Epithelial barrier dysfunction

The intestinal epithelium regulates the flow of nutrients, ions and water between the lumen and underlying tissues, limiting contact between the host and the intraluminal quantities of exogenous antigens and microbes. In a healthy subject, the transepithelial transport of small amounts of food- and microbial-antigens participates in the induction of a homeostatic immune response that allows immune tolerance to such antigens⁽ Reference Izcue, Coombes and Powrie ^{33 ,} Reference Strobel and Mowat ^{34 )}, preventing the internalisation of both pathogenic and commensal microbes⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. However, epithelial barrier dysfunction can lead to the entry of excessive dietary or microbe-derived macromolecules, which are putative contributors to the pathogenesis of a spectrum of human diseases including food allergies, coeliac disease (CeD), IBD, autoimmune diseases and the metabolic syndrome⁽ Reference Goll and Granlund ^{35 ,} Reference Cani, Bibiloni and Knauf ^{36 )}. Improving intestinal barrier function, particularly the paracellular pathway, may form a therapeutic strategy for the treatment or prevention of diseases driven by luminal antigens. Understanding how antigens are transported across the epithelium in both healthy and diseased states may assist in the development of appropriate therapies⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}.

The transport of molecules across the intestinal mucosa occurs through two distinct mechanisms: paracellular diffusion through tight junctions (TJ) between adjacent intestinal epithelial cells, and transcellular transport involving the transcytosis of materials which may or may not be mediated by membrane receptors (illustrated in Fig. 1).

Fig. 1

Potential mechanisms of enterocytic uptake of peptides. (1) Paracellular: increased permeability of tight junctions may permit the passage of peptides. (2) Passive diffusion: cell-penetrating peptides are capable of transporting peptides as cargo. (3) Endocytosis: followed by endosomal release of the peptides. (4) Carrier-mediated transport: transport via the intestinal peptide transporter PEPT1 (H⁺/di- and tri-peptide symporter). Inside the enterocyte peptides can be hydrolysed to their constituent amino acids (AA) and transported across the basolateral membrane by specific AA transporters. It is thought that the transport of peptides across the basolateral membrane is mediated through other transporters such as those suggested by Terada et al. ⁽ Reference Terada, Sawada and Saito ^{322 ,} Reference Terada and Inui ^{323 )}, Shepherd et al. ⁽ Reference Shepherd, Lister and Affleck ^{324 )} and Irie et al.⁽ Reference Irie, Terada and Okuda ^{325 )}.

Paracellular transport

The paracellular pathway involves structures joining adjacent intestinal epithelial cells and delineated by TJ, adherens junctions and desmosomes⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. The rate-limiting factor in the paracellular diffusion of molecules are the TJ, a network of transmembrane proteins (claudins⁽ Reference Furuse, Fujita and Hiiragi ^{37 )}, occludin⁽ Reference Furuse, Hirase and Itoh ^{38 )}, junctional adhesion molecule A⁽ Reference Mandell, McCall and Parkos ^{39 )} and tricellulin⁽ Reference Ikenouchi, Furuse and Furuse ^{40 )}) which control the TJ plasticity and permeability. TJ form pores that range in diameter between 0·4 and 0·9 nm to 5 and 6 nm in villi or crypts, respectively. Occludin interacts with the zonula occludens proteins (ZO-1, ZO-2) that regulate the actomyosin ring. TJ allow the diffusion of mostly cations and inert small molecules less than 600 Da such as water-soluble peptides⁽ Reference Vermeirssen, Van Camp and Verstraete ^{41 )}.

Increased paracellular permeability has been observed in IBD together with abnormal TJ structure and a down-regulation and redistribution of several TJ proteins or the subjacent adherens junction proteins⁽ Reference Arrieta, Bistritz and Meddings ^{42 ,} Reference Madsen, Malfair and Gray ^{43 )}. A variety of pathological conditions can increase paracellular permeability in which molecules of greater size can diffuse non-specifically across the intestinal epithelial layer⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. The involvement of proinflammatory cytokines in the pathophysiology of IBD is well recognised⁽ Reference Raddatz, Bockemuhl and Ramadori ^{44 ,} Reference Stallmach, Giese and Schmidt ^{45 )}, and these cytokines are implicated in epithelial barrier dysfunction that leads to increased intestinal permeability along paracellular pathways⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. Both interferon-γ (IFN-γ) and TNF-α are elevated in the mucosa of IBD patients and contribute to a proinflammatory cascade that includes barrier disruption⁽ Reference Gassler, Rohr and Schneider ^{46 ,} Reference Kucharzik, Walsh and Chen ^{47 )}. Bruewer et al. ⁽ Reference Bruewer, Luegering and Kucharzik ^{48 )} have shown that the specificity of these mediators for the disruption of specific intercellular junctional proteins under inflammatory conditions is indicated, as the junctional proteins remained unaltered in non-inflamed areas of diseased tissue. Some of the mechanisms underlying the structural and functional modifications of TJ include the endocytosis of junctional proteins⁽ Reference Utech, Ivanov and Samarin ^{49 ,} Reference Matsuda, Kubo and Furuse ^{50 )}, epithelial apoptosis⁽ Reference Bojarski, Gitter and Bendfeldt ^{51 –} Reference Begue, Wajant and Bambou ^{53 )}, reduced transcription of TJ proteins⁽ Reference Mankertz, Tavalali and Schmitz ^{54 )} and the activation of myosin light-chain kinase phosphorylation to promote cytoskeletal contraction⁽ Reference Zolotarevsky, Hecht and Koutsouris ^{55 )}. Menard et al.⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )} suggest that myosin light-chain kinase is a key molecule that stimulates the opening of TJ by phosphorylating the myosin light chains. Increased claudin-2 expression increases the number of pores that allow the paracellular movement of small molecules. Myosin light-chain kinase activation and occludin down-regulation increase paracellular transport that is characteristic of both UC and CD⁽ Reference Turner ^{56 )}.

Probiotics are live bacteria which improve the health of the host beyond their inherent nutritional value⁽ Reference Mennigen, Nolte and Rijcken ^{57 )}. It has been hypothesised that probiotics have anti-inflammatory effects in human IBD and preserve intestinal epithelial integrity⁽ Reference Gionchetti, Rizzello and Morselli ^{58 )}. A number of in vitro studies using epithelial monolayers have demonstrated that probiotics have improved epithelial barrier function following Escherichia coli infection or incubation with proinflammatory cytokines⁽ Reference Bai, Pak and Lee ^{59 –} Reference Zyrek, Cichon and Helms ^{62 )}. Several in vivo studies have demonstrated that probiotic therapy may change the expression of TJ proteins⁽ Reference Mennigen, Nolte and Rijcken ^{57 ,} Reference Ukena, Singh and Dringenberg ^{63 )} and decrease paracellular permeability by increasing the phosphorylation of TJ proteins, such as ZO-1, claudin-1, or occludin in dextran-sodium sulfate-induced colitis (acute model) mice⁽ Reference Mennigen, Nolte and Rijcken ^{57 ,} Reference Ukena, Singh and Dringenberg ^{63 ,} Reference Miyauchi, Morita and Tanabe ^{64 )} and IL-10^–/– (chronic model) mice⁽ Reference Chen, Yang and Zhang ^{65 ,} Reference Ewaschuk, Diaz and Meddings ^{66 )}. However, the underlying molecular mechanisms by which probiotics diminish paracellular permeability remain unclear⁽ Reference Chen, Yang and Zhang ^{65 )}.

