Crohn’s disease (CD) is caused by a combination of environmental and genetic factors. Dysregulation of the innate immune response to luminal microbial or nutritional antigens seems to play a major role in the pathogenesis of CD.1–6 The subsequent proinflammatory response involves primarily T helper (TH)1 and TH17 cells, characterized by the secretion of tumor necrosis factor (TNF)-α, interferon-γ, interleukin (IL)-12, and IL-23 in response to exposure to bacterial products.2,5,6 Bacteria may be found in granulomas or adherent to the intestinal mucosa2,5,6; microbial biofilms may also trigger an adaptive immune response. Some bacteria isolated from patients with CD, such as adherent–invasive Escherichia coli (AIEC), are shown to replicate in macrophages and epithelial cells under certain circumstances.7,8
Although progress has been achieved in determining the genetic and immune basis for susceptibility to the disease, understanding the contribution of potential environmental risk factors has been difficult. Exposure to infections as risk factors has been the focus of interest of many studies. Similar to other autoimmune diseases, some epidemiological studies have suggested a role for the “hygiene hypothesis,” whereby exposure to infections in childhood confers protection against disease.9–11
An alternative environmental factor, which has not been adequately explored, is the effect of diet on CD. Diet has a dramatic effect on the composition of the intestinal microbiome and gut immune status.12–14 Exclusive enteral nutrition (EEN) is an effective therapy for induction of remission in pediatric CD.15 We hypothesize that the major mechanism leading to response to dietary therapy used in children with active CD is exclusion of dietary factors that may lead, when present, to a “bacterial penetration cycle” and subsequent stimulation of an adaptive immune response. We suggest that a change in consumption of foods or additives that may impair the barrier function of the intestinal epithelium, in turn, allows adherence and invasion of nonpathogenic bacteria or bacterial antigens. Adherence of bacteria to the intestinal epithelium, penetration and replication within epithelial cells, dendritic cells, and macrophages,7,8 leads to continuous triggering of the adaptive immune system, resulting in inflammation. If disease susceptibility genes that lead to loss of autophagy or Paneth cell dysfunction are present,16 the innate immune system will be even less likely to detect and eradicate these bacteria, leading to continuous activation of the adaptive immune response, tissue damage, further loss of epithelial integrity, and increased penetration of bacteria leading to a vicious cycle of inflammation that we have termed the bacterial penetration cycle.
DIET CONTRIBUTES TO THE PATHOGENESIS OF CD
CD is clearly on the rise in countries exposed to industrialization and Western diet.17 Several factors may implicate diet in the pathogenesis of CD or in disease activity. The strongest argument for an effect of diet is the ability of EEN to induce remission in CD. Although some studies have assessed enteral nutrition in adults, most were less conclusive and with multiple confounding factors, and this practice is uncommon in adults with CD in most countries.18 Multiple pediatric studies indicated that approximately 50% to 80% of children, fed an exclusive liquid diet (no exposure to other food), irrespective of the specific type of liquid diet, will enter complete remission, often with normalization of inflammatory markers19–26; however, recent studies are very consistent with the observation that approximately 70% to 90% of children will attain remission, depending on adherence. This effect has been shown primarily in children with recent disease onset. Johnson et al22 demonstrated in a prospective controlled trial that randomized children to EEN or partial enteral nutrition (PEN) (with similar intake of calories and nutrients from a normal diet) that success was dependent on exclusion of food; however, the low rate of response to EEN in this study (42%) could raise the possibility that other factors may have affected the outcomes.
Two recent large retrospective studies have shown remission rates in up to 80% of children receiving EEN. Rubio et al25 treated 106 pediatric patients with oral or nasogastric Modulen (a polymeric liquid formula) for 8 weeks. By week 8, 34 of 45 patients (76%) achieved remission in the oral group using an intention-to-treat analysis and 52 of 61 patients (85%) achieved remission using tube feeding; the difference by route of administration was not significant. All patients showed a significant decrease in disease severity assessed by the Pediatric CD Activity Index (P < 0.0001). Of note, 72% of the patients in this study were treated at disease onset. Patients who were not fully compliant with exclusivity had a 35% lower remission rate, although this did not reach statistical significance and the number of patients in the study was small. Nevertheless, this suggests that it is the exclusion or reduction in exposure to dietary components and not the exposure to enteral nutrition alone, which is mostly likely responsible for the improvement. Data published only as an abstract from the Children’s Hospital of Philadelphia retrospectively evaluating 23 children with active disease demonstrated that remission could be achieved in 65% of children who received 80% to 90% of their caloric intake from a semi-elemental formula over 12 weeks (R. Baldassano, personal communication, 2012). Even if this study and others suggest that complete exclusivity is not required, a dramatic reduction in exposure to food elements does indicate a potential deleterious effect of food components in this setting.
