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Microbial Influences in Inflammatory Bowel Diseases

  • R. Balfour Sartor
    Correspondence
    Address requests for reprints to: R. Balfour Sartor, MD, UNC Department of Medicine/GI, CB 7032, Room 7309 Biomolecular Research Bldg, Chapel Hill, North Carolina 27599-7032. fax: (919) 843-6899.
    Affiliations
    Department of Medicine, and Department of Microbiology and Immunology, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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      The predominantly anaerobic microbiota of the distal ileum and colon contain an extraordinarily complex variety of metabolically active bacteria and fungi that intimately interact with the host’s epithelial cells and mucosal immune system. Crohn’s disease, ulcerative colitis, and pouchitis are the result of continuous microbial antigenic stimulation of pathogenic immune responses as a consequence of host genetic defects in mucosal barrier function, innate bacterial killing, or immunoregulation. Altered microbial composition and function in inflammatory bowel diseases result in increased immune stimulation, epithelial dysfunction, or enhanced mucosal permeability. Although traditional pathogens probably are not responsible for these disorders, increased virulence of commensal bacterial species, particularly Escherichia coli, enhance their mucosal attachment, invasion, and intracellular persistence, thereby stimulating pathogenic immune responses. Host genetic polymorphisms most likely interact with functional bacterial changes to stimulate aggressive immune responses that lead to chronic tissue injury. Identification of these host and microbial alterations in individual patients should lead to selective targeted interventions that correct underlying abnormalities and induce sustained and predictable therapeutic responses.

      Abbreviations used in this paper:

      AIEC (adherent/invasive E coli), DC (dendritic cells), DSS (dextran sodium sulfate), IL (interleukin), MAP (Mycobacterium avium subspecies paratuberculosis), NLR (NOD-like receptor), SCFA (short-chain fatty acids), TLR (toll-like receptors.)
      Although microbial pathogens have been postulated to cause Crohn’s disease and ulcerative colitis since their original descriptions, it is now generally accepted that commensal enteric bacteria provide the constant antigenic stimulation that continuously activates pathogenic T cells to cause chronic intestinal injury.
      • Eckburg P.B.
      • Relman D.A.
      The role of microbes in Crohn’s disease.
      • Strober W.
      • Fuss I.
      • Mannon P.
      The fundamental basis of inflammatory bowel disease.
      • Sartor R.B.
      Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis.
      • Xavier R.J.
      • Podolsky D.K.
      Unravelling the pathogenesis of inflammatory bowel disease.
      Recent evidence from a variety of disciplines, including genetics, molecular microbiology, basic immunology, experimental rodent models, immunodiagnostics, translational research, and clinical trials, combine to firmly implicate abnormal host-microbial interactions in the pathogenesis of idiopathic inflammatory bowel diseases (IBD) and their complications, including pouchitis.
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      CCFA microbial-host interactions workshop: highlights and key observations.
      This review briefly summarizes the rapidly expanding knowledge of the composition and function of enteric microbiota and etiologic theories of how commensal enteric bacteria induce, perpetuate, and reactivate IBD and chronic, immune-mediated intestinal inflammation and cause local and systemic complications. Although this review emphasizes the pathogenesis and pathophysiologic mechanisms of IBD, it briefly explores therapeutic implications.

      Composition and Metabolic Activity of Commensal Enteric Bacteria

      The incredibly complex microbiota of the distal ileum and colon provide an abundant source of potentially detrimental organisms, ligands, and antigens that can activate pathogenic innate and adaptive immune responses, respectively, and metabolic products that affect epithelial and immune functions. These bacteria and their biologically active products are intimately associated with the intestinal mucosa and induce physiologic and pathophysiologically important immune responses. Molecular detection and metabolomic techniques have revolutionized understanding the composition and metabolic activities of these largely uncultivatable organisms.
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      Diversity of the human intestinal microbial flora.
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      Metagenomic analysis of the human distal gut microbiome.
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      An obesity-associated gut microbiome with increased capacity for energy harvest.
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      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
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      Ecological and evolutionary forces shaping microbial diversity in the human intestine.
      Molecular analysis of microbial composition of fecal and mucosal samples using 16s ribosomal DNA and RNA have increased previous culture-based estimates of 200–300 colonic species to as high as 1800 genera and between 15,000 and 36,000 individual species.
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      The total microbial load of the intestine, 1013–1014 microorganisms, collectively contain at least 100 times as many genes as the human genome.
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      Metagenomic analysis of the human distal gut microbiome.
      These bacteria and fungi increase in both concentration and complexity from the proximal gastric and duodenal population of 102–103 aerobic organisms/gram luminal contents to 1011–1012 predominantly anaerobic bacteria/gram in the cecum and colon (Figure 1). Greater than 99% of the gut microbiota is composed of species within 4 bacterial divisions: Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria.
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      The role of microbes in Crohn’s disease.
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      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      The dominant Firmicutes (64% of attached colonic species) are primarily composed of the Clostridium XIV and IV groups, and Bacteroidetes account for 23% of normal species. Enterobacteriaceae such as Escherichia coli are relatively minor components of the Proteobacteria division (8% of all bacteria).
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      Few studies have been performed on the small intestinal microbiota because of sampling difficulties. A molecular analysis of mucosally associated bacteria reported enrichment of Streptococceae and Lactobacillales species (Bacillus subgroup of Firmicutes), Actinomycinaeae, and Corynebacteriaeceae (Actinobacter group), with reciprocal decreases in Clostridia and Bacteroides species.
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      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      Figure thumbnail gr1
      Figure 1Composition and luminal concentrations of dominant microbial species in various regions of the gastrointestinal tract.
      These diverse intestinal microbiota actively metabolize nonabsorbed dietary carbohydrates, exfoliated epithelial cells, and mucus to produce multiple metabolites that profoundly influence intestinal epithelial function,
      • Hooper L.V.
      • Gordon J.I.
      Commensal host-bacterial relationships in the gut.
      host energy balance,
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      • Ley R.E.
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      • et al.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      and immune response.
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      Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: implications for chronic inflammation.
      Gordon et al
      • Ley R.E.
      • Peterson D.A.
      • Gordon J.I.
      Ecological and evolutionary forces shaping microbial diversity in the human intestine.
      propose that humans are superorganisms composed of bacterial and human genes that mutualistically and interdependently determine our metabolic profile. Analysis of the metagenome of fecal samples from 2 healthy subjects showed that the intestinal microbiome is enriched with genes involved in metabolism of sucrose, starch, glycans, arabinose, mannose, xylose, and xenobiotic compounds and synthesis of methane, vitamins, isoprenoids, and short-chain fatty acids (SCFA) such as butyrate.
      • Gill S.R.
      • Pop M.
      • Deboy R.T.
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      These enzymes permit mammalian utilization of the nondigested plant polysaccharides xylan, pectin, and arabinose. Intestinal bacteria, especially by Firmicutes such as Clostridium species, and Bifidobacterium species, metabolize dietary fiber to SCFA, which accounts for up to 10% of the human energy source. Increased ratios of Firmicutes/Bacteroidetes have been documented in obese humans
      • Ley R.E.
      • Turnbaugh P.J.
      • Klein S.
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      Microbial ecology: human gut microbes associated with obesity.
      and genetically obese Ob/Ob mice.
      • Turnbaugh P.J.
      • Ley R.E.
      • Mahowald M.A.
      • et al.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      Increased production of SCFA by the microbiota of Ob/Ob vs wild-type (WT) mice and enhanced weight gain in gnotobiotic WT mice colonized with Ob/Ob mouse fecal bacteria
      • Turnbaugh P.J.
      • Ley R.E.
      • Mahowald M.A.
      • et al.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      document the potential role of the intestinal microbiota on human energy metabolism. Mammalian intestinal mucus provides a reservoir of glycans that serve as a source of bacterial nutrition during periods of fasting. Gut microbial detoxification of xenobiotic compounds could impact drug metabolism and carcinogenesis.
      • Gill S.R.
      • Pop M.
      • Deboy R.T.
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      Gut bacteria metabolize bile acids and, reciprocally, bile acids influence bacterial function and gene expression.
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      • Skilbeck C.A.
      • Nakano V.
      • et al.
      Bile salts enhance bacterial co-aggregation, bacterial-intestinal epithelial cell adhesion, biofilm formation and antimicrobial resistance of Bacteroides fragilis.

      Physiologic Microbial/Host Interactions

      In normal hosts, commensal bacteria activate a sequential program of homeostatic responses by epithelial cells, macrophages, dendritic cells (DC), T lymphocytes, and B cells/plasma cells that permit coexistence with potentially toxic microbial products.
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      • Fuss I.
      • Mannon P.
      The fundamental basis of inflammatory bowel disease.
      • Clavel T.
      • Haller D.
      Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: implications for chronic inflammation.
      Homeostatic mechanisms depend on down-regulating bacterial receptors, inducing intracellular and secreted molecules that terminate innate and adaptive immune responses, and stimulating protective molecules that mediate mucosal barrier function. Bacterial recognition is dependent on transmembrane pattern recognition receptors, including the structurally homologous toll-like receptors (TLR) and intracellular NOD-like receptor (NLR) family
      • Strober W.
      • Fuss I.
      • Mannon P.
      The fundamental basis of inflammatory bowel disease.
      • Xavier R.J.
      • Podolsky D.K.
      Unravelling the pathogenesis of inflammatory bowel disease.
      • Strober W.
      • Murray P.J.
      • Kitani A.
      • et al.
      Signalling pathways and molecular interactions of NOD1 and NOD2.
      (Figure 2). Ligation of these bacterial receptors stimulate central signaling cascades that include nuclear factor-κB (NF-κB), AKT/phosphatidylinositol-3’-kinase, and mitogen-activated protein kinase pathways (Figure 2). These pathways are inhibited by induction of inhibitory molecules, including A20, peroxisomal proliferator-activated receptor γ, inhibitor of NF-κB (IκB) α, interferon (IFN) α/β, interleukin (IL)-10, transforming growth factor (TGF)-β, and eicosanoids (PGE2, lipoxins) and blockade of IκBα polyubiquination and degradation by commensal bacteria.
      • Fukata M.
      • Chen A.
      • Klepper A.
      • et al.
      Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: role in proliferation and apoptosis in the intestine.
      • Neish A.S.
      • Gewirtz A.T.
      • Zeng H.
      • et al.
      Prokaryotic regulation of epithelial responses by inhibition of IκB-α ubiquitination.
      Optimal regulation of these inhibitory pathways requires paracrine interactions between epithelial cells and lamina propria regulatory lymphocytes that secrete TGF-β and IL-1018 (Figure 3).
      Figure thumbnail gr2
      Figure 2Recognition of bacterial ligands by membrane-bound toll-like receptors (TLR) and nod-like receptors (NLR), with signaling through conserved pathways such as NF-κB and mitogen-activated protein kinases (MAPK) signal transduction pathways.
      Figure thumbnail gr3
      Figure 3Potential mechanisms by which enteric bacteria and fungi induce chronic immune-mediated intestinal injury. (A) Pathogenic bacteria. A traditional pathogen or functional alterations in commensal bacteria (increased epithelial adherence, invasion, resistance to killing) can lead to increased bacterial stimulation of innate and adaptive immune responses. (B) Abnormal microbial composition. Decreased concentrations of protective bacteria that produce SCFA such as butyrate can enhance mucosal permeability. Conversely, increased concentrations of aggressive bacteria increase the amount of adjuvants and antigens that induce pathogenic immune responses or increase production of toxic metabolites such as hydrogen sulfide (H2S) that increase mucosal permeability and block butyrate metabolism. (C) Defective host containment of commensal bacteria. Defective secretion of antimicrobial peptides or secretory IgA can lead to mucosal bacterial overgrowth, whereas defective killing of phagocytosed bacteria can lead to persistent intracellular bacteria and ineffective clearance of bacterial antigens. Increased mucosal permeability can result in overwhelming exposure of bacterial TLR ligands and antigens that activate pathogenic innate and T-cell immune responses. (D) Defective host immunoregulation. Ineffective down-regulation of innate immune responses, either in epithelial or antigen-presenting cells (APC), can induce inflammatory responses through secretion of chemokines or proinflammatory cytokines. Dysfunction of regulatory T cells or APC can lead to overly aggressive T responses (loss of tolerance) to ubiquitous microbial antigens or induction of cross-reactive autoimmune responses because of molecular mimicry between host and microbial antigens.