Transcellular transport

The transcellular transport of large particles, including microbes, has been ascribed to M cells located in the follicle-associated epithelium of Peyer’s patches⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )} and isolated lymphoid follicles in the distal part of the intestine⁽ Reference Keita, Gullberg and Ericson ^{67 ,} Reference Keita, Salim and Jiang ^{68 )}. Dendritic cells may sample bacteria in the intestinal lumen by extending dendrites between adjacent epithelial cells⁽ Reference Rescigno, Rotta and Valzasina ^{69 )}.

Lipid-soluble oliogopeptides may enter the enterocytes by passive diffusion where they are susceptible to hydrolytic degradation by cytosolic enzymes⁽ Reference Vermeirssen, Van Camp and Verstraete ^{41 )}. As large polar molecules (for example, peptide fragments >600 Da) cannot pass through the hydrophobic cell membrane of the enterocyte they may be captured by invagination of the apical membrane. Such vesicles normally fuse with lysosomes to form phagolysosomes, in which enzymic digestion of the macromolecules occurs. Only protein that escapes hydrolysis within these structures can be drawn through the enterocytes and cross the basolateral membrane.

The transcytosis of internalised vesicles may carry specifically bound ligands (receptor-mediated transcytosis), non-specifically adsorbed ligands (adsorptive transcytosis) or fluids (fluid-phase transcytosis) from the apical membrane across the cell to the basolateral membrane⁽ Reference Shimizu, Tsunogai and Arai ^{70 ,} Reference Shen, Wan and Ekrami ^{71 )}.

Epithelial cells of the intestinal mucosa also sample large molecules greater than 600 Da in size (such as food antigens) by endocytosis at the apical membrane and transcytosis toward the lamina propria. Within the epithelial cell, proteins/peptides are digested in acidic and lysosomal compartments before being released as amino acids or partly degraded products at the basolateral pole of the enterocytes⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. Partially degraded food antigens in early endosomes bind to major histocompatibility complex (MHC) class II molecules in an intracellular endocytotic compartment (MIIC). Inward invagination of MIIC compartments leads to the formation of exosomes, small membrane vesicles (40–90 nm) bearing MHC class II/peptide complexes at their surface⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. Antigen-loaded exosomes can then fuse with the basement membrane before being released into the extracellular medium to interact with local immune cells⁽ Reference Van Niel, Mallegol and Bevilacqua ^{72 )}. Exosome-bound peptides are much more potent than free peptides to interact with dendritic cells and stimulate peptide presentation to T cells⁽ Reference Mallegol, Van Niel and Lebreton ^{73 )}.

IgA is a dimeric protective mucosal immunoglobulin secreted into the intestinal lumen as secretory IgA (SIgA), and is the most representative immunoglobulin at the mucosal surface. The major role of SIgA in healthy subjects is to restrict potentially harmful food and microbial antigens from entering the intestinal epithelia⁽ Reference Fernandez, Pedron and Tournebize ^{74 )}. However, in some pathological conditions the abnormal apical-to-basal retrotransport of SIgA immune complexes can mediate the entry of noxious antigens into the intestinal epithelial cells⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. In CeD, an enteropathy induced by the abnormal activation of T cells by gluten-derived gliadin peptides, SIgA allows the transcytosis of IgA–gliadin immune complexes through the intestinal barrier via the transferrin receptor CD71 at the apical surface⁽ Reference Matysiak-Budnik, Moura and Arcos-Fajardo ^{75 )}. In healthy individuals this receptor is confined to the basolateral membrane and gliadin peptides are taken up by non-specific endocytosis and almost exclusively degraded by the intestinal epithelia. In active CeD the retrotransport of IgA–gliadin immune complexes most likely triggers exacerbated adaptive and innate immune responses that result in mucosal lesions⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}.

Significant quantities of IgG are also secreted at the mucosal surface which suggests a protective role. The transcytosis of IgG is mediated via the neonatal Fc receptor at the surface of intestinal epithelial cells in an acidic environment. At the basolateral side of the enterocyte the neutral pH induces the dissociation of IgG immune complexes from the receptor. Although the role of the neonatal Fc receptor in humans has not been established, it has been reported to mediate passive immunity in suckling rats from the maternal milk⁽ Reference Brambell ^{76 ,} Reference Jones and Waldmann ^{77 )}. Bacteria as well as food antigens can be transported as IgG immune complexes via neonatal Fc receptors, a mechanism that is most likely involved in the defence against intestinal pathogens⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}, and a process that has been reported for commensal E. coli ⁽ Reference Yoshida, Kobayashi and Kuo ^{78 )}. However, the entry of bacteria or degraded bacterial components (for example, flagellin⁽ Reference Kobayashi, Qiao and Yoshida ^{79 )}) might precipitate an inappropriate immune response such as chronic inflammation⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}.

The overexpression of CD23, the low-affinity receptor for IgE, has been detected on both the apical and basolateral membranes of patients with gastrointestinal diseases such as IgE-dependent bovine milk allergy and enteropathy, autoimmune enteropathy, CD and UC⁽ Reference Kaiserlian, Lachaux and Grosjean ^{80 )}. Such an overexpression of CD23 at the apical surface of enterocytes can drive the transport of IgE–allergen immune complexes from the intestinal lumen (bypassing lysosomal degradation) to the lamina propria, which then triggers mast cell degranulation and the rapid onset of an allergic inflammatory response in subepithelial immunoreactive cells⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}.

Soderholm et al. ⁽ Reference Soderholm, Peterson and Olaison ^{21 )} were the first to demonstrate that protein-sized macromolecules can permeate, at an increased rate, the non-inflamed ileal mucosa of patients with CD. Although they had previously demonstrated that molecular leakage can be induced by TJ dysfunction in patients with CD⁽ Reference Soderholm, Olaison and Hedman ^{81 )}, later studies indicated that the increased endosomal uptake of antigens was mediated by the proinflammatory cytokine TNF-α, which plays a pivotal role in CD pathogenesis. This demonstrates the importance of the transcellular route of antigen uptake in the barrier dysfunction of CD, and underlines the importance of immune–epithelial interaction in the development of mucosal inflammation⁽ Reference Soderholm, Streutker and Yang ^{82 )}, which suggests that anti-TNF-α therapy may produce positive effects in patients suffering from CD⁽ Reference D’Haens ^{83 )}.

Another cytokine abundant in CD is IFN-γ. This cytokine is also thought to enhance the transcytosis of macromolecules and was first demonstrated by Terpend et al. in 1998 using ³H-labelled horseradish peroxidase in an in vitro intestinal epithelial model. This suggests that IFN-γ may enhance both paracellular and transcellular leakage⁽ Reference Terpend, Boisgerault and Blaton ^{84 )}.

Antigen transcytosis into endocytic cell compartments and finally into the cytosol is strongly enhanced in rapid antigen uptake into the cytosol enterocyte (RACE) cells of patients with CD and UC. This suggests that in the inflamed intestine both paracellular and transcellular transport pathways are increased and contribute to overstimulation of the local immune system. Such overstimulation creates a vicious cycle in which luminal antigens reach the lamina propria, interact with subepithelial immunoreactive cells, and drive the secretion of permeability-enhancing factors that further increase epithelial barrier dysfunction⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}.