In a study performed by the Porto group, Levine et al27 prospectively evaluated 150 new-onset previously untreated pediatric patients with CD and evaluated which treatment factors predicted deep remission after 12 weeks, defined as corticosteroid-free remission (defined as Pediatric CD Activity Index ≤ 7.5) and normal C-reactive protein. Although there was no difference between corticosteroids and EEN for 6 to 8 weeks in achieving clinical remission, EEN was significantly superior to corticosteroids or mesalamine for achieving deep remission, corrected for disease severity, and use of immunomodulators at 12 weeks (54% by EEN, 26% by corticosteroids; P = 0.014). Buchanan et al26 retrospectively evaluated the treatment of 105 pediatric patients with 8 weeks of EEN using Modulen; duration of disease was not specified. The remission rate was 80%, and this was accompanied by a drop in erythrocyte sedimentation rate and C-reactive protein, and improvements in weight and height z scores. In this study, remission rates were high with both ileal and colonic involvement.
This effect of EEN has been shown to be independent of fat or protein composition in the liquid diets in pediatric studies but to be dependent on exclusion of normal diet.19,22–24 Thus, rather than the composition of EEN being associated with remission of disease, it may be the exclusion or at least a significant dose reduction of certain components of the Western diet that is responsible for the observed improvement.
The presence of the single-layer epithelium as a barrier is crucial for proper functioning of the gastrointestinal tract.28 Although the epithelium must serve as an effective barrier against harmful macromolecules and microorganisms, it must also remain permeable to essential nutrients and simultaneously allow controlled exposure of the mucosal immune system to food antigens and microbial factors29 necessary for the development and regulation of the immune system.30 Impairment in the function of the intestinal barrier leads to increased permeability to luminal antigens; this has been proposed as an initiating factor in the pathogenesis of chronic human inflammatory bowel diseases (IBD). Epithelial cells are held together by apical junctional complexes, which include tight junctions and adherens junctions. These multiprotein structures are highly regulated and selective; they are bound to the cytoskeleton through adaptors (e.g., zonula occludens-1; β-catenin) and connected to similar structures in adjacent cells through a variety of transmembrane proteins (e.g., claudins, occludin, E-cadherin).31 Additional factors governing permeability to macromolecules or bacteria include the composition of the microbiome32 and the mucous layer.33
INCREASED GUT PERMEABILITY IN IBD
One of the constant features in IBD seems to be an increase in intestinal permeability that can precede inflammatory lesions and trigger mucosal inflammation.34 Increased paracellular permeability occurs in both acute and chronic ileitis and colitis in humans,35 and in IBD mouse knockout models, including mdr1a −/− and IL-10−/− mice.29 Other studies have shown an important role for cytokines, including TNF-α and interferon-γ in increasing permeability of tight junctions in intestinal epithelial cells.36 Bacteria are also hypothesized to play a critical role in regulating this process. Although a unique or specific bacterial species has not been identified as a single cause of increased permeability in human IBD, several species, such as AIEC, which is linked to the development of small intestinal CD, have been shown to impair permeability.37
Zeissig et al38 investigated mechanisms leading to increased permeability in patients with CD and found that they included upregulation of pore-forming claudin-2, along with downregulation and redistribution of claudin-5 and claudin-8, which enhance the barrier and decrease permeability. Tight junction rearrangement normalized when disease became inactive in this study.
Although it is clear that increased intestinal permeability is characteristic of active CD, it is unclear whether this is a secondary phenomenon because of inflammation or a primary cause of disease. Adding to this controversy is the finding that mice lacking occludin do not have a primary barrier dysfunction leading to intestinal inflammation.39 However, in a study of patients with CD and healthy relatives, impaired gut permeability was found in 48% of CD, 40% of healthy first-degree relatives, and only 5% of a healthy control population.40 This was not associated with NOD2 mutations. Interestingly, intestinal permeability has been found to be increased in spouses of patients with CD41 and to precede clinical and endoscopic onset of disease.42 These findings may indicate that a leaky gut may precede CD, or be environmentally induced, representing the complex role of permeability defects in IBD pathogenesis.