       Epithelial Cells

      Intestinal epithelial cells not only provide an efficient barrier to exclude viable enteric bacteria and their immunologically active adjuvants and antigens from interacting with lamina propria immune cells but actively participate as innate immune sensors of microbial pathogens and commensal organisms.
      • Clavel T.
      • Haller D.
      Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: implications for chronic inflammation.
      • Kagnoff M.F.
      • Eckmann L.
      Epithelial cells as sensors for microbial infection.
      Colonization of germ-free rodents with Enterococcus faecalis or Bacteroides vulgatus transiently activate NF-κB signaling and induce chemokine expression in colonic epithelial cells through TLR2 and TLR4 signaling, respectively.
      • Ruiz P.A.
      • Shkoda A.
      • Kim S.C.
      • et al.
      IL-10 gene-deficient mice lack TGF-β/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.
      • Haller D.
      • Holt L.
      • Schwabe R.F.
      • et al.
      Transforming growth factor-β 1 inhibits non-pathogenic gram-negative bacteria-induced NF-κB recruitment to the interleukin-6 gene promoter in intestinal epithelial cells through modulation of histone acetylation.
      Time-dependent epithelial NF-κB activation after bacterial colonization was confirmed in NF-κB-green fluorescent protein receptor mice.
      • Karrasch T.
      • Kim J.S.
      • Muhlbauer M.
      • et al.
      Gnotobiotic IL-10-/- NF-κBEGFP mice reveal the critical role of TLR/NF-κB signaling in commensal bacteria-induced colitis.
      In normal hosts, NF-κB signaling and expression of chemokines and TLR2 signaling was down-regulated 7–10 days after bacterial colonization, coinciding with SMAD signaling, suggesting that TGF-β produced by lamina propria lymphocytes inhibited bacterial-induced epithelial NF-κB activation.
      • Ruiz P.A.
      • Shkoda A.
      • Kim S.C.
      • et al.
      IL-10 gene-deficient mice lack TGF-β/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.
      An essential role for mucosal IL-10 in regulating this protection is suggested by persistent NF-κB activation and TLR2 and chemokine expression in IL-10-deficient mice, which developed chronic colitis after E faecalis monoassociation.
      • Ruiz P.A.
      • Shkoda A.
      • Kim S.C.
      • et al.
      IL-10 gene-deficient mice lack TGF-β/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.
      The critical role of commensal bacterial-NF-κB-dependent mucosal homeostatic responses by epithelial cells is demonstrated by potentiation of dextran sodium sulfate (DSS)-induced colitis in germ-free mice,
      • Kitajima S.
      • Morimoto M.
      • Sagara E.
      • et al.
      Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice.
      TLR9-deficient mice,
      • Lee J.
      • Mo J.H.
      • Katakura K.
      • et al.
      Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.
      TLR4 knockout mice,
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      • Michelsen K.S.
      • Eri R.
      • et al.
      Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis.
      and mice deficient in Myd 88, an adaptor protein required for TLR and IL-1 signaling.
      • Rakoff-Nahoum S.
      • Paglino J.
      • Eslami-Varzaneh F.
      • et al.
      Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.
      However, some studies have observed no enhancement
      • Ohkawara T.
      • Takeda H.
      • Nishihira J.
      • et al.
      Macrophage migration inhibitory factor contributes to the development of acute dextran sulphate sodium-induced colitis in Toll-like receptor 4 knockout mice.
      or even decreased mucosal injury
      • Heimesaat M.M.
      • Fischer A.
      • Siegmund B.
      • et al.
      Shift towards pro-inflammatory intestinal bacteria aggravates acute murine colitis via Toll-like receptors 2 and 4.
      in DSS-treated TLR4- and/or TLR2-deficient mice. A protective role for epithelial NF-κB signaling by either bacteria, IL-1, or TNF stimulation of TLRs, NLR, or cytokine receptors is elegantly demonstrated by conditional ablation of NEMO (IκB kinase γ) in intestinal epithelial cells causing spontaneous severe colitis.
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      This report is consistent with worsening of ischemia-reperfusion intestinal injury in mice with selective epithelial IKKβ deficiency.
      • Egan L.J.
      • Eckmann L.
      • Greten F.R.
      • et al.
      IκB-kinase β-dependent NF-κB activation provides radioprotection to the intestinal epithelium.
      Blockade of epithelial NF-κB signaling led to increased bacterial translocation across the injured epithelium, similar to TLR4-deficient mice treated with DSS,
      • Fukata M.
      • Chen A.
      • Klepper A.
      • et al.
      Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: role in proliferation and apoptosis in the intestine.
      and activation of pathogenic CD4+ T cells.
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      Important protective mechanisms of epithelial cell NF-κB activation include induction of type 1 interferons (IFNα/β) by bacterial DNA through TLR9 stimulation of NF-κB, mediated by Myd 88 and DNA-dependent protein kinase,
      • Katakura K.
      • Lee J.
      • Rachmilewitz D.
      • et al.
      Toll-like receptor 9-induced type I IFN protects mice from experimental colitis.
      and enhanced secretion of thymic stromal lymphopoietin, which inhibits DC production of IL-12/23 p40 and induction of T-helper cell-1 (Th1) and Th17 responses to helminth infection.
      • Zaph C.
      • Troy A.E.
      • Taylor B.C.
      • et al.
      Epithelial-cell-intrinsic IKK-β expression regulates intestinal immune homeostasis.
      Regulatory vs inductive TLR signaling is polarized; basolateral stimulation of TLR9 by synthetic bacterial DNA activated NF-κB, whereas physiologic apical stimulation of epithelial cell lines inhibited NF-κB signaling by blocking degradation of ubiquinated IκBα.
      • Lee J.
      • Mo J.H.
      • Katakura K.
      • et al.
      Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.
      Bacterial exposure activates protective pathways that prevent subsequent injurious responses to the same stimuli. TLR ligation down-regulates NF-κB activation upon reexposure to the same or different TLR ligands through induction of multiple inhibitors, including IRAK-M, toll-interacting protein (TOLLIP), single immunoglobulin IL-1 receptor (SIGIRR), A20, NOD2, and peroxisomal proliferator-activated receptor γ.
      • Shibolet O.
      • Podolsky D.K.
      TLRs in the Gut IV. Negative regulation of Toll-like receptors and intestinal homeostasis: addition by subtraction.
      Pretreatment of epithelial cells with flagellin, a TLR5 ligand, induced antiapoptotic molecules through NF-κB and phosphatidylinositol 3-kinase/AKT and prevented apoptosis with subsequent bacterial ligand stimulation.
      • Zeng H.
      • Wu H.
      • Sloane V.
      • et al.
      Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways.
      Bacterial colonization of germ-free mice stimulates epithelial expression of peroxisomal proliferator-activated receptor γ
      • Kelly D.
      • Campbell J.I.
      • King T.P.
      • et al.
      Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-γ and RelA.
      and antimicrobial peptides,
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      • Wong M.H.
      • Thelin A.
      • et al.
      Molecular analysis of commensal host-microbial relationships in the intestine.
      which inhibits activation of epithelial cells and decreases the concentration of mucosally associated bacteria, respectively. NOD2 regulation of α defensin production is indirectly suggested by decreased α defensin production in Crohn’s disease patients with NOD2 polymorphisms,
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      • Salzman N.H.
      • Porter E.
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      Reduced Paneth cell α-defensins in ileal Crohn’s disease.
      decreased α defensin production by NOD2-deficient mice,
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      • Ogura Y.
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      Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.
      and constitutive expression of NOD2 by Paneth cells,
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      Crohn’s disease and the NOD2 gene: a role for paneth cells.
      which are the source of α defensin production. NOD2 is an intracellular receptor that recognizes muramyl dipeptide, a peptidoglycan component with well-documented adjunctive properties.
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      Signalling pathways and molecular interactions of NOD1 and NOD2.
      Likewise, β defensin 2 expression is regulated by TLR signaling in colonic epithelial cells.
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      • Thomas L.S.
      • et al.
      Beta-defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells.
      Epithelial production of chemokines, which recruit phagocytic neutrophils, is an essential component of containing enteric pathogens and preventing bacterial translocation. In addition, repair of epithelial damage is stimulated by bacterial-induced NF-κB. Defective TLR signaling in Myd 88−/− mice leads to bacteremia, decreased mucosal hyperplasia, and death in pathogenic bacterial-infected Myd 88−/− mice.
      • Lebeis S.L.
      • Bommarius B.
      • Parkos C.A.
      • et al.
      TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium.
      Bone marrow chimeras show that this protection is mediated in part by TLR-mediated induction of chemokines by epithelial cells and repair of the mucosal barrier. Epithelial-specific blockade of NF-κB signaling decreased chemokine production and led to enhanced bacterial translocation.
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      Similarly, NOD2-deficient mice are more susceptible to oral Listeria monocytogenes infection.
      • Kobayashi K.S.
      • Chamaillard M.
      • Ogura Y.
      • et al.
      Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.
      TLR4-deficient mice treated with DSS have increased bacterial translocation to regional lymph nodes and decreased epithelial proliferation.
      • Fukata M.
      • Chen A.
      • Klepper A.
      • et al.
      Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: role in proliferation and apoptosis in the intestine.
      Finally, NF-κB and mitogen-activated protein kinase are integral mediators of epithelial restitution following damage.
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      • Steinbrecher K.A.
      • Allard B.
      • et al.
      Wound-induced p38MAPK-dependent histone H3 phosphorylation correlates with increased COX-2 expression in enterocytes.