The microbiota

The host and intestinal microbiota are in a state of symbiotic mutualism, forming what Goodacre⁽ Reference Goodacre ^{85 )} terms a human–microbe hybrid, where the human genome and the microbiome collectively define a ‘superorganism’. In the healthy human gut the composition of the microbiome is unique and consists of hundreds to thousands of bacterial species in largely four different phyla: Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria⁽ Reference Wilson, Teft and Morse ^{86 )} although they are not distributed uniformly in number, species or metabolic activity. Diet and age are known to influence the composition of the gut microbiome⁽ Reference Gill, Pop and DeBoy ^{87 ,} Reference Wu, Chen and Hoffmann ^{88 )}. Although the intestinal tract is colonised by large numbers of commensal bacteria, intestinal infections and the translocation of bacterial proinflammatory antigens (for example, lipopolysaccharide and peptidoglycan) that can provoke NF-κB-dependent immune responses are uncommon in healthy individuals. However, the development of IBD appears to be dependent on the presence of a commensal microbiota, as mice raised in germ-free environments fail to develop IBD⁽ Reference Sartor ^{89 )}. While the classic interpretation of IBD pathogenesis is a loss of mucosal tolerance to proinflammatory bacteria, someone with IBD is more likely to contract an intestinal infection because of a weakened mucosal barrier, resulting from a defective immune defence system⁽ Reference Wehkamp and Stange ^{25 ,} Reference Matsuoka and Kanai ^{90 )}.

An excessive immune response to bacteria inhabiting the intestinal lumen and their degradation products is a common feature of IBD. Despite recent progress in defining factors that exacerbate or ameliorate these diseases, their precise causes remain poorly defined⁽ Reference Jia, Whitehead and Griffiths ^{91 )}. Nevertheless, there is broad agreement that luminal microbes are of particular relevance in the development of these pathologies⁽ Reference Gersemann, Wehkamp and Stange ^{6 )}. The composition of the microbiota is altered in IBD towards fewer anti-inflammatory and greater numbers of proinflammatory bacteria⁽ Reference Bibiloni, Mangold and Madsen ^{92 –} Reference Frank, Amand and Feldman ^{94 )}. The treatment of IBD is often accompanied by substantial changes in the composition of intestinal microbiota and related immunoglobulins⁽ Reference van der Waaij, Kroese and Visser ^{95 )}. However, no single group of bacteria has been implicated to be uniquely causally related to these diseases⁽ Reference Jia, Whitehead and Griffiths ^{91 )}. It has been suggested that a toxin, such as H₂O₂ produced by bacteria, might play a significant role in provoking intestinal inflammation⁽ Reference Jia, Whitehead and Griffiths ^{91 )}. Although many studies have failed to link sulfate-reducing bacteria (SRB) and IBD⁽ Reference Pitcher, Beatty and Cummings ^{96 –} Reference Carmen Collado and Sanz ^{101 )}, it has been proposed that SRB can exacerbate IBD by generating hydrogen sulfide, as well as depleting the production of beneficial butyrate⁽ Reference Shatalin, Shatalina and Mironov ^{102 ,} Reference Marquet, Duncan and Chassard ^{103 )}. Although the work of Jia et al.⁽ Reference Jia, Whitehead and Griffiths ^{91 )} demonstrated some changes in the number of SRB in IBD patients pre- and post-treatment, their data were not unequivocal.

While there is little disagreement that patients suffering from IBD have antibodies against several microbes and microbial antigens⁽ Reference Giaffer, Clark and Holdsworth ^{104 )}, a number of researchers suggest that dysbiosis of the intestinal microbiome is a causal factor of inflammation in IBD patients, without reference to the genetic predisposition of IBD patients⁽ Reference Gersemann, Wehkamp and Stange ^{6 ,} Reference Ingersoll, Ayyadurai and Charania ^{9 ,} Reference Charrier and Merlin ^{10 ,} Reference Sartor ^{89 ,} Reference Rescigno ^{105 ,} Reference Qin, Li and Raes ^{106 )}. However, although commensal intraluminal microbiota are essential for the development and maintenance of IBD there is little evidence to support the hypothesis that intestinal microbiota are the single causal factor of the disease. Although the defence against these microbes is compromised in IBD it may be reasoned that this is due to a defective innate barrier where the production of α-defensins is reduced in ileal CD, that β-defensins are decreased in colonic CD, and the mucus layer is deficient in UC⁽ Reference Gersemann, Wehkamp and Stange ^{6 )}.

There is little evidence that the numbers of bacteria in the large intestine of human subjects with IBD are any different from those of normal subjects. However, a disequilibrium of inflammatory and non-inflammatory components of the intestinal microbiota (dysbioses) is associated with IBD⁽ Reference Frank, Amand and Feldman ^{94 ,} Reference Qin, Li and Raes ^{106 –} Reference Sokol, Lay and Seksik ^{111 )}. Whether the changes in intestinal microbiota observed in IBD are simply a consequence of chronic inflammation and its treatment, or are necessary determinants of initiation and/or perpetuation of pathogenesis, is still open to question⁽ Reference Frank, Zhu and Sartor ^{18 )}. Microbiological investigations have so far failed to identify consistent alterations of microbiota composition in IBD patients relative to healthy controls⁽ Reference Sokol, Lay and Seksik ^{111 )}. While the commensal microbiota are largely tolerated by the mucosa and ignored by the systemic immune systems of normal hosts⁽ Reference Feng and Elson ^{112 –} Reference Slack, Hapfelmeier and Stecher ^{114 )}, they are essential drivers of pathogenic mucosal and systemic inflammatory responses in genetically predisposed subjects⁽ Reference Xavier and Podolsky ^{8 ,} Reference Sartor ^{89 ,} Reference Sartor ^{115 ,} Reference Abraham and Cho ^{116 )}.

Such dysbiosis, widely reported in connection with CD⁽ Reference Frank, Amand and Feldman ^{94 ,} Reference Qin, Li and Raes ^{106 –} Reference Sokol, Lay and Seksik ^{111 )}, is not solely the result of environmental effects, such as treatment history or diet. Complex interactions exist between the host genotype and the enteral microbial community⁽ Reference Frank, Zhu and Sartor ^{18 )}. Such interactions may arise as a consequence of direct genetic effects on microbial composition, perhaps through altered Paneth cell function⁽ Reference Cadwell, Patel and Maloney ^{5 ,} Reference Cadwell, Liu and Brown ^{117 ,} Reference Wehkamp, Salzman and Porter ^{118 )}, or as a direct result of the pathogenic process. However, the question of whether dysbiosis contributes to CD pathogenesis or is an innocuous by-product remains to be established. Furthermore, how dysfunction or inflammation of the mucosal barrier can lead to dysbiosis is also unclear. However, other than in exceptional cases, it is unlikely that disease-associated dysbioses will satisfy all of the criteria that have been proposed to prove causality⁽ Reference Hill ^{119 –} Reference Fredricks and Relman ^{121 )}.

In a study of forty twin pairs focusing on disease incidence in genetically matched individuals that were concordant or discordant for either CD or UC, it was demonstrated that the microbiota of CD patients differed from those of healthy individuals, whereas the microbiota of patients with UC was similar to healthy controls⁽ Reference Willing, Dicksved and Halfvarson ^{107 )}. Also the microbial profiles of patients with CD, predominantly affecting the ileum, were different to those where the disease affected the colon. Although other studies have shown that the microbial profile of IBD patients differs in inflamed and non-inflamed states⁽ Reference Sepehri, Kotlowski and Bernstein ^{122 –} Reference Walker, Sanderson and Churcher ^{124 )}, the faecal microbiota cannot be differentiated between patients with the active disease and those in remission⁽ Reference Willing, Dicksved and Halfvarson ^{107 )}. In terms of microbial profile the disease phenotype was a more significant factor than genotype. Although significant differences in the microbial profile between inflamed and non-inflamed mucosal biopsy sites have been demonstrated, such differences varied so greatly between individuals that no obvious bacterial signature could be positively associated with the inflamed intestine⁽ Reference Walker, Sanderson and Churcher ^{124 )}. Although a dysbiosis is observed in IBD patients, relative to healthy controls, it may result from a disturbed intestinal environment rather than be the direct cause of disease. The complex mutualistic interaction between the microbiota and the host suggests that the relationship is bi-directional⁽ Reference Quigley ^{125 )} and that any observed inflammatory changes may be secondary to, rather than causative of, the disease process; this is a factor that must be considered in future studies⁽ Reference Sartor ^{126 )}.