EFFECTS OF DIET ON INTESTINAL PERMEABILITY, MICROBIAL PENETRATION, AND VULNERABILITY TO BACTERIA OR BACTERIAL ANTIGENS
Two different plausible mechanisms may associate barrier defects with CD. In the first scenario, permeability is primarily increased, leading to enhancement of antigen exposure of bacterial products, which then leads to loss of tolerance, and inflammation.1–4 A second appealing scenario starts with increased permeability leading more directly to bacterial translocation (as opposed to general antigen exposure), which in turn triggers a normative adaptive immune response in an attempt to contain this process. Thus, candidates for a causative effect of dietary-induced barrier disruption as an environmental trigger should be constituents with a greater representation in Western diet than in diets from the developing world; these can then impair the previously mentioned mechanisms involved in controlling permeability.
Several different food components commonly present in the Western diets have been shown to affect epithelial cell permeability in studies (Fig. 1). Sodium caprate, a medium-chain fatty acid found in dairy products, has been shown to increase permeability in rat ileum43 and subsequently in human ileum samples taken from patients with CD.44 Lammers et al, demonstrated that gliadin, the toxic component of gluten that instigates inflammation in celiac disease, also induces a direct increase in small intestinal permeability by binding to the chemokine receptor CXCR3 expressed in small intestinal epithelium, which leads to MyD88-dependent zonulin release. Zonulin disrupts the ZO1 junctional proteins binding to occludins and claudins.44 The effect on transepithelial resistance was confined to the jejunum and ileum, in the intestinal epithelium from patients without celiac disease.45,46 Excessive consumption of wheat could thus have a dose-dependent detrimental effect on the gut barrier, primarily in the small intestine, where zonulin receptors are expressed.
Regulatory roles of mucin proteins such as Muc2 and Muc3 have been previously demonstrated to be important in IBD.47 Muc2 is the major mucin present in the intestinal mucus barrier, and it has been shown that its deficiency leads to inflammation in the colon. Detergents and emulsifiers added to food products may affect or impair the mucus barrier.48 Carboxymethylcellulose, present in a variety of industrialized milk products, breads, sauces, and sausages, has been shown to enable bacterial adherence to intestinal epithelium. Carboxymethylcellulose-treated IL-10−/− mice demonstrated a massive bacterial overgrowth, distention of spaces between villi with bacterial infiltration, and adherence to the mucosa.49 The exact mechanism for this effect was not determined but could be related to effects on the epithelial barrier.
Lastly, translocation of E. coli across M cells and Peyer's patches in patients with CD has been shown to be inhibited by certain plant fibers but increased with low concentrations of polysorbate 80, an emulsifier commonly used in processed foods.49 Escherichia coli translocation across human Peyer's patches was reduced by 45% by soluble plantain nonstarch polysaccharide and was increased 2-fold by the emulsifier polysorbate 80. Translocation across Caco2 cells was increased nearly 60-fold. All these products have been shown to predominantly affect the permeability or translocation of bacteria on small intestinal epithelial cells, whereas evidence for an effect of dietary components on colonic mucosa is not available at present.
EFFECTS OF DIET ON THE GUT MICROBIOME: ANOTHER POTENTIAL LINK BETWEEN DIET AND DISEASE
Recent advances in culture-independent techniques, mainly through sequencing of variable regions in the 16S ribosomal-RNA microbial gene, provide robust tools to define associations between the gut microbiome and disease.50–52 Dysbiosis, an “imbalance” in intestinal microbes related to disease, is proposed to contribute to a variety of human inflictions, including obesity and metabolic disorders, diabetes, autoimmune conditions, cancer, and IBD.30,53–55
How diet contributes to disease through dysbiosis is an important gap in scientific knowledge with clear potential for novel interventions to prevent many, if not most, chronic conditions affecting humans. Understanding the role of microbes and diet play in CD will provide critical insight into disease pathogenesis.