       Mucosal Immune Responses

      Commensal enteric bacteria stimulate tolerogenic mucosal immune responses in normal hosts leading to diminished responses to TLR ligands in lamina propria macrophages, a low IL-12/23/IL-10 ratio in DC, regulatory responses in T cells, and secretory IgA rather than IgG by plasma cells.
      • Strober W.
      • Fuss I.
      • Mannon P.
      The fundamental basis of inflammatory bowel disease.
      • Xavier R.J.
      • Podolsky D.K.
      Unravelling the pathogenesis of inflammatory bowel disease.
      Germ-free (sterile) mice have underdeveloped mucosal and systemic immune systems with decreased cellularity of the Peyer’s patches, lamina propria, mesenteric lymph nodes, and spleen and diminished mucosal immune function.
      • Cebra J.J.
      Influences of microbiota on intestinal immune system development.
      Intestinal macrophages have diminished responses to bacterial stimuli such as lipopolysaccharide (LPS; endotoxin) because of low expression of bacterial receptors, including CD14 and TLR4, relative to monocytes but retain phagocytic activity.
      • Smythies L.E.
      • Sellers M.
      • Clements R.H.
      • et al.
      Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity.
      DC from WT mice secrete large amounts of IL-10 relative to IL-12/23 p40 in response to LPS.
      • Hoentjen F.
      • Sartor R.B.
      • Ozaki M.
      • et al.
      STAT3 regulates NF-{κ}B recruitment to the IL-12p40 promoter in dendritic cells.
      Epithelial cell-derived thymic stromal lymphopoietin inhibits LPS-stimulated IL-12/23 p40 production,
      • Zaph C.
      • Troy A.E.
      • Taylor B.C.
      • et al.
      Epithelial-cell-intrinsic IKK-β expression regulates intestinal immune homeostasis.
      illustrating the importance of epithelial cell-DC interactions. Although NF-κB signaling to the nucleus is intact in these DC, binding of the active p65 NF-κB subunit to the IL-12/23 p40 promoter is inhibited by IL-10 in a histone acetylase-dependent manner.
      • Hoentjen F.
      • Sartor R.B.
      • Ozaki M.
      • et al.
      STAT3 regulates NF-{κ}B recruitment to the IL-12p40 promoter in dendritic cells.
      In vivo, bacterial colonization can induce constitutive IL-23 expression to a greater extent in the ileum than in the colon.
      • Becker C.
      • Wirtz S.
      • Blessing M.
      • et al.
      Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells.
      The ability of commensal bacteria to induce regulatory adaptive immune responses is variable, with defective oral tolerance measured by splenic IgG responses in germ-free mice restored to normal by bacterial colonization or feeding LPS
      • Wannemuehler M.J.
      • Kiyono H.
      • Babb J.L.
      • et al.
      Lipopolysaccharide (LPS) regulation of the immune response: LPS converts germfree mice to sensitivity to oral tolerance induction.
      but no difference in high- or low-dose oral tolerance measured by T-cell proliferation between germ-free vs specific pathogen-free mice.
      • Walton K.L.W.
      • Galanko J.A.
      • Sartor R.B.
      • et al.
      T cell-mediated oral tolerance is intact in germ-free mice.
      Cong et al identified cecal bacterial antigen responsive CD4+ TR1 cells that secreted IL-10, suppressed Th1 responses in vitro, and prevented experimental colitis when cotransferred with IFN-γ-secreting CD4+ Th1 lymphocytes to T cell-deficient mice.
      • Cong Y.
      • Weaver C.T.
      • Lazenby A.
      • et al.
      Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora.
      Immunoglobulin recognizing commensal bacteria is produced in normal hosts, but profiles favor protective secretory IgA rather than pathogenic IgG or IgM.
      • Macpherson A.
      • Khoo U.Y.
      • Forgacs I.
      • et al.
      Mucosal antibodies in inflammatory bowel disease are directed against intestinal bacteria.
      It has been postulated that NOD2 regulates homeostasis of Peyer’s patches and mucosal lymphoid aggregates, the site of T- and B-cell stimulation. Although NOD2-deficient mice do not develop macroscopically or clinically evident colitis, their Peyer’s patches are increased in size and number, contain more CD4+ T cells, and spontaneously produce proinflammatory cytokines.
      • Barreau F.
      • Meinzer U.
      • Chareyre F.
      • et al.
      CARD15/NOD2 is required for Peyer’s patches homeostasis in mice.
      These changes are accompanied by increased mucosal permeability and enhanced translocation of bacteria. These observations are consistent with either down-regulation of TLR2 ligand-stimulated IL-12/23 p40 following activation of NOD2 by muramyl dipeptide
      • Watanabe T.
      • Kitani A.
      • Murray P.J.
      • et al.
      NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses.
      • van Beelen A.J.
      • Zelinkova Z.
      • Taanman-Kueter E.W.
      • et al.
      Stimulation of the intracellular bacterial sensor NOD2 programs dendritic cells to promote interleukin-17 production in human memory T cells.
      or altered composition of enteric microbiota because of defective secretion of antimicrobial α defensins in the absence of NOD2.
      • Kobayashi K.S.
      • Chamaillard M.
      • Ogura Y.
      • et al.
      Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.
      In summary, the net effect of commensal bacteria and their adjuvants and antigens is to maintain mucosal homeostasis by inhibiting pathogenic innate and adaptive immune responses, inducing secreted antimicrobial peptides, and promoting epithelial restitution.

      Microbial Influences in Chronic Immune-Mediated Intestinal Inflammation

       Clinical Evidence

      An important role for microbial agents in the pathogenesis of Crohn’s disease and pouchitis is suggested by clinical, experimental, and therapeutic studies, but less convincing evidence is available for ulcerative colitis (Table 1). Crohn’s disease and ulcerative colitis preferentially occur in the colon and distal ileum, which contain the highest intestinal bacterial concentrations. Moreover, the composition and function of the microbiota in Crohn’s disease, ulcerative colitis, and pouchitis are abnormal,
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      which is detailed in the Theories of Pathogenesis section. Functional alterations are most evident in adherent, invasive Escherichia coli that colonize the ileum of Crohn’s disease patients.
      • Barnich N.
      • Darfeuille-Michaud A.
      Adherent-invasive Escherichia coli and Crohn’s disease.
      Fluorescent in situ hybridization (FISH) studies demonstrate dramatically increased mucosally associated bacteria in active Crohn’s disease and to a lesser extent ulcerative colitis,
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      although similar increases in nonspecific colitis and lack of abnormalities in quiescent IBD samples suggest a secondary process. Similarly, bacteria invade mucosal ulcers and fistulae in active Crohn’s disease,
      • Cartun R.W.
      • Van Kruiningen H.J.
      • Pedersen C.A.
      • et al.
      An immunocytochemical search for infectious agents in Crohn’s disease.
      • Liu Y.
      • Van Kruiningen H.J.
      • West A.B.
      • et al.
      Immunocytochemical evidence of Listeria, Escherichia coli, and Streptococcus antigens in Crohn’s disease.
      and bacterial DNA is present within granulomas,
      • Ryan P.
      • Kelly R.G.
      • Lee G.
      • et al.
      Bacterial DNA within granulomas of patients with Crohn’s disease—detection by laser capture microdissection and PCR.
      whereas bacteria and LPS are translocated to the portal vein in active IBD. The presence of luminal, mucosally associated and translocating bacteria provides antigens and TLR ligands that could stimulate pathogenic immune responses that induce and perpetuate intestinal inflammation in genetically susceptible hosts. A pathogenic role of luminal constituents is suggested by prevention and treatment of Crohn’s disease by diversion of the fecal strain and reactivation of histologic and biochemical inflammation within 1 week following reinfusion of ileostomy contents.
      • D’Haens G.R.
      • Geboes K.
      • Peeters M.
      • et al.
      Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum.
      The ability of metronidazole
      • Rutgeerts P.
      • Hiele M.
      • Geboes K.
      • et al.
      Controlled trial of metronidazole treatment for prevention of Crohn’s recurrence after ileal resection.
      and ornidazole
      • Rutgeerts P.
      • Van Assche G.
      • Vermeire S.
      • et al.
      Ornidazole for prophylaxis of postoperative Crohn’s disease recurrence: a randomized, double-blind, placebo-controlled trial.
      to decrease postoperative recurrence of ileal Crohn’s disease suggests a role for anaerobic bacteria in this recurrence. In addition, adherent/invasive E coli strains also have been found in increased frequency in postoperative recurrent ileal Crohn’s disease.
      • Darfeuille-Michaud A.
      • Neut C.
      • Barnich N.
      • et al.
      Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease.
      Crohn’s disease-related genetic polymorphisms in the intracellular bacterial recognition receptor, NOD2
      • Hugot J.P.
      • Chamaillard M.
      • Zouali H.
      • et al.
      Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • et al.
      A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.
      ; TLR2, 4, and 5
      • Pierik M.
      • Joossens S.
      • Van Steen K.
      • et al.
      Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases.
      ; the autophagy gene ATG 16L1, which regulates intracellular microbial processing and killing
      • Hampe J.
      • Franke A.
      • Rosenstiel P.
      • et al.
      A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn’s disease in ATG16L1.
      • Rioux J.D.
      • Xavier R.J.
      • Taylor K.D.
      • et al.
      Genome-wide association study identifies new susceptibility loci for Crohn’s disease and implicates autophagy in disease pathogenesis.
      ; and NCF4, which mediates NADPH-dependent bacterial killing in phagocytic cells,
      • Rioux J.D.
      • Xavier R.J.
      • Taylor K.D.
      • et al.
      Genome-wide association study identifies new susceptibility loci for Crohn’s disease and implicates autophagy in disease pathogenesis.
      further support the hypothesis that a subset of Crohn’s disease patients have defective innate immune responses to microbial agents.
      • Korzenik J.R.
      Is Crohn’s disease due to defective immunity?.
      Table 1Clinical Evidence Implicating Enteric Microbiota in the Pathogenesis of IBD
      Crohn’s disease, ulcerative colitis, and pouchitis are localized to intestinal segments with highest bacterial concentrations
      • Eckburg P.B.
      • Relman D.A.
      The role of microbes in Crohn’s disease.
      Abnormal microbial composition in pouchitis, Crohn’s disease, and ulcerative colitis
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      • Iwaya A.
      • Iiai T.
      • Okamoto H.
      • et al.
      Change in the bacterial flora of pouchitis.
      Enhanced E coli virulence in Crohn’s disease
      • Darfeuille-Michaud A.
      • Neut C.
      • Barnich N.
      • et al.
      Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease.
      Altered metabolic activity of microbiota in Crohn’s disease, ulcerative colitis, and pouchitis
      • Marchesi J.R.
      • Holmes E.
      • Khan F.
      • et al.
      Rapid and noninvasive metabonomic characterization of inflammatory bowel disease.
      • Smith F.M.
      • Coffey J.C.
      • Kell M.R.
      • et al.
      A characterization of anaerobic colonization and associated mucosal adaptations in the undiseased ileal pouch.
      Increased mucosal-associated bacteria in IBD patients
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      Enhanced mucosal invasion and translocation in active Crohn’s disease and ulcerative colitis
      • Liu Y.
      • Van Kruiningen H.J.
      • West A.B.
      • et al.
      Immunocytochemical evidence of Listeria, Escherichia coli, and Streptococcus antigens in Crohn’s disease.
      Polymorphisms of genes that encode bacterial receptors and processing in Crohn’s disease
      • Hugot J.P.
      • Chamaillard M.
      • Zouali H.
      • et al.
      Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease.
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • et al.
      A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.
      • Hampe J.
      • Franke A.
      • Rosenstiel P.
      • et al.
      A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn’s disease in ATG16L1.
      Fecal stream diversion prevents and treats Crohn’s disease and pouchitis; inflammation recurs upon restoration of fecal flow
      • D’Haens G.R.
      • Geboes K.
      • Peeters M.
      • et al.
      Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum.
      Serologic and T-cell responses to enteric microbial antigens in patients with Crohn’s disease, ulcerative colitis, and pouchitis
      • Mow W.S.
      • Vasiliauskas E.A.
      • Lin Y.C.
      • et al.
      Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.
      • Targan S.R.
      • Karp L.C.
      Defects in mucosal immunity leading to ulcerative colitis.
      Antibiotics treat pouchitis and Crohn’s colitis/ileocolitis
      • Sartor R.B.
      Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics and prebiotics.
      Probiotics prevent relapse of pouchitis and ulcerative colitis
      • Gionchetti P.
      • Rizzello F.
      • Venturi A.
      • et al.
      Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial.
      • Kruis W.
      • Fric P.
      • Pokrotnieks J.
      • et al.
      Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine.
      Crohn’s disease and ulcerative colitis patients exhibit T-cell and serologic responses to a number of enteric bacterial and fungal species.
      • Mow W.S.
      • Landers C.J.
      • Steinhart A.H.
      • et al.
      High-level serum antibodies to bacterial antigens are associated with antibiotic-induced clinical remission in Crohn’s disease: a pilot study.
      • Duchmann R.
      • Kaiser I.
      • Hermann E.
      • et al.
      Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD).
      Antimicrobial serologic responses are particularly well characterized, have provided insights into phenotypic and genetic subsets of disease, and demonstrate great promise for predicting aggressiveness of disease and response to therapy. Approximately 50% of Crohn’s disease patients have positive serologic responses to either OmpC (E coli), I2 (Pseudomonas fluorescence), Cbir 1 flagellin (Clostridium species), or Saccharomyces cerevisiae, which cross-react with Candida albicans.
      • Mow W.S.
      • Landers C.J.
      • Steinhart A.H.
      • et al.
      High-level serum antibodies to bacterial antigens are associated with antibiotic-induced clinical remission in Crohn’s disease: a pilot study.
      • Targan S.R.
      • Landers C.J.
      • Yang H.
      • et al.
      Antibodies to CBir1 flagellin define a unique response that is associated independently with complicated Crohn’s disease.
      Approximately 80% of Crohn’s disease patients display positive serologic responses to S cerevisiae, OmpC, or I2, and quantitation of these serologies correlates with the aggressiveness and need for surgery in both prospective and retrospective studies.
      • Mow W.S.
      • Landers C.J.
      • Steinhart A.H.
      • et al.
      High-level serum antibodies to bacterial antigens are associated with antibiotic-induced clinical remission in Crohn’s disease: a pilot study.
      • Dubinsky M.C.
      • Lin Y.C.
      • Dutridge D.
      • et al.
      Serum immune responses predict rapid disease progression among children with Crohn’s disease: immune responses predict disease progression.
      Similarly, approximately 60% of ulcerative colitis patients exhibit antibodies to neutrophils with an immunohistochemical perinuclear staining pattern (pANCA). These antibodies react with enteric bacteria.
      • Seibold F.
      • Brandwein S.
      • Simpson S.
      • et al.
      pANCA represents a cross-reactivity to enteric bacterial antigens.
      Of potential diagnostic importance, antimicrobial serologies in serum stored from Israeli soldiers predicted the onset of Crohn’s disease and ulcerative colitis many years later.
      • Israeli E.
      • Grotto I.
      • Gilburd B.
      • et al.
      Anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies as predictors of inflammatory bowel disease.
      Furthermore, serologic profiles correlated with therapeutic responses to fecal diversion and antibiotics in Crohn’s disease patients,
      • Mow W.S.
      • Landers C.J.
      • Steinhart A.H.
      • et al.
      High-level serum antibodies to bacterial antigens are associated with antibiotic-induced clinical remission in Crohn’s disease: a pilot study.
      • Spivak J.
      • Landers C.J.
      • Vasiliauskas E.A.
      • et al.
      Antibodies to I2 predict clinical response to fecal diversion in Crohn’s disease.
      indicating the possibility of identifying patient subsets by immunologic responses to discrete bacterial antigens. Genetic regulation of immune responses to bacteria is suggested by hereditable phenotypes of OmpC serologies in asymptomatic relatives of Crohn’s disease patients
      • Mei L.
      • Targan S.R.
      • Landers C.J.
      • et al.
      Familial expression of anti-Escherichia coli outer membrane porin C in relatives of patients with Crohn’s disease.
      and association of antibacterial immunoglobulin responses with NOD2 genetic variants.
      • Papadakis K.A.
      • Yang H.
      • Ippoliti A.
      • et al.
      Anti-flagellin (CBir1) phenotypic and genetic Crohn’s disease associations.
      • Devlin S.M.
      • Yang H.
      • Ippoliti A.
      • et al.
      NOD2 variants and antibody response to microbial antigens in Crohn’s disease patients and their unaffected relatives.