It has previously been reported that a reduced abundance of Faecalibacterium prausnitzii, a bacterium thought to exert anti-inflammatory effects⁽ Reference Sokol, Pigneur and Watterlot ^{127 )}, is common in ileal CD patients⁽ Reference Willing, Halfvarson and Dicksved ^{108 ,} Reference Swidsinski, Loening-Baucke and Vaneechoutte ^{128 )}. However, F. prausnitzii only represents one of many core members of the microbiota that are less abundant in this disease phenotype. This has led to the suggestion that the oral replenishment of Roseburia, Alistipes, Collinsella and members of the Ruminococcaceae family may be beneficial⁽ Reference Willing, Dicksved and Halfvarson ^{107 )}.

In humans there are at least eighteen mucin-type glycoproteins; however, mucin 2 (MUC2) is the predominant component of the mucin layer in both the small and large intestines⁽ Reference Maldonado-Contreras and McCormick ^{129 )}. When comparing the thinning of the mucus layer in patients suffering from IBD with healthy controls there is evidence that Streptococcus is associated with CD (80% of all bacteria) and that Lactobacillus is associated with UC (90% of all bacteria)⁽ Reference Fyderek, Strus and Kowalska-Duplaga ^{130 ,} Reference Corfield ^{131 )}. In a study by Joossens et al. ⁽ Reference Joossens, Huys and Cnockaert ^{132 )} a faecal microbiota dysbiosis was identified in patients with CD that was characterised by a decreased presence of butyrate-producing bacteria in conjunction with mucin degradation; although the patients’ relatives possessed similarly enhanced mucin degradation they did not share the depleted butyrate-producing bacteria.

As the mucosal barrier is the primary defence of the host against intestinal bacteria the shift from normobiosis observed in relatives of CD patients to the dysbiosis seen in CD patients might be an intermediate step towards CD and disease-associated dysbioses⁽ Reference Joossens, Huys and Cnockaert ^{132 )}. Although Joossens et al. ⁽ Reference Joossens, Huys and Cnockaert ^{132 )} did not study the overall butyrate-producing or mucin degradation capacity of the microbiota in this cohort, there was a functional overlap between dysbiosis in patients with CD and their unaffected relatives at risk. Quigley emphasises the importance of this study suggesting that it indicates a role of the microbiota in CD that is independent of the genetic background and diet, features that the CD patients would have shared with their unaffected relatives⁽ Reference Quigley ^{125 )}. However, Quigley fails to mention the finding of Joossens et al.⁽ Reference Joossens, Huys and Cnockaert ^{132 )} that the CD patients exhibited mucin degradation, a fact that weakens the argument that the microbiota in CD are independent of the genetic background and diet.

The immune system

Epithelial innate immunity includes pattern recognition receptors on the intestinal surfaces such as Toll-like receptors and nucleotide-binding oligomerisation domain (NOD)-containing molecules. Recognition of bacteria by the vertebrate innate immune systems relies upon the detection of invariant molecules by specialised receptors. The view is now emerging that activation of both Toll-like receptors and NOD by different bacterial agonists is important in an inflammatory response⁽ Reference Kufer and Sansonetti ^{133 )}. It appears that NOD1 and NOD2 receptors detect the peptidoglycan components of bacterial cell walls, and the nucleotide-binding domain, leucine-rich (NLR) proteins ipaf and Naip detect bacterial flagellin⁽ Reference Wilmanski, Petnicki-Ocwieja and Kobayashi ^{134 ,} Reference Girardin, Travassos and Herve ^{135 )}. NOD1 specifically senses diaminopimelic acid-containing muramyl peptides⁽ Reference Girardin, Boneca and Carneiro ^{136 ,} Reference Chamaillard, Hashimoto and Horie ^{137 )} and NOD2 detects muramyl dipeptide (MDP)⁽ Reference Girardin, Boneca and Viala ^{138 ,} Reference Inohara, Ogura and Fontalba ^{139 )}, a motif found in almost all bacteria. Nod1 and Nod2 mutations have been associated with IBD in human subjects⁽ Reference Le Bourhis, Benko and Girardin ^{140 )}, with Nod2 being identified as the first susceptibility gene for CD⁽ Reference Hugot, Chamaillard and Zouali ^{141 ,} Reference Ogura, Bonen and Inohara ^{142 )}. It is hypothesised that any impairment to NOD2 function in innate immune responses to bacterial peptides, such as a dysfunctional PepT1 peptide transporter, may lead to defective sensing of bacterial proinflammatory peptides, abnormal bacterial survival and chronic inflammation of the intestinal mucosa⁽ Reference Gruber, Lichti and Rath ^{7 )}. The question of whether a defective PepT1 mechanism is a causative factor for the disease pathogenesis or severity of the IBD has yet to be determined⁽ Reference Gruber, Lichti and Rath ^{7 )}.

Constitutive and inducible antimicrobial peptides such as defensins and cathelicidins interact with secreted mucins and play an important role in intestinal defence⁽ Reference Wehkamp, Schmid and Stange ^{143 ,} Reference Bevins, Martin-Porter and Ganz ^{144 )}. Antimicrobial peptides, predominantly the defensins in mammals, possess a broad spectrum of antimicrobial activity. Antimicrobial peptides are hydrophobic peptides possessing positively charged domains that can interact with and disrupt cell membranes causing cell lysis, which leads to the efflux of ions and nutrients. In the small intestine, the most abundant constitutive defensins are the α-defensins HD5 and HD6 found in the Paneth cells. Following stimulation of the pattern recognition receptors by bacterial products (for example, lipopolysaccharide, which activates Toll-like receptor-4, and MDP, which activates NOD2)⁽ Reference Ayabe, Satchell and Wilson ^{145 )}, defensins are released into the lumen. In comparison with vertebrate cell membranes, bacterial cells possess high concentrations of negatively charged phospholipids, which defensins selectively and preferentially bind to⁽ Reference Khan and Islam ^{146 )}. Together with antimicrobial lysozymes, Paneth cells are key to intestinal defence.

Most dietary protein is completely digested and absorbed as amino acids, dipeptides or tripeptides. However, some proteins are resistant to both the acidic pH of the stomach and enzyme proteolysis⁽ Reference Astwood, Leach and Fuchs ^{147 )}, such that large immunogenic peptides or intact proteins may reach the small intestine⁽ Reference Mahé, Messing and Thuillier ^{148 )}. For example, β-lactoglobulin, a major bovine milk allergen, and gluten/gliadins, a major factor underlying CeD, are both partially resistant to digestive enzymes⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. The ineffective digestion of gliadins which are high in proline content produce large irreducible immunogenic peptides that may activate the lamina propria CD4⁺ T cells in coeliac patients⁽ Reference Shan, Molberg and Parrot ^{149 ,} Reference Shan, Qiao and Arentz-Hansen ^{150 )}. The risk of developing food allergies from incomplete protein digestion has been reported in mice given antiulcer medication known to impair protein digestion where even low doses of ovalbumin resulted in the development of IgE-mediated food allergy⁽ Reference Diesner, Knittelfelder and Krishnamurthy ^{151 )}. Such observations indicate that immunogenic proteins and peptides present in the lumen may serve as potential candidates for intestinal absorption and immune stimulation⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}.

