviations: IL, interleukin; LPS, lipopolysaccharide; SNP, single nucleotide polymorphism.

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and possibly for regulating bacterial trafficking in the intestinal tract.

NOD1In addition to NOD2, evidence is now emerging to argue for a role of other intracellular bacterial sensors in Crohn’s disease, including NOD1 (also known as CARD4). NOD1 detects the tripeptide motif, GM-TriDAP, found within the peptidoglycan of Gram-negative bac-teria.137 A complex intronic insertion–deletion poly-morphism in NOD1 (designated ND1+32656*1) has been suggested to be involved in Crohn’s disease susceptibility in a British138 and Spanish population.139 However, these findings were not replicated in a larger British cohort,140 a Swedish cohort and two Scottish cohorts.141,142 A meta-analysis of eight published studies revealed a possible association of this NOD1 polymorphism with the early onset of Crohn’s disease.143

NLRP3Recently, polymorphisms in NLRP3 (also known as Cryopyrin, CIAS1, NALP3, and Pypaf1) or in regions in close proximity to the NLRP3 gene, have been implicated in the susceptibility to Crohn’s disease. NLRP3 has been previously associated with a number of auto inflammatory disorders collectively known as cryopyrin-associated

periodic syndromes.144 Six polymorphisms in the region 4.7 kb downstream of the NLRP3 gene have been found to be linked to Crohn’s disease susceptibility in a Belgian and Canadian popula tion of European ancestry.145 PBMCs from patients who are homo zygous for either one of these six polymorphisms have reduced NLRP3 expression and IL-1β production after LPS stimula-tion.145 However, no association between these poly-morphisms in NLRP3 and Crohn’s disease was found in a British study.146 Another polymorphism (Q705K) in the NLRP3 gene itself, in combination with a poly-morphism (C10X) in the NFκB inhibitory element CARD8 (also known as TUCAN), has been associ-ated with Crohn’s disease in Swedish men (Table 7).147 Activation of NLRP3 by a diverse panel of ligands leads to the formation of an inflammasome complex con-sisting of caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD), which medi-ates the processing of IL-1β and IL-18. Given that IL-1β is one of the cytokines upregulated in patients with Crohn’s disease,148 it is possible that dysregulation of NLRP3 may contribute to the development of Crohn’s disease. Murine studies have demonstrated that Nlrp3 has a critical role in host defenses against S. aureus, S. Typhimurium, and L. monocytogenes and for main-taining epithelial prolifera tion in the intestine.149–151

Table 7 | Adaptors of pattern-recognition receptors or related proteins implicated in the pathogenesis of Crohn’s disease

Protein Known function Relative expression in Crohn’s disease compared with controls



ATG16L1 Autophagy; controls endotoxin-induced inflammation

Similar ATG16L1 mRNA levels in colonic mucosa of patients with Crohn’s disease and controls, and in inflamed or non-inflamed tissues from patients with Crohn’s disease156,189

T300A (WD-repeat domain)153–156 Abnormal Paneth cells characterized by disorganized and diminished granules; defective muramyl-dipeptide-induced killing of S. Typhimurium in epithelial but not macrophages or dendritic cells; dendritic cells fail to induce MHC class II or generate antigen-specific CD4+ T cells116,131,166

CARD8 (also known as TUCAN, CARDINAL)

Inhibition of NOD2-mediated NFκB activation and IL-1β and IL-8 production

Upregulated in colon of Crohn’s disease patients and co-localizes with NOD2190

C10X147,191 Truncated CARD8 due to a premature stop codon191

CARD9 Adaptor protein for NOD2, dectin-1, and RIG-I; mediates the production of proinflammatory cytokines and TH17 responses

NA rs10870077192 NA

CD14 Its ligand is LPS; co-receptor for TLR4 and amplifies cellular sensitivity to LPS