Although initial analyses of the human gut microbiome revealed remarkable variability within and between individuals,56 certain patterns appeared, such as phylum-level changes and reduced community diversity in obese individuals.57 A pioneering study in this rapidly expanding field, using 16S ribosomal RNA sequencing, showed a correlation between dietary patterns (herbivore, omnivore, and carnivore) of 59 mammalian species and their microbiome.58 However, it remained unclear whether these differences reflect true effects of diet on microbes or coevolution of mammalian hosts with their microbes. A subsequent study by the same group suggests that diet does affect microbial composition because simple inheritance of microbes is not driven only by evolution. Using a group of 18 humans with meticulous dietary records, this study showed that changing a diet alters not only the composition of the microbiome but also its function (using broad microbial gene sequencing and pathway analyses).12
Specific effects of diet on microbial composition and function are best documented using animal models where many factors can be controlled. Germ-free mice are especially attractive because in other settings, the microbiome is still very complex. For example, these mice can be colonized by a single microbial species, such as Bacteroides thetaiotaomicron, a human gut bacterium, to better define host–diet–microbe interactions. This approach has revealed the importance of pathways involved in metabolism of dietary components (e.g., vitamin B12) in establishing bacterial colonization.59 Variation in dietary factors, especially polysaccharides, can influence the ability of commensal microbes to compete and colonize the gut.60
Advanced modeling reveals that 60% of the variability in species in the gut is determined by diet.61 Specific examples include effects of breastfeeding, weaning, introduction of solids, and supplementation with fish oil and other nutrients.62,63 Other cases, such as glutamine supplementation in very low birth weight infants, did not seem to affect composition.64 More broad effects have been shown by comparing the gut microbiota between children in rural Africa and Europe, showing more Bacteroidetes and less Firmicutes in those exposed to African diet.13
Experiments in animal models of colitis have provided some insight, but many mechanisms still remain unclear. For example, removal of dietary iron from TNFΔARE/WT mice (these mice develop spontaneous ileitis because of dysregulation of TNF-α) prevents occurrence of inflammation, possibly through changes in microbial composition, endoplasmic reticulum stress response, and apoptosis, all related to luminal iron exposure.65 Using another animal IBD model (IL-10−/− cell transfer; develop colitis), Kajiura et al14 showed changes in microbial composition and reduced cytokine secretion when mice were fed elemental diet. Steck et al66 demonstrated that colonization of IL-10−/− mice with Enterococcus faecalis was associated with loss of epithelial integrity that was independent of inflammation. Specifically, intestinal epithelial cells isolated from IL-10−/− mice colonized with E. faecalis had significantly reduced levels of the extracellular domain of E-cadherin. The levels of occludin and JAM-A were unchanged. The reduction in E-cadherin levels was shown to result from proteolytic cleavage of E-cadherin by gelatinase, secreted by E. faecalis, thus facilitating bacterial translocation and increased permeability, as demonstrated in Ussing chamber studies. Hypothetically, dysbiosis favoring E. faecalis colonization could lead to an increase in gelatinase-mediated permeability and inflammation. However, a study in human subjects with CD demonstrated an increase in E. faecalis in subjects during enteral nutrition, which is counterintuitive to this specific dysbiosis hypothesis.14 Devkota et al recently demonstrated in an IL-10−/− mouse model that exposure to saturated milk fats altered taurine conjugation of bile acids, which in turn led to expansion of a proinflammatory colitogenic bacterium, Bilophila wadsworthia, likely through an increase in organic sulfur. This study demonstrated that food-induced changes in host bile acid composition may alter the microbiome and lead to intestinal inflammation. Saturated milk fats are frequently incorporated into processed food.67
MICROBES, IMMUNE RESPONSES, AND CD
The role of microbes in IBD pathogenesis is well established and reviewed in detail elsewhere.35,68,69 Several studies describe changes in microbial diversity and composition in patients with IBD50,52,70; however, mechanisms leading to these changes and whether these are primary or secondary remain unclear. Although no single microbial species has been identified as a causative agent of IBD, AIEC strains, isolated from patients with CD,seem to play a role in disease pathogenesis, at least in a subset of patients.71,72 Some mechanisms linking AIEC to pathways involved in gut inflammation further support a role for these microbes. For example, defects in control of intracellular bacteria, such as mutations in autophagy,73 are genetically linked to CD and regulate responses to AIEC. Impaired autophagy in macrophages leads to increased bacterial survival in mononuclear cells and proinflammatory responses that include the secretion of TNF-α.74 Another potential link and research focus between bacteria, immune responses, and CD is the role of Paneth cells, specialized epithelial cells found in intestinal crypts that secrete antimicrobial peptides to remove bacteria from the mucosa. Defects in this defense mechanism, which are linked to autophagy and NOD2, can lead to continuous stimulation of the adaptive immune system by resident microbes that gain access after diet-related barrier disruption and then are not properly cleared from crypts.75,76
MAINTENANCE OF REMISSION