Intestinal alkaline phosphatase (IAP) is expressed throughout the gastrointestinal tract and has an essential role in intestinal homeostasis through interactions with the microbiota resident in the gut⁽ Reference Estaki, DeCoffe and Gibson ^{152 )}. IAP appears to have four major interactions in the gut: (a) the dephosphorylation of toxic pro-inflammatory microbial ligands such the lipopolysaccharides, components of the cell wall of Gram-negative bacteria, the presence of which in the blood can stimulate a strong inflammatory response; (b) the regulation of bicarbonate secretion and increasing the pH distal to the stomach; (c) modulation of long-chain fatty acid absorption; and (d) the regulation of the microbial ecosystem within the gut by forming a complex relationship between microbiota, diet and the intestinal mucosal surface, and the translocation of microbes across the gut wall⁽ Reference Estaki, DeCoffe and Gibson ^{152 ,} Reference Lalles ^{153 )}. IAP also dephosphorylates other pro-inflammatory ligands released from damaged cells such as extracellular nucleotides, for example, ATP. The detoxification of such ligands is essential in the prevention of inflammatory conditions such as IBD. IAP is reported as having a protective effect by ameliorating inflammation from increased permeability of the intestinal endothelia that results from vascular endothelial growth factor affecting the pericellular TJ. Such abnormal intestinal permeability can be partially reduced by IAP down-regulating vascular endothelial growth factor expression and regulating specific TJ proteins for example, claudin-2⁽ Reference Wang, Chen and Zhu ^{154 )}. Compared with normal subjects, epithelial IAP mRNA expression is reduced in patients with UC and CD⁽ Reference Martínez-Augustin, López-Posadas and González ^{155 )} and is especially marked in severe cases of CeD⁽ Reference Molnar, Vannay and Sziksz ^{156 )}. Animal studies have shown that orally administered IAP may reduce inflammation by down-regulating the immune response, specifically reducing pro-inflammatory cytokines, for example, TNF-α⁽ Reference Estaki, DeCoffe and Gibson ^{152 ,} Reference Tuin, Poelstra and de Jager-Krikken ^{157 )}. In an uncontrolled trial by Lukas et al.⁽ Reference Lukas, Drastich and Konecny ^{158 )}, IAP was administered intraduodenally daily over a period of 7 d to patients with UC and was associated with short-term improvement in disease activity scores and clinical effects. The gastrointestinal administration of IAP appears to ameliorate both gut inflammation and favours intestinal tissue regeneration, whereas enteral and systemic administration of IAP attenuates systemic inflammation only⁽ Reference Lalles ^{153 )}.

Genetic disruption

IBD is a multigenic disease where an increased number of inherited risk alleles is associated with an increased risk of developing the disease, earlier onset and greater severity (for example, fibrostenotic or fistulising symptoms and/or the necessity for surgical intervention)⁽ Reference Weersma, Stokkers and van Bodegraven ^{159 ,} Reference Ellinghaus, Bethune and Petersen ^{160 )}. However, genetic susceptibility does not necessitate the development of the disease, as studies with monozygous twins discordant for IBD suggest that diverse environmental interactions (for example, diet and gastrointestinal tract microbiome) also play a role in the development of this disease⁽ Reference Willing, Dicksved and Halfvarson ^{107 ,} Reference Willing, Halfvarson and Dicksved ^{108 ,} Reference Qin ^{161 )}. Using ordinal regression analysis, Weersma et al. ⁽ Reference Weersma, Stokkers and van Bodegraven ^{159 )} reported that individuals with six CD-associated risk alleles (OR 7·56) were unlikely to develop the condition whereas individuals with seven risk alleles (OR 25·6) were much more likely to develop CD. The genetic associations with IBD have been extensively reviewed by a number of researchers⁽ Reference Ferguson ^{162 –} Reference Boirivant and Cossu ^{169 )}. Genome-wide association studies have resulted in the rapid discovery of susceptibility genes with over 163 IBD genes associated with these diseases⁽ Reference Satokari ^{170 –} Reference Thompson and Lees ^{172 )}. Model-selection analysis has indicated that 110 of the 163 susceptibility loci are associated with both CD and UC, with thirty being specific to CD and twenty-three specific to UC⁽ Reference Jostins, Ripke and Weersma ^{171 )}. More recently thirty-six loci specific to CD⁽ Reference Rivas, Beaudoin and Gardet ^{173 )} and 100 specific to UC have been reported⁽ Reference Thompson and Lees ^{172 )}. These loci encode genes which are involved in a number of homeostatic systems, the disruption of which provide a mechanistic description of IBD. Dysfunctional genes that are implicated in epithelial barrier function, bacterial recognition and adaptive immune response are given Table 1.

Table 1

Dysfunctional genes implicated in epithelial barrier function, bacterial recognition and adaptive immune responseFootnote *

IBD5, inflammatory bowel disease 5; DLG5, drosophila discs large homolog; ITLN1, intelectin-1; XBP1, X-box binding protein 1; MDR1, multidrug resistance protein 1; MALB1, Mal region B mutants; CDH1, cadherin 1; HNF4A, hepatocyte nuclear factor 4α; NOD1 and 2, nucleotide-binding oligomerisation domain; CARD9, caspase recruitment domain-containing protein 9; TLR4, Toll-like receptor 4; ATG16L1, autophagy-related protein 16-1; IRGM, immunity-related GTPase family M protein; LRRK2, leucine-rich repeat kinase 2; IL23R, IL23 receptor; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; HLA, human leucocyte antigen; MST1, macrophage stimulating 1; PTPN2, protein tyrosine phosphatase, non-receptor type 2.

* An additional sixteen genes and loci are implicated in inflammatory bowel disease after Graham & Xavier⁽ Reference Graham and Xavier ^{164 )}: RNF186 (ring finger protein 186); SP110 (nuclear body protein 110); SP140 (nuclear body protein 140); MST1 (macrophage stimulating 1 (hepatocyte growth factor-like)); FUT2 (fucosyltransferase); SLC22A4 (solute carrier family); GSDMB (gasdermin B); ORMDL3 (orosomucoid like 3); TNFAIP3 (TNFα-induced protein 3); SLC6A7 (solute carrier family); IL10RA (IL10 receptor α); IL18RAP (IL18 receptor accessory protein); MUC19 (mucin 19); CUL2 (cullin 2); PTPN22 (protein tyrosine phosphatase, non-receptor type 22); C1orf106 (chromosome 1 open reading frame 106).

Advanced genomic techniques have identified other loci and polymorphisms that are associated with IBD and highlight other cellular pathways that may contribute to the onset or progression of the disease⁽ Reference Khor, Gardet and Xavier ^{174 )}. A further sixteen genes and loci implicated in IBD suggest connections between cellular metabolism, inflammation and mucosal microbial communities (see notes for Table 1)⁽ Reference Graham and Xavier ^{164 )}. A comprehensive review of SNP with either susceptibility or protective effects in IBD has recently been published⁽ Reference Neuman and Nanau ^{165 )}. It appears there is significant overlap in genes associated with autoimmune and autoinflammatory diseases that indicate common immunological mechanisms and unique disease-specific pathways which lead to the complex pathophysiology of IBD⁽ Reference Graham and Xavier ^{164 )}. A number of the susceptibility genes identified with IBD (for example, ATG16L1) are common variants with high prevalence in the healthy population⁽ Reference Weersma, Stokkers and van Bodegraven ^{159 )}. Indeed many of the SNP implicated in IBD by genome-wide association studies are not independently causative of the disease phenotype; they exist as linked disequilibrium with as yet to be discovered variants that are functional⁽ Reference Graham and Xavier ^{164 )}. In the future, genetic screening for IBD-related SNP, combined with an assessment of the intestinal microbiome and other environmental factors (for example, diet), might allow clinicians to identify patients at risk of IBD and improve differential diagnosis and optimise treatment efficacy of the disease⁽ Reference Neuman and Nanau ^{165 ,} Reference Viennois, Baker and Xiao ^{175 )}.