       Evidence in Rodent Models

      Rodent models of spontaneous or induced intestinal inflammation provide compelling evidence that commensal enteric bacterial antigens continuously drive chronic, immune-mediated colitis and ileitis (Table 2).
      • Strober W.
      • Fuss I.
      • Mannon P.
      The fundamental basis of inflammatory bowel disease.
      • Sartor R.B.
      Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis.
      • Cong Y.
      • Weaver C.T.
      • Lazenby A.
      • et al.
      Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora.
      • Hansen J.
      • Sartor R.B.
      Insights from animal models.
      Genetically engineered mice and rats with systemic immunoregulatory defects can display an intestinal phenotype of diarrhea, wasting, rectal prolapse, and inflammation usually limited to the colon, with occasional ileal, duodenal, and gastric antral involvement.
      • Hansen J.
      • Sartor R.B.
      Insights from animal models.
      The predominant colonic phenotype suggests involvement of the complex anaerobic commensal enteric microbiota. Commensal bacteria are convincingly implicated by the observation that most germ-free (sterile) susceptible rodents have no intestinal inflammation or immune activation but rapidly develop disease and pathogenic immune responses after colonization with specific pathogen-free enteric bacteria.
      • Sellon R.K.
      • Tonkonogy S.
      • Schultz M.
      • et al.
      Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.
      • Rath H.C.
      • Herfarth H.H.
      • Ikeda J.S.
      • et al.
      Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human β2 microglobulin transgenic rats.
      • Veltkamp C.
      • Tonkonogy S.L.
      • de Jong Y.P.
      • et al.
      Continuous stimulation by normal luminal bacteria is essential for the development and perpetuation of colitis in Tg(ε26) mice.
      • Taurog J.D.
      • Richardson J.A.
      • Croft J.T.
      • et al.
      The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats.
      Two exceptions are IL-2-deficient
      • Schultz M.
      • Tonkonogy S.L.
      • Sellon R.K.
      • et al.
      IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation.
      and Samp-1/Yit
      • Bamias G.
      • Okazawa A.
      • Rivera-Nieves J.
      • et al.
      Commensal bacteria exacerbate intestinal inflammation but are not essential for the development of murine ileitis.
      mice, which exhibit mild colitis and ileitis, respectively, in the germ-free state but have more aggressive inflammation when colonized with normal enteric bacteria. Germ-free mice have no viable bacteria, but killed bacteria and microbial products in autoclaved or radiated food provide antigenic stimuli. Killed bacteria or purified bacterial peptidoglycan-polysaccharide polymers induce chronic T cell-mediated granulomatous enterocolitis when injected into susceptible rats,
      • Sartor R.B.
      • Cromartie W.J.
      • Powell D.W.
      • et al.
      Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments.
      • Mourelle M.
      • Salas A.
      • Guarner F.
      • et al.
      Stimulation of transforming growth factor β1 by enteric bacteria in the pathogenesis of rat intestinal fibrosis.
      and luminal bacterial products can potentiate colitis.
      • Sartor R.B.
      • Bond T.M.
      • Schwab J.H.
      Systemic uptake and intestinal inflammatory effects of luminal bacterial cell wall polymers in rats with acute colonic injury.
      Potentiated acute mucosal injury in germ-free mice treated with DSS,
      • Kitajima S.
      • Morimoto M.
      • Sagara E.
      • et al.
      Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice.
      a model with no requirement for T cells,
      • Dieleman L.A.
      • Ridwan B.U.
      • Tennyson G.S.
      • et al.
      Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice.
      emphasizes the protective role of bacterial-induced TLR signaling through NF-κB in epithelial cell homeostasis.
      • Rakoff-Nahoum S.
      • Paglino J.
      • Eslami-Varzaneh F.
      • et al.
      Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.
      In the chronic colitis and ileitis models, enteric bacteria induce Th1, Th17, or Th2 responses.
      • Sellon R.K.
      • Tonkonogy S.
      • Schultz M.
      • et al.
      Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.
      • Rath H.C.
      • Herfarth H.H.
      • Ikeda J.S.
      • et al.
      Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human β2 microglobulin transgenic rats.
      • Schultz M.
      • Tonkonogy S.L.
      • Sellon R.K.
      • et al.
      IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation.
      • Bamias G.
      • Okazawa A.
      • Rivera-Nieves J.
      • et al.
      Commensal bacteria exacerbate intestinal inflammation but are not essential for the development of murine ileitis.
      • Kim S.C.
      • Tonkonogy S.L.
      • Albright C.A.
      • et al.
      Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria.
      A functional role for bacterial-activated effector T cells is demonstrated by the ability of cecal bacterial antigen or Cbir 1 flagellin-activated CD4+ cells, but not nonspecifically activated anti-CD3 antibody stimulated CD4+ cells, to adoptively transfer chronic colitis to T cell-deficient recipients.
      • Cong Y.
      • Weaver C.T.
      • Lazenby A.
      • et al.
      Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora.
      • Lodes M.J.
      • Cong Y.
      • Elson C.O.
      • et al.
      Bacterial flagellin is a dominant antigen in Crohn’s disease.
      Studies in gnotobiotic rodents selectively colonized (monoassociated) with individual bacteria show both bacterial- and host-specific responses. For example, Bacteroides vulgatus but not E coli, Enterococcus faecalis, and multiple other bacterial species induce colitis in monoassociated HLA B27 transgenic rats.
      • Rath H.C.
      • Herfarth H.H.
      • Ikeda J.S.
      • et al.
      Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human β2 microglobulin transgenic rats.
      • Rath H.C.
      • Wilson K.H.
      • Sartor R.B.
      Differential induction of colitis and gastritis in HLA-B27 transgenic rats selectively colonized with Bacteroides vulgatus and Escherichia coli.
      In contrast, E coli or E faecalis induced colitis with bacterial antigen-specific CD4+ T-cell responses in monoassociated IL-10−/− mice, whereas B vulgatus has no effect, and none of these 3 bacterial species induce colitis in monoassociated bone marrow-transplanted CD3ε transgenic mice.
      • Kim S.C.
      • Tonkonogy S.L.
      • Albright C.A.
      • et al.
      Different host genetic backgrounds determine disease phenotypes induced by selective bacterial colonization.
      In a single inbred host, 2 different bacterial strains caused different phenotypes of disease and show additive effects. E coli induces relatively mild cecal-predominant colitis in monoassociated IL-10−/− mice, whereas E faecalis causes more severe, late-onset distal colonic inflammation.
      • Kim S.C.
      • Tonkonogy S.L.
      • Albright C.A.
      • et al.
      Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria.
      Dual association with both species causes more aggressive, early-onset pancolitis.
      • Kim S.C.
      • Tonkonogy S.L.
      • Karrasch T.
      • et al.
      Dual association of gnotobiotic IL-10-/- mice with two nonpathogenic commensal bacteria induces aggressive pancolitis.
      As with human IBD, experimental intestinal inflammation is associated with increased mucosal adherence
      • Hale L.P.
      • Gottfried M.R.
      • Swidsinski A.
      Piroxicam treatment of IL-10-deficient mice enhances colonic epithelial apoptosis and mucosal exposure to intestinal bacteria.
      and translocation of viable bacteria and bacterial products across the injured mucosa to regional lymph nodes and portal and systemic blood.
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      • Yamada T.
      • Deitch E.
      • Specian R.D.
      • et al.
      Mechanisms of acute and chronic intestinal inflammation induced by indomethacin.
      • Gardiner K.R.
      • Erwin P.J.
      • Anderson N.H.
      • et al.
      Colonic bacteria and bacterial translocation in experimental colitis.
      • Hobson C.H.
      • Butt T.J.
      • Ferry D.M.
      • et al.
      Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis.
      Numerous studies show the efficacy of antibiotics, probiotics, and prebiotics in experimental colitis.
      • Sartor R.B.
      Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics and prebiotics.
      • Sheil B.
      • Shanahan F.
      • O’Mahony L.
      Probiotic effects on inflammatory bowel disease.
      Observations that broad-spectrum antibiotic combinations can both prevent onset and reverse established colitis and that selective antibiotics attenuate the onset of disease when used prophylactically but not treat active inflammation can help guide clinical studies.
      • Rath H.C.
      • Schultz M.
      • Freitag R.
      • et al.
      Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice.
      • Hoentjen F.
      • Harmsen H.J.
      • Braat H.
      • et al.
      Antibiotics with a selective aerobic or anaerobic spectrum have different therapeutic activities in various regions of the colon in interleukin-10 gene deficient mice.
      Table 2Evidence Implicating Enteric Microbiota in the Pathogenesis of Chronic Experimental Intestinal Inflammation
      Luminal commensal bacteria are required for chronic inflammation in most rodent models
      • Sartor R.B.
      Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis.
      Systemic immunoregulatory defects can have a colonic phenotype
      • Kuhn R.
      • Lohler J.
      • Rennick D.
      • et al.
      Interleukin-10-deficient mice develop chronic enterocolitis.
      Bacterial antigens stimulate pathogenic T-cell responses
      • Cong Y.
      • Brandwein S.L.
      • McCabe R.P.
      • et al.
      CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease.
      Increased mucosal association and translocation of bacteria
      • Swidsinski A.
      • Loening-Baucke V.
      • Lochs H.
      • et al.
      Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice.
      Broad spectrum antibiotics, probiotics, and prebiotics prevent and treat disease (99)
      Defective bacterial signaling in epithelial cells induce chronic, immune-mediated intestinal inflammation
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      Defective immunoregulation, mucosal barrier function, or bacterial killing induce chronic, immune-mediated intestinal inflammation or potentiate acute mucosal injury
      • Kobayashi K.S.
      • Chamaillard M.
      • Ogura Y.
      • et al.
      Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.
      • Olson T.S.
      • Reuter B.K.
      • Scott K.G.
      • et al.
      The primary defect in experimental ileitis originates from a nonhematopoietic source.
      • Hermiston M.L.
      • Gordon J.I.
      Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin.
      • Kuhn R.
      • Lohler J.
      • Rennick D.
      • et al.
      Interleukin-10-deficient mice develop chronic enterocolitis.