Several small randomized controlled trials have shown an effect of PEN on the maintenance of nutrition.77–79 These studies differ from the above-mentioned studies in which the starting point was quiescent disease and the outcome was relapse (a scenario more likely to succeed than induction of remission during active disease). Most of these studies used similar designs (50% of daily calories as enteral nutrition compared with free diet) and found that patients consuming elemental or semi-elemental formulas for a year had fewer relapses. Yamomoto et al evaluated postoperative recurrence in patients on free diet or in patients receiving 50% of daily requirements using an elemental formula and a low-fat diet for 1 year. Clinical recurrence occurred in 5% of the PEN versus 35% in the free diet group (P = 0.048). Endoscopic recurrence occurred in 30% of PEN patients versus 70% of patients in the free diet group (P = 0.027).78 This group used the same methodology to investigate 40 patients in clinical remission, half received PEN and a low-fat diet and half received free diet for 1 year. The relapse rate among patients on free diet was 65% versus 25% receiving PEN (P = 0.03). Mucosal cytokines were significantly lower at 1 year in the PEN group.79
CONNECTING THE DOTS
A growing body of evidence links disease susceptibility genes to bacterial involvement in the pathogenesis of CD. Patients with ileal disease phenotypes carry mutations or polymorphisms that are associated with abnormal Paneth cell function or defective autophagy.80–82 The effect of EN on induction of remission and in maintenance has been demonstrated with different formulations (elemental, semi-elemental, and polymeric formulas). An attractive but speculative hypothesis for the observed increase in the prevalence of IBD, and CD especially, may be that a persistent change in dietary intake leads to breakdown of the epithelial barrier. This might allow adherence, translocation, and penetration of bacteria that under normal conditions would be nonpathogenic, or of bacterial antigens, in susceptible individuals (e.g., genetically determined Paneth cell dysfunction or defective autophagy). Persistent exposure to adherent or penetrating bacteria may then trigger the adaptive immune system, resulting in inflammation, further breakdown of the epithelial barrier, and increasing migration and sensitization to these bacteria; this then can induce a vicious cycle (bacterial penetration cycle, Fig. 2). EEN, especially in early stages of small intestinal disease, might act by decreasing exposure to these offending agents and subsequent decline in penetrating or resident bacteria, and epithelial restitution,79 reversing this cycle. Alternatively, EEN might act through removal of dietary components, which affect microbial composition, decrease a proinflammatory response, promote restitution of the epithelial barrier, and likewise allowing termination of this vicious disease-forming cycle before a critical threshold is reached. The limited data available from studies involving PEN as maintenance therapy (and the unpublished report on 80%–90% EN with limited diet for induction of remission) could also be consistent with a protective effect of reduction in exposure to “offending” dietary components. Maintenance therapy with liquid nutrition and a low-fat diet are likely to reduce the daily intake of other (potentially “deleterious”) food components, especially industrialized or processed foods. This same mechanism could act by decreasing intestinal permeability or maintaining a less inflammatory microbiome.
If this scenario is true, and given that EEN induces disease remission and mucosal healing,83 it might be sufficient to withdraw specific offending agents, to achieve remission, at least in selected patients, early in the disease cycle. This mechanism would also explain the lower success rates of PEN to induce remission with continued exposure to regular diet in the previously mentioned studies.22,25
However, identification of agents or combinations of dietary components that may affect intestinal permeability or the microbiome is still very challenging and difficult to assess in human subjects.
In conclusion, dietary factors may have a plausible role as environmental factors in the pathogenesis of CD. Research into the environmental causes of IBD lags behind the research into the genetic and immune components of this disease and may offer insight and also new therapeutic targets for intervention in the future.
1. Barrett JC, Hansoul S, Nicolae DL, et al.. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet. 2008;40:955–962.
2. Peeters H, Bogaert S, Laukens D, et al.. CARD15 variants determine a disturbed early response of monocytes to adherent-invasive Escherichia coli
strain LF82 in Crohn's disease. Int J Immunogenet. 2007;34:181–191.
3. Thompson NP, Driscoll R, Pounder RE, et al.. Genetics versus environment in inflammatory bowel disease: results of a British twin study. BMJ. 1996;312:95–96.
4. Cho JH, Weaver CT. The genetics of inflammatory bowel disease. Gastroenterology. 2007;133:1327–1339.
5. Martin HM, Campbell BJ, Hart CA, et al.. Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer. Gastroenterology. 2004;127:80–93.
6. Ryan P, Kelly RG, Lee G, et al.. Bacterial DNA within granulomas of patients with Crohn's disease—detection by laser capture microdissection and PCR. Am J Gastroenterol. 2004;99:1539–1543.
7. Bringer MA, Billard E, Glasser AL, et al.. Replication of Crohn's disease-associated AIEC within macrophages is dependent on TNF-alpha secretion. Lab Invest. 2012;92:411–419.
8. Lapaquette P, Glasser AL, Huett A, et al.. Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. Cell Microbiol. 2010;12:99–113.
9. Lashner BA, Loftus EV Jr. True or false? The hygiene hypothesis for Crohn's disease. Am J Gastroenterol. 2006;101:1003–1004.