Increased CD14-positive macrophages in the lamina propria;103 similar numbers of CD14-positive PBMC in Crohn’s disease and controls183

–159C/T (promoter region) with or without NOD2 mutations108,193

Increased susceptibility to S. pneumonia and septic shock194,195

IRGM Autophagy; controls mitochondrial morphology and cell death

Relative expression of deletion (risk) haplotype and reference (protective) haplotype varies in different cell lines158

rs13361189; rs4958847; a 20.1 kb deletion upstream of IRGM157,158

siRNA-knockdown of IRGM results in defective killing and autophagy of intracellular bacteria158,160

MAL (TIRAP) Adaptor protein for TLR4; mediates the production of proinflammatory cytokines

NA rs671492G 196 NA

Abbreviations: ATG16L1, autophagy-related 16-like 1 gene; IRGM, immunity-related GTPase M; LPS, lipopolysaccharide; MAL, MyD88 adaptor-like protein; MHC, major histocompatibility complex; NA, not available; NFκB, nuclear factor κB; PBMC, peripheral blood mononuclear cell; siRNA, small interfering RNA; TLR, Toll-like receptor; TUCAN, tumor-up-regulated CARD-containing antagonist of caspase 9.

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Investigations into how impaired bacterial recognition and killing, as a result of NLR poly morphisms, drives disease susceptibility may provide insight into the etiopathogenesis of Crohn’s disease.

Autophagy and defensin-mediated bacterial killingAutophagy is a cellular process responsible for the degrada tion of cytosolic components including bac-teria, long-lived proteins and damaged organelles.152 Genetic studies have revealed that an SNP in ATG16L1 (autophagy-related 16-like 1 gene) is associated with Crohn’s disease,153–156 while a large deletion of a 20.1 kb region in the promoter of another autophagy-related gene known as IRGM (immunity-related GTPase M) has also been shown to confer susceptibility to Crohn’s disease (Table 7).157,158 A number of studies have collectively shown that ATG16L1 or IRGM are important for the killing of intracellular bacteria such as AIEC, S. Typhimurium, and M. tuberculosis.128,154,158–160 For example, Lapaquette and colleagues observed that siRNA-mediated knock-down of ATG16L1 or IRGM in HeLa cells results in increased proliferation of AIEC.160 Another IRGM SNP (–261T), located in the promoter region distinct from the SNPs associated with Crohn’s disease, has been shown to confer protection against tuberculosis.161 Interestingly, a study using transfection experiments showed that NOD2 with the frameshift mutation is unable to recruit ATG16L1 to the cell membrane and initiate autophagy in response to Shigella flexneri.162 Moreover, dendritic cells from patients carrying NOD2 mutations associated with Crohn’s disease failed to induce autophagy,131 pro-viding evidence to support the nexus between NLR and autophagy in the context of Crohn’s disease.

Defective bacterial killing in Crohn’s disease has also been linked to impaired α-defensin and β-defensin production, which are antimicrobial peptides that regulate intestinal homeostasis. For example, the level of α-defensins (HD5 and HD6) (which are produced primarily by crypt-residing epithelial cells of the small intestine known as Paneth cells) have been shown to be lower in patients with ileal Crohn’s disease com-pared with healthy controls.163,164 Furthermore, biopsy samples from patients with ileal Crohn’s disease who have the NOD2 mutation demonstrate even lower levels of HD5 and HD6 mRNA compared with corresponding controls.165 In addition to the association between NOD2 mutations and α-defensin production, patients with the Crohn’s-disease-associated ATG16L1 mutation were also found to have abnormal Paneth cells, character-ized by dis organized and/or diminished numbers of granules.166 This finding suggests a possible intimate relationship between autophagy and defensin-mediated bacterial killing. Taken together, these studies indi-cate that impaired clearance of extracellular and intra-cellular bacteria is intricately linked to the pathogenesis of Crohn’s disease.