      Theories of Pathogenesis

      Four broad mechanisms have been proposed to drive pathogenic immunologic responses to luminal microbial antigens (Table 3,Figure 3). These mechanisms increase exposure of bacterial antigens to mucosal T cells or alter host immune responses to commensal bacteria.
      Table 3Theories of the Pathogenesis of IBD
      • 1
        Microbial pathogens induce intestinal inflammation
        • Traditional pathogen: Mycobacterium avium subspecies  paratuberculosis
          • Chiodini R.J.
          Crohn’s disease and the mycobacterioses: a review and comparison of two disease entities.
          • Sartor R.B.
          Does Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease?.
        • Functional alterations of commensal bacteria:   enteroadherent/invasive Escherichia coli, toxigenic   Bacteroides fragilis or Staphylococcus aureus
          • Darfeuille-Michaud A.
          • Neut C.
          • Barnich N.
          • et al.
          Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease.
          • Rabizadeh S.
          • Rhee K.J.
          • Wu S.
          • et al.
          Enterotoxigenic Bacteroides fragilis: a potential instigator of colitis.
          • Yang P.C.
          • Liu T.
          • Wang B.Q.
          • et al.
          Rhinosinusitis derived Staphylococcal enterotoxin B possibly associates with pathogenesis of ulcerative colitis.
      • 2
        Dysbiosis of commensal microbiota
        • Decreased ratio of protective/aggressive commensal   bacterial species
          • Frank D.N.
          • St. Amand A.L.
          • Feldman R.A.
          • et al.
          Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
        • Decreased species that produce short-chain fatty acids
          • Frank D.N.
          • St. Amand A.L.
          • Feldman R.A.
          • et al.
          Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
        • Overgrowth of aggressive commensal microbial species:   hydrogen sulfide reducing bacteria, Candida albicans
          • Smith F.M.
          • Coffey J.C.
          • Kell M.R.
          • et al.
          A characterization of anaerobic colonization and associated mucosal adaptations in the undiseased ileal pouch.
      • 3
        Host genetic defects in containing commensal microbiota
        • Defective bacterial killing: intracellular killing, secreted   antimicrobial peptides
          • Wehkamp J.
          • Salzman N.H.
          • Porter E.
          • et al.
          Reduced Paneth cell α-defensins in ileal Crohn’s disease.
          • Rioux J.D.
          • Xavier R.J.
          • Taylor K.D.
          • et al.
          Genome-wide association study identifies new susceptibility loci for Crohn’s disease and implicates autophagy in disease pathogenesis.
        • Defective mucosal barrier function
          • Olson T.S.
          • Reuter B.K.
          • Scott K.G.
          • et al.
          The primary defect in experimental ileitis originates from a nonhematopoietic source.
          • Hermiston M.L.
          • Gordon J.I.
          Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin.
      • 4
        Defective host immunoregulation
        • Ineffective epithelial homeostatic mechanisms
          • Nenci A.
          • Becker C.
          • Wullaert A.
          • et al.
          Epithelial NEMO links innate immunity to chronic intestinal inflammation.
        • Abnormal antigen processing/presentation
          • Rioux J.D.
          • Xavier R.J.
          • Taylor K.D.
          • et al.
          Genome-wide association study identifies new susceptibility loci for Crohn’s disease and implicates autophagy in disease pathogenesis.
          • Liu B.
          • Holt L.
          • Sartor R.B.
          APC regulate TH1/TH17-mediated colitis in IL-10 deficient mice.
        • Defective regulatory function: innate, acquired (loss of   tolerance)
          • Kraus T.A.
          • Toy L.
          • Chan L.
          • et al.
          Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease.
          • Liu B.
          • Holt L.
          • Sartor R.B.
          APC regulate TH1/TH17-mediated colitis in IL-10 deficient mice.
          • Izcue A.
          • Coombes J.L.
          • Powrie F.
          Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation.
          • Beckwith J.
          • Cong Y.
          • Sundberg J.P.
          • et al.
          Cdcs1, a major colitogenic locus in mice, regulates innate and adaptive immune response to enteric bacterial antigens.
        • Overly aggressive T-cell responses to enteric microbiota
          • Kim S.C.
          • Tonkonogy S.L.
          • Albright C.A.
          • et al.
          Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria.
          • Kontoyiannis D.
          • Pasparakis M.
          • Pizarro T.T.
          • et al.
          Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies.
        • Autoimmune responses: molecular mimicry between   microbial/host antigens
          • Lamhonwah A.M.
          • Ackerley C.
          • Onizuka R.
          • et al.
          Epitope shared by functional variant of organic cation/carnitine transporter, OCTN1, Campylobacter jejuni and Mycobacterium paratuberculosis may underlie susceptibility to Crohn’s disease at 5q31.
          • Polymeros D.
          • Bogdanos D.P.
          • Day R.
          • et al.
          Does cross-reactivity between Mycobacterium avium paratuberculosis and human intestinal antigens characterize Crohn’s disease?.

       Microbial Pathogen

       Mycobacterium avium subspecies paratuberculosis

      Mycobacterium avium subspecies paratuberculosis (MAP) causes spontaneous granulomatous enterocolitis in ruminants such as cattle with clinical manifestations of diarrhea and wasting, making this obligate intracellular pathogen a credible etiologic agent of Crohn’s disease.
      • Chiodini R.J.
      Crohn’s disease and the mycobacterioses: a review and comparison of two disease entities.
      • Sartor R.B.
      Does Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease?.
      It was first cultured from resected Crohn’s disease tissues in 1984.
      • Chiodini R.J.
      • Van Kruiningen H.J.
      • Thayer W.R.
      • et al.
      Possible role of mycobacteria in inflammatory bowel disease I. An unclassified Mycobacterium species isolated from patients with Crohn’s disease.
      The vast majority of studies reports increased recovery or detection of MAP in Crohn’s disease tissues by slow growing culture, polymerase chain reaction (PCR) of an insertion element, IS900, FISH, or serology compared with ulcerative colitis or controls, although the reported detection rate in Crohn’s disease ranges from 0% to 100%.
      • Sartor R.B.
      Does Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease?.
      • Behr M.A.
      • Schurr E.
      Mycobacteria in Crohn’s disease: a persistent hypothesis.
      • Autschbach F.
      • Eisold S.
      • Hinz U.
      • et al.
      High prevalence of Mycobacterium avium subsp. paratuberculosis IS900 DNA in gut tissues from individuals with Crohn’s disease.
      However, a preliminary report of a National Institutes of Health-sponsored blinded study showed no difference in the rate of culture recovery by 2 independent laboratories
      • Sartor R.B.
      • Blumberg R.S.
      • Braun J.
      • et al.
      CCFA microbial-host interactions workshop: highlights and key observations.
      and no detection of MAP 16s rRNA.
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      Proposed mechanisms of transmission include infected milk, meat, and water. Some studies detected MAP in commercial milk samples,
      • Millar D.
      • Ford J.
      • Sanderson J.
      • et al.
      IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows’ milk in England and Wales.
      a major city water supply,
      • Mishina D.
      • Katsel P.
      • Brown S.T.
      • et al.
      On the etiology of Crohn’s disease.
      and even human breast milk,
      • Naser S.A.
      • Schwartz D.
      • Shafran I.
      Isolation of Mycobacterium avium subsp paratuberculosis from breast milk of Crohn’s disease patients.
      although a recent report failed to find epidemiologic support for exposure through contaminated milk or drinking water.
      • Abubakar I.
      • Myhill D.J.
      • Hart A.R.
      • et al.
      A case-control study of drinking water and dairy products in Crohn’s disease—further investigation of the possible role of Mycobacterium avium paratuberculosis.
      This obligate intracellular pathogen that evades killing within phagolysosomes
      • Rumsey J.
      • Valentine J.F.
      • Naser S.A.
      Inhibition of phagosome maturation and survival of Mycobacterium avium subspecies paratuberculosis in polymorphonuclear leukocytes from Crohn’s disease patients.
      has been postulated to selectively infect hosts with defective innate immune killing, such as NOD2 polymorphisms.
      • Sartor R.B.
      Does Mycobacterium avium subspecies paratuberculosis cause Crohn’s disease?.
      • Behr M.A.
      • Schurr E.
      Mycobacteria in Crohn’s disease: a persistent hypothesis.
      However, no association between MAP serologies and NOD2 polymorphisms was seen in a large population-based study in Manitoba
      • Bernstein C.N.
      • Wang M.H.
      • Sargent M.
      • et al.
      Testing the interaction between NOD-2 status and serological response to Mycobacterium paratuberculosis in cases of inflammatory bowel disease.
      or between MAP cultures and NOD2 polymorphisms in Sardinia.
      • Sechi L.A.
      • Gazouli M.
      • Ikonomopoulos J.
      • et al.
      Mycobacterium avium subsp. paratuberculosis, genetic susceptibility to Crohn’s disease, and Sardinians: the way ahead.
      Furthermore, MAP was not preferentially recovered from ileal Crohn’s disease,
      • Autschbach F.
      • Eisold S.
      • Hinz U.
      • et al.
      High prevalence of Mycobacterium avium subsp. paratuberculosis IS900 DNA in gut tissues from individuals with Crohn’s disease.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      as would be expected if NOD2 patients were infected. Similarly, polymorphisms in the natural resistance-associated macrophage protein-1 (NRAMP-1), which is associated with M tuberculosis infection, were not associated with MAP detection.
      • Sechi L.A.
      • Gazouli M.
      • Sieswerda L.E.
      • et al.
      Relationship between Crohn’s disease, infection with Mycobacterium avium subspecies paratuberculosis and SLC11A1 gene polymorphisms in Sardinian patients.
      Finally, despite uncontrolled reports of long-standing cures of Crohn’s disease by triple antimycobacterial antibiotics,
      • Gui G.P.
      • Thomas P.R.
      • Tizard M.L.
      • et al.
      Two-year-outcomes analysis of Crohn’s disease treated with rifabutin and macrolide antibiotics.
      a well-designed, 2-year prospective trial of clarithromycin, rifabutin, and ethambutol failed to show sustained response.
      • Selby W.
      • Pavli P.
      • Crotty B.
      • et al.
      Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn’s disease.
      These studies and lack of potentiation of disease by corticosteroids and anti-TNF antibodies suggest that MAP is not the causative agent of most Crohn’s disease patients, although infection of a subset of patients with intracellular killing defects caused by ATG 16L1, NCF4, or other genes yet to be reported needs to be investigated.