10. Ekbom A. Appendicectomy and childhood hygiene: different sides of the same coin? Gut. 1998;43:451.
11. Bernstein CN, Rawsthorne P, Cheang M, et al.. A population-based case control study of potential risk factors for IBD. Am J Gastroenterol. 2006;101:993–1002.
12. Muegge BD, Kuczynski J, Knights D, et al.. Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. Science. 2011;332:970–974.
13. De Filippo C, Cavalieri D, Di Paola M, et al.. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci U S A. 2010;107:14691–14696.
14. Kajiura T, Takeda T, Sakata S, et al.. Change of intestinal microbiota with elemental diet and its impact on therapeutic effects in a murine model of chronic colitis. Dig Dis Sci. 2009;54:1892–1900.
15. Critch J, Day AS, Otley A, et al.. Use of enteral nutrition for the control of intestinal inflammation in pediatric Crohn disease. J Pediatr Gastroenterol Nutr. 2012;54:298–305.
16. Fritz T, Niederreiter L, Adolph T, et al.. Crohn's disease: NOD2, autophagy and ER stress converge. Gut. 2011;60:1580–1588.
17. Molodecky NA, Soon IS, Rabi DM, et al.. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.
18. Lochs H, Steinhardt HJ, Klaus-Wentz B, et al.. Comparison of enteral nutrition and drug treatment in active Crohn's disease. Results of the European Cooperative Crohn's Disease Study. IV. Gastroenterology. 1991;101:881–888.
19. Ludvigsson JF, Krantz M, Bodin L, et al.. Elemental versus polymeric enteral nutrition in paediatric Crohn's disease: a multicentre randomized controlled trial. Acta Paediatr. 2004;93:327–335.
20. Teahon K, Smethurst P, Pearson M, et al.. The effect of elemental diet on intestinal permeability and inflammation in Crohn's disease. Gastroenterology. 1991;101:84–89.
21. Sanderson IR, Boulton P, Menzies I, et al.. Improvement of abnormal lactulose/rhamnose permeability in active Crohn's disease of the small bowel by an elemental diet. Gut. 1987;28:1073–1076.
22. Johnson T, Macdonald S, Hill SM, et al.. Treatment of active Crohn's disease in children using partial enteral nutrition with liquid formula: a randomised controlled trial. Gut. 2006;55:356–361.
23. Dziechciarz P, Horvath A, Shamir R, et al.. Meta-analysis: enteral nutrition in active Crohn's disease in children. Aliment Pharmacol Ther. 2007;26:795–806.
24. Khoshoo V, Reifen R, Neuman MG, et al.. Effect of low- and high-fat, peptide-based diets on body composition and disease activity in adolescents with active Crohn's disease. JPEN J Parenter Enteral Nutr. 1996;20:401–405.
25. Rubio A, Pigneur B, Garnier-Lengline H, et al.. The efficacy of exclusive nutritional therapy in paediatric Crohn's disease, comparing fractionated oral vs. continuous enteral feeding. Aliment Pharmacol Ther. 2011;33:1332–1339.
26. Buchanan E, Gaunt WW, Cardigan T, et al.. The use of exclusive enteral nutrition for induction of remission in children with Crohn's disease demonstrates that disease phenotype does not influence clinical remission. Aliment Pharmacol Ther. 2009;30:501–507.
27. Levine A, Sladek M, Shaoul R, et al.. Exclusive enteral nutrition is superior to corticosteroids and mesalamine for achieving normal CRP remission in new onset paediatric Crohns disease. J Crohns Colitis. 2012;6:S111.
28. Clayburgh DR, Shen L, Turner JR. A porous defense: the leaky epithelial barrier in intestinal disease. Lab Invest. 2004;84:282–291.
29. Collett A, Higgs NB, Gironella M, et al.. Early molecular and functional changes in colonic epithelium that precede increased gut permeability during colitis development in mdr1a(-/-) mice. Inflamm Bowel Dis. 2008;14:620–631.
30. Clemente JC, Ursell LK, Parfrey LW, et al.. The impact of the gut microbiota on human health: an integrative view. Cell. 2012;148:1258–1270.
31. Laukoetter MG, Bruewer M, Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex. Curr Opin Gastroenterol. 2006;22:85–89.
32. Ohland CL, Macnaughton WK. Probiotic bacteria and intestinal epithelial barrier function. Am J Physiol Gastrointest Liver Physiol. 2010;298:G807–G819.