ConclusionsStudies using culture-based and molecular methodolo-gies have clearly identified compositional changes in

bacterial populations (dysbiosis) of the intestinal tract in patients with Crohn’s disease. However, an impor-tant question remains unanswered—what is the origin of dysbiosis? The answer to this question would provide vital clues towards understanding the pathogenesis of Crohn’s disease. One possibility could be that dys-biosis is driven by microbial factors (such as the occur-rence of a recent infection) and/or by host factors in the form of inappropriate immune responses (under-pinned by genetic polymorphisms). Indeed, gastro-enteritis has been shown to be a predisposing factor for Crohn’s disease. On the other hand, specific bacteria have also been associated with Crohn’s disease, many of which are equipped with a repertoire of virulence factors that provide them with the capacity to facili-tate invasion, intracellular replication and to modulate immune responses.

Given that Crohn’s disease is a disease represented by multiple phenotypes affecting many different sites along the gastrointestinal tract, it is equally plausible to argue that each putative pathogen implicated in the literature is the causative agent of one of the diverse subsets of this disease. For example, AIEC is a possible cause of ileal Crohn’s disease. It is also possible to argue that certain pathogens can induce disease only in the presence of specific host genetic polymorphisms. The importance of the role of host responses is convincingly supported by the link between mutations in genes encoding TLRs, NLRs and autophagy proteins and susceptibility to Crohn’s disease. Mutations in a gene encoding a spe-cific TLR or NLR results in defective recognition and killing of specific groups of bacteria, an observation that further argues for the notion that individual pathogens may trigger specific Crohn’s disease phenotypes in susceptible individuals.

It is clear that deciphering this complex, multifactorial disease requires a holistic investigative effort to under-stand the bacterial profile, host genetics and immuno-logic defects in patients who are newly diagnosed, prior to the onset of chronic inflammation. In conclu-sion, interaction between the microbiota and pattern- recognition receptors is vital for gut homeostasis, and dysregulation of this balance due to a specific bacterial agent or dysbiosis is likely to result in the onset of Crohn’s disease in susceptible individuals.

Review criteria

PubMed was used to identify references. No date restrictions were applied to the search and only articles published in English were reviewed. The following search terms were used individually or in combination to search for appropriate articles: “autophagy”, “bacteria”, “Campylobacter”, “CD”, “Crohn’s”, “Escherichia”, “Helicobacter”, “IBD”, “inflammatory bowel disease” “Mycobacterium”, “NOD”, “NLR”, “pathogen”, “Toll”, and “TLR”. Reference lists from articles and reviews were examined to identify additional relevant references.

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AcknowledgmentsWe apologize to our colleagues whose work was not cited due to space limitations. We thank the National Health and Medical Research Council (NHMRC) of Australia and the Broad Medical Foundation for funding our research studies in Crohn’s disease. We would also like to acknowledge Dr P. Tourlomousis and Dr C. Bryant (University of Cambridge) for providing thoughtful feedback on the manuscript. S. M. M. is a recipient of a Cambridge International Scholarship, and N. O. K. is a recipient of a NHMRC Postdoctoral Training Fellowship.

Author contributionsAll authors contributed equally to all aspects of this Review.

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The role of bacteria and pattern-recognition receptors in Crohn’s diseaseSi Ming Man, Nadeem O. Kaakoush & Hazel M. Mitchell

Nat. Rev. Gastroenterol. Hepatol. 8, 152–168 (2011); doi:10.1038/nrgastro.2011.3

In the Review by Man et al. published in the March 2011 issue of Nature Reviews Gastroenterology and Hepatology, MyD88 was incorrectly labeled as TRIF and shown in yellow instead of purple in Figure 2. The corresponding sentence in the figure legend should have read: “TLR9 recognizes endosomal CpG DNA and signals through the MyD88 and MAPKK pathways, both of which lead to the production of proinflammatory cytokines.” The error has been corrected for the HTML and PDF versions of the article.

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