       Functional changes in commensal bacteria

      Barnich and Darfeuille-Michaud described adherent/invasive E coli (AIEC) that persist within epithelial cells and macrophages and selectively colonize the ileum of Crohn’s disease patients.
      • Barnich N.
      • Darfeuille-Michaud A.
      Adherent-invasive Escherichia coli and Crohn’s disease.
      AIEC were recovered from 65% of chronically inflamed ileal resections and 36% of mucosal biopsy specimens of the neoterminal ileum of patients with early postresection recurrent Crohn’s disease and 22% of endoscopically normal Crohn’s disease biopsy specimens in contrast to 3.7% of colonic biopsy specimens from the same patients and 6% of normal control ileal biopsy specimens.
      • Neut C.
      • Bulois P.
      • Desreumaux P.
      • et al.
      Changes in the bacterial flora of the neoterminal ileum after ileocolonic resection for Crohn’s disease.
      • Darfeuille-Michaud A.
      • Boudeau J.
      • Bulois P.
      • et al.
      High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease.
      These observations were confirmed by Baumgart et al,
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      who documented adherent E coli in 38% of patients with active ileal Crohn’s disease but low recovery in colonic Crohn’s disease. Recovered E coli strains adhered to and invaded epithelial cell lines and expressed virulence factors that more closely related to avian and uropathic E coli strains.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      In addition, AIEC persist and even replicate within macrophages and secrete large amounts of TNF.
      • Glasser A.L.
      • Boudeau J.
      • Barnich N.
      • et al.
      Adherent invasive Escherichia coli strains from patients with Crohn’s disease survive and replicate within macrophages without inducing host cell death.
      Combined with defective antimicrobial peptide function in Crohn’s disease,
      • Wehkamp J.
      • Salzman N.H.
      • Porter E.
      • et al.
      Reduced Paneth cell α-defensins in ileal Crohn’s disease.
      • Nuding S.
      • Fellermann K.
      • Wehkamp J.
      • et al.
      Reduced mucosal antimicrobial activity in Crohn’s disease of the colon.
      these functional changes can help explain the dramatic increase in mucosally associated Enterobacteriaceae species, including E coli, in Crohn’s disease.
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      AIEC adhesion to epithelial cells depends on type 1 pili expression by bacteria and CEACAM 6 expression on the apical surface of ileal epithelial cells
      • Barnich N.
      • Carvalho F.A.
      • Glasser A.L.
      • et al.
      CEACAM6 acts as a receptor for adherent-invasive E coli, supporting ileal mucosa colonization in Crohn’s disease.
      (Figure 4). Epithelial CEACAM 6 expression is stimulated by IFN-γ, TNF, and AIEC adherence and is selectively increased in ileal Crohn’s disease, providing a mechanism for the preferential recovery of AIEC from this area. Molecular deletion studies have implicated a number of genes (fliC, NIP1, OmpC, yfg L) that regulate type 1 pilus expression and mediate epithelial cell adherence/invasion and another gene (htr A) that mediates intracellular replication of AIEC strains within macrophages.
      • Mow W.S.
      • Landers C.J.
      • Steinhart A.H.
      • et al.
      High-level serum antibodies to bacterial antigens are associated with antibiotic-induced clinical remission in Crohn’s disease: a pilot study.
      • Rolhion N.
      • Barnich N.
      • Claret L.
      • et al.
      Strong decrease in invasive ability and outer membrane vesicle release in Crohn’s disease-associated adherent-invasive Escherichia coli strain LF82 with the yfgL gene deleted.
      • Boudeau J.
      • Barnich N.
      • Darfeuille-Michaud A.
      Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn’s disease is involved in bacterial invasion of intestinal epithelial cells.
      • Rolhion N.
      • Carvalho F.A.
      • Darfeuille-Michaud A.
      OmpC and the sigma(E) regulatory pathway are involved in adhesion and invasion of the Crohn’s disease-associated Escherichia coli strain LF82.
      E coli strains from ileal Crohn’s disease do not produce toxins but express several virulence genes (col V, hcp, eae, stx 1, rat A) that mediate epithelial adherence and iron acquisition and closely resemble genes identified in enteroadherent/invasive E coli strains isolated from Boxer dogs with chronic granulomatous colitis.
      • Simpson K.W.
      • Dogan B.
      • Rishniw M.
      • et al.
      Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs.
      These genes are found in uropathic E coli and avian infections but not in human enteric pathogenic E coli strains. Although serotypes of E coli strains from the Boxer dogs and French, American, and Canadian Crohn’s disease patients are different, they preferentially belong to phylogenic group B2 and D that are associated with uropathic and avian virulent E coli strains.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      • Simpson K.W.
      • Dogan B.
      • Rishniw M.
      • et al.
      Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs.
      • Kotlowski R.
      • Bernstein C.N.
      • Sepehri S.
      • et al.
      High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease.
      E coli strains likely acquire these virulence genes by horizontal transfer from pathogens.
      Figure thumbnail gr4
      Figure 4Pathogenesis of epithelial attachment and tissue injury with adherent/invasive E coli (AIEC). AIEC induce carcinoembryonic antigen cell adhesion molecule (CEACAM) 6, either directly or by stimulating production of TNF and interferon γ (IFNγ), and attach, invade, and stimulate macrophages and T cells in the lamina propria. Modified from Abraham C and Cho J, N Engl J Med 2007;357:708–710.
      Although AIEC do not produce toxins, enterotoxins from pathogens and commensal bacterial species can cause clinical and experimental intestinal inflammation. Clostridium difficile toxin can reactivate quiescent IBD
      • Issa M.
      • Vijayapal A.
      • Graham M.B.
      • et al.
      Impact of Clostridium difficile on inflammatory bowel disease.
      and induce acute experimental epithelial injury.
      • Kim H.
      • Rhee S.H.
      • Pothoulakis C.
      • et al.
      Inflammation and apoptosis in Clostridium difficile enteritis is mediated by PGE(2) up-regulation of Fas ligand.
      Likewise, enterotoxigenic Bacteroides fragilis can induce experimental colitis and IL-17 production.
      • Rabizadeh S.
      • Rhee K.J.
      • Wu S.
      • et al.
      Enterotoxigenic Bacteroides fragilis: a potential instigator of colitis.
      Sinus infection by enterotoxin B-producing Staphylococci has been postulated to stimulate inflammation in ulcerative colitis patients.
      • Yang P.C.
      • Liu T.
      • Wang B.Q.
      • et al.
      Rhinosinusitis derived Staphylococcal enterotoxin B possibly associates with pathogenesis of ulcerative colitis.
      Functional alterations other than toxin elaboration, such as superoxide generation
      • Huycke M.M.
      • Abrams V.
      • Moore D.R.
      Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA.
      and quorum sensing,
      • Nallapareddy S.R.
      • Singh K.V.
      • Sillanpaa J.
      • et al.
      Endocarditis and biofilm-associated pili of Enterococcus faecalis.
      can affect the ability of bacteria to induce experimental inflammation. In addition, E coli isolated from IBD patients express serine protease autotransporters similar to uropathic strains that are postulated to facilitate mucosal invasion.
      • Kotlowski R.
      • Bernstein C.N.
      • Sepehri S.
      • et al.
      High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease.
      In unpublished studies, we demonstrated that serine proteases, such as gelatinase, promote experimental colitis and in vitro bacterial translocation across epithelial monolayers (Haller D, Liu B, and Sartor RB, unpublished data).
      Virulence factors and toxin production must be specifically investigated because bacterial culture and 16s ribosomal DNA or RNA analysis will only identify bacterial species rather than pathophysiologically important functional changes. Understanding the molecular mechanisms of reciprocal regulation of bacterial and host epithelial genes is a key area of scientific investigation with both physiologic and pathophysiologic relevance.