Interestingly Hu et al.⁽ Reference Hu, Smith and Ma ^{176 )} generated mice in which the PepT1 gene was disrupted by the insertion of a lacZ reporter gene under the control of the endogenous PepT1 promoter. Although the Pept1-null mice lacked expression of PepT1 protein in the intestine and kidney tissues in which this peptide transporter is normally expressed, the Pept1-deficient mice were found to be viable, fertile, grew to normal size and weight, and were without any obvious abnormalities⁽ Reference Hu, Smith and Ma ^{176 )}.

Other environmental factors

With the sudden emergence and dramatic increase in IBD during the last century⁽ Reference Russel ^{177 )}, a variety of environmental factors has been implicated with the onset of IBD, including: food storage in refrigerators, smoking, the use of non-steroidal anti-inflammatory drugs, and infections⁽ Reference Steel, Nussberger and Romero ^{178 ,} Reference Boll, Markovich and Weber ^{179 )}. Diet and food additives have long been suspected as major factors in IBD pathogenesis⁽ Reference Gruber, Lichti and Rath ^{7 )} and in this context two new fields of study have emerged: nutrigenetics, which recognises the effect of genetic variation on nutrient requirements, and nutrigenomics, which describes the impact of nutrient regulation of gene expression⁽ Reference Ferguson ^{162 )}. Although Qin⁽ Reference Qin ^{161 )} suggests a multitude of possible dietary factors affecting IBD, he singles out saccharin and/or sucralose to be a key causative factor in the disease. Proposing a unified hypothesis regarding the aetiology for IBD, Qin suggests that saccharin inhibits both the activity of β-glucuronidase itself as well as the growth of β-glucuronidase-positive bacteria in the gastrointestinal tract which are necessary for the deconjugation of biliary bilirubin⁽ Reference Qin ^{180 )}, that in turn leads to damage of the protective mucus layer and the underlying gut tissue by the poorly inactivated digestive proteases⁽ Reference Qin ^{181 )} (the bacteria–protease–mucus–barrier hypothesis)⁽ Reference Qin ^{161 )}.

Peptide transporters

The principal transporter for the absorption of di- and tripeptides arising from the digestion of both exogenous and endogenous proteins in the intestinal lumen is the high-capacity, low-affinity PepT1 protein⁽ Reference Smith, Clemencon and Hediger ^{26 )}. Peptide transporters are integral membrane proteins that mediate the cellular uptake of di- and tripeptides. In vertebrates there are two peptide transporter proteins: PepT1 expressed predominantly in brush-border membranes of the small intestine and PepT2 in the kidney and lung. Although PepT1 is highly expressed in the small intestine⁽ Reference Ogihara, Saito and Shin ^{182 )} there is little or no expression in the healthy colon⁽ Reference Ismair, Vavricka and Kullak-Ublick ^{183 )}. These transport proteins operate as electrogenic proton/peptide symporters with a broad substrate specificity, possibly transporting 400 dipeptides and 8000 tripeptides composed of l-α amino acids⁽ Reference Weitz, Harder and Casagrande ^{184 )}, but not free amino acids or peptides with more than three amino acid residues⁽ Reference Daniel ^{185 ,} Reference Bailey, Boyd and Collier ^{186 )}. The electrochemical gradient across the apical enterocyte membrane is dependent upon the Na-proton exchanger NHE3 and allows the absorption of di- and tripeptides against a concentration gradient, enabling higher intracellular than extracellular peptide concentrations⁽ Reference Spanier ^{187 ,} Reference Watanabe, Kato and Ito ^{188 )}.

Transport is enantio-selective and involves a variable proton-to-substrate stoichiometry for the uptake of neutral and mono- or polyvalently charged peptides. The peptide transporter proteins can also transport many therapeutic drugs (for example, β-lactam antibiotics, selected angiotensin-converting enzyme inhibitors, and peptidase inhibitors) and thereby determine their bioavailability and pharmacokinetics⁽ Reference Weitz, Harder and Casagrande ^{184 )}. In addition, PepT1 has an important role in the innate immune response to bacteria by mediating the transepithelial transport of bacterial antigens⁽ Reference Chen, Yang and Zhang ^{65 )}. Microbial peptides imported by PepT1, for example MDP, induce NOD2-dependent activation of the NF-κB pathway⁽ Reference Gruber, Lichti and Rath ^{7 )} with submucosal macrophages that in turn release proinflammatory cytokines, for example, IL-8 and the monocyte chemoattractant protein-1 (MCP-1)⁽ Reference Vavricka, Musch and Fujiya ^{189 ,} Reference Zucchelli, Torkvist and Bresso ^{190 )}. PepT1 polymorphisms in the SLC15A1 gene have been associated with IBD⁽ Reference Strober, Asano and Fuss ^{191 )} and NOD2 polymorphisms with CD⁽ Reference Zucchelli, Torkvist and Bresso ^{190 )}. The clinical relevance of intestinal uptake in disease has recently been reviewed by Freeman⁽ Reference Freeman ^{192 )}.

Despite the comprehensive analysis of the structure and functions of PepT1, with many hundreds of publications over the last 40 years, its overall importance in amino acid absorption from the gastrointestinal tract is still largely unknown⁽ Reference Nassl, Rubio-Aliaga and Fenselau ^{193 )}. Using mice lacking PepT1 (Pept1 ^–/–) the extent that PepT1 deletion is compensated for by changes in expression and function of the amino acid transporters in intestinal epithelial cells and the role of the transporter in amino acid absorption and metabolism have been characterised⁽ Reference Nassl, Rubio-Aliaga and Fenselau ^{193 )}. The intragastric administration of ¹⁵N-labelled proteins and the concomitant analysis of plasma and tissue amino acid levels have indicated that the role of PepT1 in the overall intestinal amino acid absorption is negligible when low amounts of protein are ingested. However, under conditions of a high protein load reaching the intestine, the maximum rate of hydrolysis in the lumen or at the brush-border membrane may be reached, leading to di- and tripeptides becoming available for PepT1 transport⁽ Reference Nassl, Rubio-Aliaga and Fenselau ^{193 )}. Nassl et al.⁽ Reference Nassl, Rubio-Aliaga and Fenselau ^{193 )} suggested that when a high-protein diet is administered to Pept1 ^–/– mice it may induce changes in body amino acid homeostasis that resemble a state of amino acid imbalance, with amino acids that are related to the urea cycle being over-represented. This is suggestive of an altered hepatic detoxification capacity in animals deficient in PepT1.

Not all researchers share the same view; for example, the enteropathogenic E. coli (EPEC), a food-borne pathogen implicated in the pathophysiology of infantile diarrhoea⁽ Reference Nataro and Kaper ^{194 )}, may also induce PepT1 expression in colonocytes⁽ Reference Nguyen, Dalmasso and Powell ^{195 )}. Nguyen et al.⁽ Reference Nguyen, Dalmasso and Powell ^{195 )} demonstrated that: EPEC transcriptionally induces functional PepT1 expression in the lipid rafts (LR) of colonocytes; that it induces PepT1 expression by intimately attaching to host cell membranes through LR; that the transcription factor Cdx2 is crucial for EPEC-induced PepT1 expression; and that PepT1 which are associated with LR have a role in bacterial–epithelial interaction and bacteria-induced intestinal inflammation. It is proposed that EPEC is a causal factor of human colonic PepT1 expression, activating signalling molecules within the LRs, resulting from changes in conformation and/or composition of LR, and consequently reducing the binding affinity of EPEC for LR. PepT1 appears to attenuate EPEC-triggered proinflammatory responses in intestinal epithelial cells, and therefore colonic PepT1 expression might be a host protective mechanism that modulates bacterial–epithelial interaction and inflammatory responses to pathogens⁽ Reference Nguyen, Dalmasso and Powell ^{195 )}, a finding that is in line with that of other researchers⁽ Reference Smith, Clemencon and Hediger ^{26 ,} Reference Merlin, Steel and Gewirtz ^{196 –} Reference Foster and Zheng ^{199 )}.