33. Wine E, Terebiznik M, Jones N. Microbial interactions with gut epithelium. In: Kleinman RE, Sanderson IR, Goulet O, et al., eds. Pediatric Gastrointestinal Disease. 5th ed. Hamilton, Canada: BC Decker Inc; 2008:373–390.
34. Vetrano S, Rescigno M, Cera MR, et al.. Unique role of junctional adhesion molecule-a in maintaining mucosal homeostasis in inflammatory bowel disease. Gastroenterology. 2008;135:173–184.
35. Sartor RB. Microbial influences in inflammatory bowel diseases. Gastroenterology. 2008;134:577–594.
36. Wang F, Graham WV, Wang Y, et al.. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005;166:409–419.
37. Wine E, Ossa JC, Gray-Owen SD, et al.. Adherent-invasive Escherichia coli
, strain LF82 disrupts apical junctional complexes in polarized epithelia. BMC Microbiol. 2009;9:180.
38. Zeissig S, Burgel N, Gunzel D, et al.. Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease. Gut. 2007;56:61–72.
39. Schulzke JD, Gitter AH, Mankertz J, et al.. Epithelial transport and barrier function in occludin-deficient mice. Biochim Biophys Acta. 2005;1669:34–42.
40. Fries W, Renda MC, Lo Presti MA, et al.. Intestinal permeability and genetic determinants in patients, first-degree relatives, and controls in a high-incidence area of Crohn's disease in Southern Italy. Am J Gastroenterol. 2005;100:2730–2736.
41. Breslin NP, Nash C, Hilsden RJ, et al.. Intestinal permeability is increased in a proportion of spouses of patients with Crohn's disease. Am J Gastroenterol. 2001;96:2934–2938.
42. Irvine EJ, Marshall JK. Increased intestinal permeability precedes the onset of Crohn's disease in a subject with familial risk. Gastroenterology. 2000;119:1740–1744.
43. Soderholm JD, Oman H, Blomquist L, et al.. Reversible increase in tight junction permeability to macromolecules in rat ileal mucosa in vitro by sodium caprate, a constituent of milk fat. Dig Dis Sci. 1998;43:1547–1552.
44. Soderholm JD, Olaison G, Peterson KH, et al.. Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease. Gut. 2002;50:307–313.
45. Lammers KM, Lu R, Brownley J, et al.. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology. 2008;135:194–204.
46. Drago S, El Asmar R, Di Pierro M, et al.. Gliadin, zonulin and gut permeability: effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scand J Gastroenterol. 2006;41:408–419.
47. Van der Sluis M, De Koning BA, De Bruijn AC, et al.. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology. 2006;131:117–129.
48. Swidsinski A, Ung V, Sydora BC, et al.. Bacterial overgrowth and inflammation of small intestine after carboxymethylcellulose ingestion in genetically susceptible mice. Inflamm Bowel Dis. 2009;15:359–364.
49. Roberts CL, Keita AV, Duncan SH, et al.. Translocation of Crohn's disease Escherichia coli across M-cells: contrasting effects of soluble plant fibres and emulsifiers. Gut. 2010;59:1331–1339.
50. Qin J, Li R, Raes J, et al.. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010;464:59–65.
51. Nelson KE, Weinstock GM, Highlander SK, et al.. A catalog of reference genomes from the human microbiome. Science. 2010;328:994–999.
52. Michail S, Durbin M, Turner D, et al.. Alterations in the gut microbiome of children with severe ulcerative colitis. Inflamm Bowel Dis. 2012;18:1799–1808.
53. Henao-Mejia J, Elinav E, Jin C, et al.. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature. 2012;482:179–185.
54. Sobhani I, Tap J, Roudot-Thoraval F, et al.. Microbial dysbiosis in colorectal cancer (CRC) patients. PLoS One. 2011;6:e16393.
55. Boerner BP, Sarvetnick NE. Type 1 diabetes: role of intestinal microbiome in humans and mice. Ann N Y Acad Sci. 2011;1243:103–118.
56. Eckburg PB, Bik EM, Bernstein CN, et al.. Diversity of the human intestinal microbial flora. Science. 2005;308:1635–1638.
57. Turnbaugh PJ, Hamady M, Yatsunenko T, et al.. A core gut microbiome in obese and lean twins. Nature. 2009;457:480–484.
58. Ley RE, Hamady M, Lozupone C, et al.. Evolution of mammals and their gut microbes. Science. 2008;320:1647–1651.
59. Goodman AL, McNulty NP, Zhao Y, et al.. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe. 2009;6:279–289.
60. Sonnenburg ED, Zheng H, Joglekar P, et al.. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations. Cell. 2010;141:1241–1252.