       Dysbiosis

       Changes in composition

      Numerous investigators have used molecular techniques to demonstrate changes in the composition of the mucosally associated and fecal microbiota in patients with Crohn’s disease, ulcerative colitis, and pouchitis.
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      • Gophna U.
      • Sommerfeld K.
      • Gophna S.
      • et al.
      Differences between tissue-associated intestinal microfloras of patients with Crohn’s disease and ulcerative colitis.
      • Manichanh C.
      • Rigottier-Gois L.
      • Bonnaud E.
      • et al.
      Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach.
      • Iwaya A.
      • Iiai T.
      • Okamoto H.
      • et al.
      Change in the bacterial flora of pouchitis.
      • Conte M.P.
      • Schippa S.
      • Zamboni I.
      • et al.
      Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease.
      • Martinez-medina M.
      • Aldeguer X.
      • Gonzalez-Huix F.
      • et al.
      Abnormal microbiota composition in the ileocolonic mucosa of Crohn’s disease patients as revealed by polymerase chain reaction-denaturing gradient gel electrophoresis.
      Most studies demonstrate decreased microbial diversity in active IBD, increased numbers of Enterobacteriaceae, including E coli, and decreased Firmicutes, with selectively decreased Clostridium species. A comprehensive study of 190 resected tissue samples by Frank et al showed decreased numbers of the phyla Firmicutes and Bacteroidetes with concomitant increases in Proteobacteria and Actinobacteria.
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      Decreases in Firmicutes were largely due to decreases in Clostridium XIVa and IV groups within the Lachnospiraceae subgroups. The absolute numbers of Proteobacteria, including Enterobacteriaceae such as E coli, were not increased, although their relative numbers were substantially more abundant because of diminished Lachnospiraceae and Bacteroidetes groups that predominate in normal controls.
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      No fundamental differences were seen between Crohn’s disease and ulcerative colitis specimens. Decreased Clostridia concentrations in Crohn’s disease are a common feature of many studies,
      • Hooper L.V.
      • Gordon J.I.
      Commensal host-bacterial relationships in the gut.
      • Gophna U.
      • Sommerfeld K.
      • Gophna S.
      • et al.
      Differences between tissue-associated intestinal microfloras of patients with Crohn’s disease and ulcerative colitis.
      • Manichanh C.
      • Rigottier-Gois L.
      • Bonnaud E.
      • et al.
      Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach.
      • Martinez-medina M.
      • Aldeguer X.
      • Gonzalez-Huix F.
      • et al.
      Abnormal microbiota composition in the ileocolonic mucosa of Crohn’s disease patients as revealed by polymerase chain reaction-denaturing gradient gel electrophoresis.
      • Swidsinski A.
      • Weber J.
      • Loening-Baucke V.
      • et al.
      Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease.
      although the decreased Bacteroides in 2 recent studies
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      conflict with Bacteroides species being predominant organisms in mucosal IBD specimens in several FISH studies
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Swidsinski A.
      • Weber J.
      • Loening-Baucke V.
      • et al.
      Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease.
      • Kleessen B.
      • Kroesen A.J.
      • Buhr H.J.
      • et al.
      Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls.
      and molecular studies.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      Similarly, decreased total numbers of mucosally adherent bacteria in the recent 16s rDNA studies
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      are opposite those of denaturing gradient gel electrophoresis and FISH-based observations.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      However, almost all investigators agree that E coli comprise an increased proportion of the fecal and mucosally associated microbiota of Crohn’s disease patients,
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      • Kotlowski R.
      • Bernstein C.N.
      • Sepehri S.
      • et al.
      High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease.
      invade the mucosa,
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Sasaki M.
      • Sitaraman S.V.
      • Babbin B.A.
      • et al.
      Invasive Escherichia coli are a feature of Crohn’s disease.
      and are present within granulomas
      • Cartun R.W.
      • Van Kruiningen H.J.
      • Pedersen C.A.
      • et al.
      An immunocytochemical search for infectious agents in Crohn’s disease.
      • Ryan P.
      • Kelly R.G.
      • Lee G.
      • et al.
      Bacterial DNA within granulomas of patients with Crohn’s disease—detection by laser capture microdissection and PCR.
      and adjacent to fistulae and ulcers.
      • Cartun R.W.
      • Van Kruiningen H.J.
      • Pedersen C.A.
      • et al.
      An immunocytochemical search for infectious agents in Crohn’s disease.
      • Liu Y.
      • Van Kruiningen H.J.
      • West A.B.
      • et al.
      Immunocytochemical evidence of Listeria, Escherichia coli, and Streptococcus antigens in Crohn’s disease.
      However, no consensus has emerged on general differences between the microbiota of Crohn’s disease and ulcerative colitis and whether these changes are primary vs secondary events. Some studies show similarities in Crohn’s disease and ulcerative colitis,
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Kotlowski R.
      • Bernstein C.N.
      • Sepehri S.
      • et al.
      High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease.
      whereas other experienced investigators show disease-specific changes.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      • Darfeuille-Michaud A.
      • Boudeau J.
      • Bulois P.
      • et al.
      High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease.
      • Gophna U.
      • Sommerfeld K.
      • Gophna S.
      • et al.
      Differences between tissue-associated intestinal microfloras of patients with Crohn’s disease and ulcerative colitis.
      • Sasaki M.
      • Sitaraman S.V.
      • Babbin B.A.
      • et al.
      Invasive Escherichia coli are a feature of Crohn’s disease.
      • Sokol H.
      • Lepage P.
      • Seksik P.
      • et al.
      Molecular comparison of dominant microbiota associated with injured versus healthy mucosa in ulcerative colitis.
      Likewise, some investigators describe differences between microbial populations in active and inactive disease,
      • Swidsinski A.
      • Ladhoff A.
      • Pernthaler A.
      • et al.
      Mucosal flora in inflammatory bowel disease.
      • Baumgart M.
      • Dogan B.
      • Rishniw M.
      • et al.
      Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum.
      • Darfeuille-Michaud A.
      • Boudeau J.
      • Bulois P.
      • et al.
      High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease.
      whereas others see no difference between active and inactive IBD.
      • Bibiloni R.
      • Mangold M.
      • Madsen K.L.
      • et al.
      The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients.
      Studies in rodents indicate that bacterial composition changes with colonic inflammation and/or infection,
      • Swidsinski A.
      • Loening-Baucke V.
      • Lochs H.
      • et al.
      Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice.
      • Tsang J.
      • Brown R.S.
      • Andersen G.L.
      • et al.
      Selective expansion of colitogenic commensal bacterial species in SPF IL-10-/- mice.
      • Lupp C.
      • Robertson M.L.
      • Wickham M.E.
      • et al.
      Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae.
      • Bibiloni R.
      • Simon M.A.
      • Albright C.
      • et al.
      Analysis of the large bowel microbiota of colitic mice using PCR/DGGE.
      suggesting that the inflamed mucosa and/or the altered inflammatory milieu selectively affect growth and adherence of different bacterial species. Several studies document selective expansion of Enterobacteriaceae,
      • Tsang J.
      • Brown R.S.
      • Andersen G.L.
      • et al.
      Selective expansion of colitogenic commensal bacterial species in SPF IL-10-/- mice.
      • Lupp C.
      • Robertson M.L.
      • Wickham M.E.
      • et al.
      Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae.
      whereas others describe expansion of Bacteroides species.
      • Swidsinski A.
      • Loening-Baucke V.
      • Lochs H.
      • et al.
      Spatial organization of bacterial flora in normal and inflamed intestine: a fluorescence in situ hybridization study in mice.
      • Bibiloni R.
      • Simon M.A.
      • Albright C.
      • et al.
      Analysis of the large bowel microbiota of colitic mice using PCR/DGGE.
      To date, no consistent differences in the probiotic genera Lactobacillus and Bifidobacterium have been identified.
      • Iwaya A.
      • Iiai T.
      • Okamoto H.
      • et al.
      Change in the bacterial flora of pouchitis.
      • Scanlan P.D.
      • Shanahan F.
      • O’Mahony C.
      • et al.
      Culture-independent analyses of temporal variation of the dominant fecal microbiota and targeted bacterial subgroups in Crohn’s disease.
      Most investigators have characterized IBD by bacterial phylotype, although the fungal diversity of the distal intestine is quite complex.
      • Scupham A.J.
      • Presley L.L.
      • Wei B.
      • et al.
      Abundant and diverse fungal microbiota in the murine intestine.
      Candida albicans stimulates anti-Saccharomyces cerevisiae antibodies (ASCA),
      • Standaert-Vitse A.
      • Jouault T.
      • Vandewalle P.
      • et al.
      Candida albicans is an immunogen for anti-Saccharomyces cerevisiae antibody markers of Crohn’s disease.
      suggesting that fungi could serve as an antigenic source for pathogenic immune responses.

       Metabolic consequences of altered intestinal microbiota

      Altered composition of luminal microbiota can lead to physiologically important changes in the intestinal environment. Enteric bacteria metabolize dietary constituents to produce both beneficial and detrimental products. For example, Clostridia and Bacteroides species preferentially produce butyrate and other SCFA that are the preferred energy substrates of colonic epithelial cells. Therefore, decreased concentrations of Clostridium groups IV and XIVa
      • Frank D.N.
      • St. Amand A.L.
      • Feldman R.A.
      • et al.
      Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases.
      could explain the observed decreased SCFA concentrations in fecal extracts of IBD patients.
      • Marchesi J.R.
      • Holmes E.
      • Khan F.
      • et al.
      Rapid and noninvasive metabonomic characterization of inflammatory bowel disease.
      Similarly, overgrowth of sulfate-reducing bacterial species in ulcerative colitis and ileal J pouches
      • Smith F.M.
      • Coffey J.C.
      • Kell M.R.
      • et al.
      A characterization of anaerobic colonization and associated mucosal adaptations in the undiseased ileal pouch.
      could enhance hydrogen sulfide production, which blocks butyrate utilization by colonocytes.
      • Roediger W.E.
      • Duncan A.
      • Kapaniris O.
      • et al.
      Reducing sulfur compounds of the colon impair colonocyte nutrition: implications for ulcerative colitis.
      Both pathways are relevant to the hypothesis that ulcerative colitis is the consequence of epithelial nutrient deficiency.
      • Roediger W.E.
      • Duncan A.
      • Kapaniris O.
      • et al.
      Reducing sulfur compounds of the colon impair colonocyte nutrition: implications for ulcerative colitis.
      Future studies should correlate luminal metabolic alterations, quantitative changes in enteric microbial composition, and expression of metabolically relevant genes by intestinal bacteria. Bacteria profoundly influence gut metabolism by interacting with human metabolic pathways.
      • Schmidt C.
      • Giese T.
      • Ludwig B.
      • et al.
      Expression of interleukin-12-related cytokine transcripts in inflammatory bowel disease: elevated interleukin-23p19 and interleukin-27p28 in Crohn’s disease but not in ulcerative colitis.
      Compositional and functional changes in the enteric microbiome have been linked to the pathogenesis of obesity,
      • Turnbaugh P.J.
      • Ley R.E.
      • Mahowald M.A.
      • et al.
      An obesity-associated gut microbiome with increased capacity for energy harvest.
      • Ley R.E.
      • Turnbaugh P.J.
      • Klein S.
      • et al.
      Microbial ecology: human gut microbes associated with obesity.
      establishing a precedent for metabolic changes contributing to IBD.

       Defective Mucosal Barrier Function and Microbial Killing

      Mucosal homeostasis depends on efficient exclusion of commensal bacteria by secretion of luminal antimicrobial peptides and complexing IgA/IgM, a relatively impermeable mucosal barrier, extrusion of xenotoxins, rapid repair of epithelial defects, down-regulation of innate and adaptive immune responses to bacteria and secondary phagocytosis, and killing of bacteria that translocate across the epithelial barrier (Figure 3C) (see Physiologic Microbial/Host Interactions section).
      • Sartor R.B.
      Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis.
      • Clavel T.
      • Haller D.
      Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: implications for chronic inflammation.
      Defects in any of these homeostatic mechanisms could result in enhanced microbial antigenic exposure that overwhelms immune tolerance, leading to pathogenic T-cell responses and chronic intestinal inflammation.