Apically expressed colonic PepT1 may be a host defence mechanism via its ability to modulate bacterial–epithelial interactions and colonic inflammation⁽ Reference Smith, Clemencon and Hediger ^{26 )}. Colonic PepT1 expressed in IBD may absorb small proinflammatory peptides derived from bacterial peptidoglycans (for example, N-formylmethionylleucyl-phenylalanine (fMLP)⁽ Reference Merlin, Steel and Gewirtz ^{196 )}, MDP⁽ Reference Vavricka, Musch and Chang ^{197 )} and l-Ala-γ-d-Glu-meso-diaminopimelic acid (Tri-DAP)⁽ Reference Dalmasso, Hang and Ingersoll ^{198 )}) that interact with NOD-like receptors and determine the activation level of inflammatory pathways such as the NF-κB and MAPK⁽ Reference Dalmasso, Hang and Ingersoll ^{198 )}. These pathways lead to proinflammatory cytokine/chemokine production and the subsequent migration of neutrophils into regions of inflammation and bacterial infection⁽ Reference Smith, Clemencon and Hediger ^{26 ,} Reference Foster and Zheng ^{199 )}.

Dalmasso et al. ⁽ Reference Dalmasso, Nguyen and Charrier-Hisamuddin ^{11 )} used Tri-DAP to induce inflammation in human colonic HT29-Cl.19A cells. Similar to fMLP and MDP, Tri-DAP is a natural peptide released during peptidoglycan degradation of Gram-negative bacteria, a bacterial tripeptide that may pass through the PepT1 transporter. Although it is still unclear if the peptide induces PepT1 expression in colonocytes, this suggests that bacterial products might induce or regulate colonic PepT1 expression⁽ Reference Dalmasso, Yan and Charrier ^{200 )}, and that once PepT1 is expressed in the colon in IBD, then PepT1 could then be involved in the transport of bacterial peptides that aggravate inflammation. However, the studies of Dalmasso et al. ⁽ Reference Dalmasso, Nguyen and Charrier-Hisamuddin ^{11 )} did indicate that colonic epithelia only respond to peptidoglycan motifs such as Tri-DAP when such products are present in the cytosol and most importantly that colonocytes fail to transport Tri-DAP or are inert to Tri-DAP under normal physiological conditions when PepT1 is not expressed in the colon.

Although the peptide/histidine transporters PhT1 and PhT2 have been found in the villous epithelia of the human small intestine⁽ Reference Bhardwaj, Herrera-Ruiz and Eltoukhy ^{201 )}, their relevance in the absorption of peptides and peptidomimetics has not been established⁽ Reference Smith, Clemencon and Hediger ^{26 )}. However, it appears that neither of these transporters is involved in the absorption of the proinflammatory peptide fMLP⁽ Reference Wu and Smith ^{202 )}.

The multidrug resistance 1 gene (MDR1), which encodes for the membrane-bound efflux transporter P-glycoprotein 170 (P-gp), has been associated with IBD and thought to protect the intestinal epithelia from the uptake of endogenous and exogenous toxins by transporting drugs and xenobiotics into the lumen⁽ Reference Gruber, Lichti and Rath ^{7 ,} Reference Tsuji, Nakashima and Deguchi ^{203 )}. Although several SNP of this gene have been reported, its relevance to the pathogenesis of IBD varies across ethnic groups⁽ Reference Walker, Thwaites and Simmons ^{204 –} Reference Potocnik, Ferkolj and Glavac ^{207 )}.

Epithelial barrier dysfunction, peptides and Crohn’s disease

The studies of Cadwell et al. ⁽ Reference Cadwell, Patel and Maloney ^{5 )} have demonstrated that a common enteric viral pathogen, norovirus, can induce a mutation in the CD susceptibility gene Atg16L1, producing intestinal pathologies in mice. These pathologies, activated by virus-plus-susceptibility gene interaction that mimic aspects of CD, were dependent on IFN-γ and TNF-α, and were preventable by treatment with broad-spectrum antibiotics⁽ Reference Cadwell, Patel and Maloney ^{5 )}. Sabbah et al.⁽ Reference Sabbah, Chang and Harnack ^{208 )} have demonstrated that NOD2 can also function as a cytoplasmic viral pattern recognition receptor that can sense viral single-stranded RNA and activate IFN production. As a result it has been suggested that the CD-associated gene NOD2 may recognise viral RNA in addition to bacterial peptidoglycan and raises the possibility that a viral infection can interact with CD susceptibility genes⁽ Reference Cadwell, Patel and Maloney ^{5 )}. Both Garrett et al. ⁽ Reference Garrett, Gallini and Yatsunenko ^{209 )} and Cadwell et al. ⁽ Reference Cadwell, Patel and Maloney ^{5 )} give clear insight into the complex interaction between gene and pathogen, which individually may display only poor association with disease incidence and severity. In animal models with induced pathology, reproducing the full disease may require combinations of specific alleles of multiple genes with certain environmental agents. Not all patients with CD present with identical symptoms or pathologies; the disease varies with time and also with age, sex, ethnicity, temporal trends and geographical distributions⁽ Reference Russel ^{177 ,} Reference Molodecky, Soon and Rabi ^{210 )}. Some therapeutic interventions may alleviate symptoms of one patient but not in others⁽ Reference Cadwell, Patel and Maloney ^{5 )}. Such complex IBD diseases may represent a combinatorial confluence of pathological responses, each with overlapping but non-identical genetic and environmental causes and therefore require different therapeutic responses⁽ Reference Cadwell, Patel and Maloney ^{5 )}.

In CD, a transcription factor-4-mediated defect in Paneth cell differentiation has been linked to a specific absence of the α-defensins, especially in patients with Nod2 mutations⁽ Reference Gersemann, Wehkamp and Stange ^{6 )}. Consequently, the deficient mucosal barrier allows luminal microbes to invade the mucosa and trigger a secondary inflammatory response⁽ Reference Wehkamp and Stange ^{25 )}.

The up-regulated expression of PepT1 in patients with IBD⁽ Reference Merlin, Si-Tahar and Sitaraman ^{211 )} and NOD2 mutations associated with CD⁽ Reference Girardin, Boneca and Viala ^{138 )} may result from defective sensing of bacterial peptidoglycan-derived peptides such as MDP (a NOD2 agonist)⁽ Reference Ismair, Vavricka and Kullak-Ublick ^{183 )}. As a result of NOD2 mutations, which result in a NOD2 deficiency, a loss of microbial surveillance and the unmonitored import of microbial proinflammatory peptides may be caused that contribute to the onset of CD.