61. Faith JJ, McNulty NP, Rey FE, et al.. Predicting a human gut microbiota's response to diet in gnotobiotic mice. Science. 2011;333:101–104.
62. Palmer C, Bik EM, Digiulio DB, et al.. Development of the human infant intestinal microbiota. PLoS Biol. 2007;5:e177.
63. Andersen AD, Molbak L, Michaelsen KF, et al.. Molecular fingerprints of the human fecal microbiota from 9 to 18 months old and the effect of fish oil supplementation. J Pediatr Gastroenterol Nutr. 2011;53:303–309.
64. van den Berg A, van Elburg RM, Westerbeek EA, et al.. The effect of glutamine-enriched enteral nutrition on intestinal microflora in very low birth weight infants: a randomized controlled trial. Clin Nutr. 2007;26:430–439.
65. Werner T, Wagner SJ, Martinez I, et al.. Depletion of luminal iron alters the gut microbiota and prevents Crohn's disease-like ileitis. Gut. 2011;60:325–333.
66. Steck N, Hoffmann M, Sava IG, et al.. Enterococcus faecalis metalloprotease compromises epithelial barrier and contributes to intestinal inflammation. Gastroenterology. 2011;141:959–971.
67. Devkota S, Wang Y, Musch MW, et al.. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice. Nature. 2012;487:104–108.
68. Neish AS. Microbes in gastrointestinal health and disease. Gastroenterology. 2009;136:65–80.
69. Kau AL, Ahern PP, Griffin NW, et al.. Human nutrition, the gut microbiome and the immune system. Nature. 2011;474:327–336.
70. Peterson DA, Frank DN, Pace NR, et al.. Metagenomic approaches for defining the pathogenesis of inflammatory bowel diseases. Cell Host Microbe. 2008;3:417–427.
71. Darfeuille-Michaud A, Boudeau J, Bulois P, et al.. High prevalence of adherent-invasive Escherichia coli
associated with ileal mucosa in Crohn's disease. Gastroenterology. 2004;127:412–421.
72. Carvalho FA, Barnich N, Sivignon A, et al.. Crohn's disease adherent-invasive Escherichia coli
colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM. J Exp Med. 2009;206:2179–2189.
73. Abraham C, Medzhitov R. Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology. 2011;140:1729–1737.
74. Lapaquette P, Bringer MA, Darfeuille-Michaud A. Defects in autophagy favour adherent-invasive Escherichia coli persistence within macrophages leading to increased pro-inflammatory response. Cell Microbiol. 2012;14:791–807.
75. Cadwell K, Patel KK, Maloney NS, et al.. Virus-plus-susceptibility gene interaction determines Crohn's disease gene Atg16L1 phenotypes in intestine. Cell. 2010;141:1135–1145.
76. Caprilli R, Lapaquette P, Darfeuille-Michaud A. Eating the enemy in Crohn's disease: an old theory revisited. J Crohns Colitis. 2010;4:377–383.
77. Takagi S, Utsunomiya K, Kuriyama S, et al.. Effectiveness of an ‘half elemental diet' as maintenance therapy for Crohn's disease: a randomized-controlled trial. Aliment Pharmacol Ther. 2006;24:1333–1340.
78. Yamamoto T, Nakahigashi M, Umegae S, et al.. Impact of long-term enteral nutrition on clinical and endoscopic recurrence after resection for Crohn's disease: a prospective, non-randomized, parallel, controlled study. Aliment Pharmacol Ther. 2007;25:67–72.
79. Yamamoto T, Nakahigashi M, Saniabadi AR, et al.. Impacts of long-term enteral nutrition on clinical and endoscopic disease activities and mucosal cytokines during remission in patients with Crohn's disease: a prospective study. Inflamm Bowel Dis. 2007;13:1493–1501.
80. Rioux JD, Xavier RJ, Taylor KD, et al.. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39:596–604.
81. Kuballa P, Huett A, Rioux JD, et al.. Impaired autophagy of an intracellular pathogen induced by a Crohn's disease associated ATG16L1 variant. PLoS One. 2008;3:e3391.
82. Wehkamp J, Harder J, Weichenthal M, et al.. NOD2 (CARD15) mutations in Crohn's disease are associated with diminished mucosal alpha-defensin expression. Gut. 2004;53:1658–1664.
83. Borrelli O, Cordischi L, Cirulli M, et al.. Polymeric diet alone versus corticosteroids in the treatment of active pediatric Crohn's disease: a randomized controlled open-label trial. Clin Gastroenterol Hepatol. 2006;4:744–753.