       Mucosal barrier defects

      Enhanced mucosal permeability is a characteristic of Crohn’s disease and ulcerative colitis, but controversy exists over the primary or secondary origins of these changes. Permeability defects, either spontaneous or after exposure to nonsteroidal anti-inflammatory drugs in asymptomatic relatives,
      • Hollander D.
      • Vadheim C.M.
      • Brettholz E.
      • et al.
      Increased intestinal permeability in patients with Crohn’s disease and their relatives A possible etiologic factor.
      • Hilsden R.J.
      • Meddings J.B.
      • Sutherland L.R.
      Intestinal permeability changes in response to acetylsalicylic acid in relatives of patients with Crohn’s disease.
      support a primary genetically detrimental defect. Molecular mechanisms for increased permeability include up-regulation of pore-forming Claudin 2, down-regulation and redistribution of tight junction components Claudins 5 and 6, and increased epithelial apoptosis in mild to moderate Crohn’s disease.
      • 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.
      TNF up-regulated Claudin 2. IL-13, which is increased in ulcerative colitis, also induced Claudin 2 expression and epithelial apoptosis.
      • Heller F.
      • Florian P.
      • Bojarski C.
      • et al.
      Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution.
      Increased mucosal permeability is a characteristic of preclinical stages of colitis in IL-10−/− mice
      • Madsen K.L.
      • Malfair D.
      • Gray D.
      • et al.
      Interleukin-10 gene-deficient mice develop a primary intestinal permeability defect in response to enteric microflora.
      and ileitis in Samp-1/Yit mice.
      • Olson T.S.
      • Reuter B.K.
      • Scott K.G.
      • et al.
      The primary defect in experimental ileitis originates from a nonhematopoietic source.
      In Samp-1/Yit and mdr-1-deficient mice, bone marrow reconstitution studies suggest that the genetic defect is due to epithelial defects.
      • Olson T.S.
      • Reuter B.K.
      • Scott K.G.
      • et al.
      The primary defect in experimental ileitis originates from a nonhematopoietic source.
      • Panwala C.M.
      • Jones J.C.
      • Viney J.L.
      A novel model of inflammatory bowel disease: mice deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis.
      A primary role for epithelial barrier function is unequivocally demonstrated by focal intestinal inflammation that corresponds to epithelial expression of dominant negative N-cadherin in transgenic mice using an epithelial promoter.
      • Hermiston M.L.
      • Gordon J.I.
      Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin.
      Both the multidrug resistance-1 (mdr-1) gene that encodes p-glycoprotein and OCTN 1/2, an organic cadion transporter in the IBD 5 gene complex, are postulated to clear microbial xenotoxins that permeate epithelial cells. Mdr-1 polymorphisms are associated with ulcerative colitis,
      • Ho G.T.
      • Nimmo E.R.
      • Tenesa A.
      • et al.
      Allelic variations of the multidrug resistance gene determine susceptibility and disease behavior in ulcerative colitis.
      mdr-1 deletion induces murine colitis,
      • Panwala C.M.
      • Jones J.C.
      • Viney J.L.
      A novel model of inflammatory bowel disease: mice deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis.
      and IBD 5 polymorphisms are associated with Crohn’s disease.
      • Rioux J.D.
      • Daly M.
      • Silverberg M.
      • et al.
      Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn’s disease.
      The importance of effective barrier exclusion is demonstrated by increased translocation of luminal bacteria, induction of pathogenic T-cell responses, and onset of severe colitis in mice with intestinal epithelial cell-targeted deletion of IKKγ (NEMO).
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      These mice exhibited increased epithelial apoptosis and disrupted epithelial integrity as a consequence of blocking epithelial NF-κB signaling. Spontaneous colitis in Muc-2-deficient mice
      • van der Sluis M.
      • De Koning B.A.
      • De Bruijn A.C.
      • et al.
      Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection.
      and potentiated DSS-induced colitis in intestinal trefoil factor knockout mice illustrates the importance of the mucus barrier.
      • Mashimo H.
      • Wu D.C.
      • Podolsky D.K.
      • et al.
      Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.

       Defective microbial killing

      Crohn’s disease patients exhibit defective microbial killing that results in increased exposure to commensal bacteria and activation of compensatory pathogenic T cells.
      • Korzenik J.R.
      Is Crohn’s disease due to defective immunity?.
      Crohn’s disease patients have defective antimicrobial peptide production, most notably α defensin 5 in ileal disease
      • Wehkamp J.
      • Salzman N.H.
      • Porter E.
      • et al.
      Reduced Paneth cell α-defensins in ileal Crohn’s disease.
      and human β defensin 2 copy numbers in Crohn’s colitis
      • Fellermann K.
      • Stange D.E.
      • Schaeffeler E.
      • et al.
      A chromosome 8 gene-cluster polymorphism with low human β-defensin 2 gene copy number predisposes to Crohn’s disease of the colon.
      with functional abnormalities in killing Bacteroides vulgatus, E coli and Enterococcus faecalis.
      • Nuding S.
      • Fellermann K.
      • Wehkamp J.
      • et al.
      Reduced mucosal antimicrobial activity in Crohn’s disease of the colon.
      NOD2 polymorphisms in Crohn’s disease are associated with selective decreased α defensin production by Paneth cells
      • Wehkamp J.
      • Salzman N.H.
      • Porter E.
      • et al.
      Reduced Paneth cell α-defensins in ileal Crohn’s disease.
      and defective clearance of intracellular pathogens by colonic epithelial cells.
      • Hisamatsu T.
      • Suzuki M.
      • Reinecker H.C.
      • et al.
      CARD15/NOD2 functions as an anti-bacterial factor in human intestinal epithelial cells.
      Decreased α defensin production in ileal Crohn’s disease is a consequence of reduced expression of Tcf-4, a Wnt-signaling pathway transcription factor, whose activity is independent of NOD2 genotype.
      • Wehkamp J.
      • Wang G.
      • Kubler I.
      • et al.
      The Paneth cell {α}-defensin deficiency of ileal Crohn’s disease is linked to Wnt/Tcf-4.
      Reduced expression of Tcf-4 in heterozygous mice led to decreased Paneth cell production of α defensins and bacterial killing.
      • Wehkamp J.
      • Wang G.
      • Kubler I.
      • et al.
      The Paneth cell {α}-defensin deficiency of ileal Crohn’s disease is linked to Wnt/Tcf-4.
      Mutations in 2 additional Crohn’s disease-related genes that regulate intracellular bacterial killing, ATG 16L1 and NCF 2, further implicate defective innate immune responses in Crohn’s disease.
      • Hampe J.
      • Franke A.
      • Rosenstiel P.
      • et al.
      A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn’s disease in ATG16L1.
      • Rioux J.D.
      • Xavier R.J.
      • Taylor K.D.
      • et al.
      Genome-wide association study identifies new susceptibility loci for Crohn’s disease and implicates autophagy in disease pathogenesis.
      ATG 16L1 mediates autophagy, which contributes to killing and processing of intracellular bacteria. Autophagy is stimulated by a variety of pathways, including TLR 4 signaling through receptor-interacting protein (RIP-1) and p38 mitogen-activated protein kinase, independent of Myd 88.
      • Xu Y.
      • Jagannath C.
      • Liu X.-D.
      • et al.
      Toll-like receptor 4 is a sensor for autophagy associated with innate immunity.
      The importance of TLR signaling through Myd 88 in bacterial clearance is documented by severe colitis and bacteremia in Citrobacter rodentium-infected Myd 88-deficient mice, which exhibit defective epithelial repair, neutrophil recruitment, and adaptive immune responses.
      • Lebeis S.L.
      • Bommarius B.
      • Parkos C.A.
      • et al.
      TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium.
      A Crohn’s disease-like phenotype in some patients with primary functional defects in neutrophil killing demonstrates a fundamental role for bacterial killing by phagocytic cells.
      • Korzenik J.R.
      Is Crohn’s disease due to defective immunity?.
      Together, these observations support the concept that defective innate immune responses in Crohn’s disease could lead to defective bacterial clearance with activation of compensatory pathogenic T cells by microbial antigens.
      • Korzenik J.R.
      Is Crohn’s disease due to defective immunity?.
      • Marks D.J.
      • Harbord M.W.
      • MacAllister R.
      • et al.
      Defective acute inflammation in Crohn’s disease: a clinical investigation.

       Defective Immunoregulation

      Disruption of any of the coordinated homeostatic mechanisms in intestinal epithelial cells or lamina propria innate or adaptive immune cells described in the Physiologic Microbial/Host Interactions section can lead to pathogenic immune responses to commensal bacteria and subsequent chronic intestinal inflammation
      • Sartor R.B.
      Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis.
      (Figure 3D). Blockade of bacterial signaling through NF-κB in intestinal epithelial cells leads to spontaneous colitis and CD4+ T-cell activation in epithelial IKKγ (NEMO)-deficient mice
      • Nenci A.
      • Becker C.
      • Wullaert A.
      • et al.
      Epithelial NEMO links innate immunity to chronic intestinal inflammation.
      and potentiated, chemically induced colitis in TLR 4, TLR 9, or Myd 88-deficient mice.
      • Fukata M.
      • Chen A.
      • Klepper A.
      • et al.
      Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: role in proliferation and apoptosis in the intestine.
      • Lee J.
      • Mo J.H.
      • Katakura K.
      • et al.
      Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.
      • Rakoff-Nahoum S.
      • Paglino J.
      • Eslami-Varzaneh F.
      • et al.
      Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.
      Genetic polymorphisms of TLR 1, 2, and 6 are not associated with increased incidence of Crohn’s disease or ulcerative colitis, but function as modifying genes, with an increased frequency of extensive colitis.
      • Pierik M.
      • Joossens S.
      • Van Steen K.
      • et al.
      Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases.
      Likewise, a polymorphism in the CD14 promoter, which regulates LPS binding, is associated with steroid use in ulcerative colitis.
      • Griga T.
      • Wilkens C.
      • Schmiegel W.
      • et al.
      Association between the promoter polymorphism T/C at position −159 of the CD14 gene and anti-inflammatory therapy in patients with inflammatory bowel disease.
      Immunologic tolerance to bacterial antigens is primarily mediated by IL-10 and TGF-β, produced by regulatory T cells and CD25+ T reg cells. Targeted deletion of either IL-10, IL-10 receptor, TGF-β, SMAD 3, which mediates TGF-β signaling, or IL-2, which is the ligand for CD25, induces colitis.
      • Strober W.
      • Fuss I.
      • Mannon P.
      The fundamental basis of inflammatory bowel disease.
      • Hansen J.
      • Sartor R.B.
      Insights from animal models.
      Immune activation in IL-10−/− mice is completely dependent on commensal bacteria
      • Sellon R.K.
      • Tonkonogy S.
      • Schultz M.
      • et al.
      Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice.
      • Kim S.C.
      • Tonkonogy S.L.
      • Albright C.A.
      • et al.
      Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria.
      with partial bacterial dependency in IL-2−/− and TGF-β-deficient mice.
      • Schultz M.
      • Tonkonogy S.L.
      • Sellon R.K.
      • et al.
      IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation.
      Macrophages, DC, and B lymphocytes have immunoregulatory activities, including IL-10 production. Gp 96 stress protein, a recently described innate tolerogenic molecule, is decreased in Crohn’s disease.
      • Schreiter K.
      • Hausmann M.
      • Spoettl T.
      • et al.
      Glycoprotein (gp) 96 expression: induced during differentiation of intestinal macrophages but impaired in Crohn’s disease.
      Although there is no direct evidence of genetic or functional defects in regulatory T-cell function in human IBD, Crohn’s disease and ulcerative colitis patients exhibit T-cell
      • Duchmann R.
      • Kaiser I.
      • Hermann E.
      • et al.
      Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD).
      and serologic responses
      • Targan S.R.
      • Landers C.J.
      • Yang H.
      • et al.
      Antibodies to CBir1 flagellin define a unique response that is associated independently with complicated Crohn’s disease.
      • Mow W.S.
      • Vasiliauskas E.A.
      • Lin Y.C.
      • et al.
      Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease.
      to a wide variety of microbial antigens and fail to develop oral tolerance.
      • Kraus T.A.
      • Toy L.
      • Chan L.
      • et al.
      Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease.
      Although most evidence supports abnormally aggressive T-cell responses to commensal bacterial antigens as the pathogenic mechanisms of IBD rather than autoimmune responses, some data suggest molecular mimicry between microbial and host antigens.
      • Lamhonwah A.M.
      • Ackerley C.
      • Onizuka R.
      • et al.
      Epitope shared by functional variant of organic cation/carnitine transporter, OCTN1, Campylobacter jejuni and Mycobacterium paratuberculosis may underlie susceptibility to Crohn’s disease at 5q31.
      • Rahbar A.
      • Bostrom L.
      • Soderberg-Naucler C.
      Detection of cytotoxic CD13-specific autoantibodies in sera from patients with ulcerative colitis and Crohn’s disease.
      • Ebringer A.
      • Rashid T.
      • Tiwana H.
      • et al.
      A possible link between Crohn’s disease and ankylosing spondylitis via Klebsiella infections.