The expression of PepT1 in the colon of patients with UC or CD may be a response to the absorption of chemotactic bacterial di- and tripeptides that cause an aggravated inflammation/immune response⁽ Reference Charrier and Merlin ^{10 ,} Reference Smith, Clemencon and Hediger ^{26 )}. The expression of PepT1 in human colonocytes has also been linked to leptin, an adipocyte-secreted hormone. High concentrations of leptin were found in inflamed colonic mucosa which in turn triggered the colonic expression of PepT1 via the cAMP response element-binding (CREB) and Cdx2 transcription factors. Such increased expression of colonic PepT1 may thus enhance the uptake of the small bacterial di- and tripeptides that perpetuate intestinal inflammation. Such findings may provide important new insights into the mechanisms of intestinal inflammation and its treatment⁽ Reference Nduati, Yan and Dalmasso ^{212 )}.

Although the mechanism of colonic PepT1 expression in IBD remains unknown, it has been suggested that its expression is most probably induced at a transcriptional level, where specific transcriptional regulation by signalling pathway(s) may be activated⁽ Reference Nguyen, Dalmasso and Powell ^{195 ,} Reference Merlin, Si-Tahar and Sitaraman ^{211 ,} Reference Ziegler, Fernández-Estívariz and Gu ^{213 ,} Reference Shimakura, Terada and Shimada ^{214 )}. However, Vavricka et al.⁽ Reference Vavricka, Musch and Fujiya ^{189 )} demonstrated both in vitro (in human colonic Caco2/bbe monolayers) and in vivo (in mouse intestine), that TNF-α and IFN-γ increased the activity and the total and apical membrane protein expression of PepT1 protein in a concentration- and time-dependent fashion. As no changes in PepT1 mRNA were observed, it may be concluded that the increased PepT1 activity and expression were post-transcriptionally regulated⁽ Reference Vavricka, Musch and Fujiya ^{189 )}. Current research into the expression of PepT1 transporters in the colon of patients with some form of IBD suggests that the presence of PepT1 in the colon is due to the bacterial load of the colon being higher than that of the ileum, and that such a high bacterial load creates a concomitantly high luminal concentration of bacterial peptides, which PepT1 transporters absorb, thus stimulating an exaggerated proinflammatory immune response.

It has also been suggested that PepT1 expression is normally restricted to the small intestine because the concentrations of bacterial di- and tripeptides are much lower in the small intestine than in the colon and that the human small intestine contains only low numbers of prokaryotes⁽ Reference Ingersoll, Ayyadurai and Charania ^{9 )}. However, this statement can be challenged in light of the evidence that the numbers of bacteria present in the distal ileum may be as high as 10⁹/ml of digesta⁽ Reference Metges ^{215 )}. Although less than the densities reported by Whitman et al. for the large intestine (10¹¹–10¹²/ml digesta⁽ Reference Whitman, Coleman and Wiebe ^{216 )}), they are in sufficient numbers that the presence of substantial quantities of bacterial di- and tripeptides in the ileum cannot be discounted, and are not evidential as to the lack of inflammation in the healthy human small intestine. Ingersol et al. ⁽ Reference Ingersoll, Ayyadurai and Charania ^{9 )} suggest that the presence of colonic PepT1 transporters is solely due to the higher numbers of bacteria in the large bowel as the profile of PepT1 expression along the normal human digestive tract is such that bacterial peptides have little access to PepT1 and minimises the intracellular uptake of bacterial peptides. As PepT1 expression is altered in patients with IBD and commensal bacteria colonising the human colon produce significant amounts of proinflammatory di- and tripeptides, the transport of the peptides by PepT1 may lead to an increased intracellular accumulation of prokaryotic peptides that trigger downstream proinflammatory effects⁽ Reference Ingersoll, Ayyadurai and Charania ^{9 )}.

Epithelial barrier dysfunction, peptides and ulcerative colitis

In experiments with conventionally raised T-bet ^–/– ×Rag2 ^–/– knockout mice lacking an adaptive immune system, the loss of the transcription factor T-bet results in a spontaneous and highly penetrant colitis that shares histological features with UC in humans⁽ Reference Garrett, Lord and Punit ^{217 )}. T-bet ^–/– × Rag2 ^–/– UC (TRUC) is associated with altered colonic barrier function, elevated TNF-α levels and dysfunctional dendritic cells. Both the T-bet-deficient genetic background and the microbiota are required for disease initiation⁽ Reference Garrett, Lord and Punit ^{217 )}. Once the disease is established, the microbiota from the afflicted mice are vertically transmissible and cause intestinal inflammation in wild-type mice. TRUC is transmissible to wild-type hosts when they are cross-fostered or co-housed with TRUC mice. In a later paper, Garrett et al. ⁽ Reference Garrett, Gallini and Yatsunenko ^{209 )} demonstrated that the presence of Proteus mirabilis and Klebsiella pneumoniae contributes to disease pathogenesis of colitis in TRUC mice and that TRUC-derived strains, in conjunction with an endogenous microbial community, incite colitis in wild-type mice⁽ Reference Garrett, Gallini and Yatsunenko ^{209 )}. Their results may provide mechanistic insights about how intestinal microbial communities, working in concert with specific colitogenic agents, contribute to the initiation and perpetuation of IBD in susceptible human hosts, and provide the foundation for proof-of-concept tests of preventative or therapeutic measures⁽ Reference Garrett, Gallini and Yatsunenko ^{209 )}.

Dietary protein is, to the host, foreign protein that may contain peptides that would trigger an immune response if exposed to the immune system in the enterocyte/lamina propria/bloodstream. The bacterial di- and tripeptides fMLP and MDP have been found to elicit an immune response; therefore it is probably safe to assume that there are other hydrolysis-resistant dietary peptides which could also elicit a similar response. Therefore any leakage of the apical/basolateral membranes would also elicit a similar response that results in inflammation. A high intake of dairy products or low dietary fibre intake has been reported to be associated with the relapse of patients with UC⁽ Reference Davies and Rhodes ^{218 –} Reference Samuelsson, Ekbom and Zack ^{221 )}. However, more recently Jowett et al. ⁽ Reference Jowett, Seal and Phillips ^{14 )} detected no association between the intake of milk or dairy products and relapse of UC, neither did they find any protective effect from increased dietary fibre. However, they did find that the consumption of meat (particularly red meat and processed meat), protein, and alcohol were linked to increased relapse in patients with UC⁽ Reference Jowett, Seal and Phillips ^{14 )}. Speculation that the high sulfur or sulfate compounds in many of these foods was the trigger associated with the relapse of UC has led to a number of studies⁽ Reference Jowett, Seal and Phillips ^{14 ,} Reference Pithadia and Jain ^{222 )} not least of which were those of Marquet et al. ⁽ Reference Marquet, Duncan and Chassard ^{103 )}, Shatalin et al. ⁽ Reference Shatalin, Shatalina and Mironov ^{102 )} and Jia et al. ⁽ Reference Jia, Whitehead and Griffiths ^{91 )} that highlighted the toxicity of hydrogen sulfide, possibly mediated through the impaired utilisation of butyrate in colonocytes⁽ Reference Roediger, Duncan and Kapaniris ^{223 )}. Carbohydrates have also been reported to result in colonic inflammation⁽ Reference Sakamoto, Kono and Wakai ^{224 )} and promote UC in some individuals⁽ Reference Pithadia and Jain ^{222 )}. Although enteral feeding to control dietary intake has been effective in the treatment of CD, it is ineffective in UC⁽ Reference King, Woolner and Hunter ^{225 )}.

Large numbers of peptides have been isolated from both milk⁽ Reference Choi, Sabikhi and Hassan ^{226 –} Reference Meisel ^{228 )} and meat⁽ Reference Schmid ^{229 –} Reference Minkiewicz, Dziuba and Michalska ^{232 )} and the possible existence of small proinflammatory peptides similar to the previously identified proinflammatory bacterial peptides cannot be excluded. Recently, Chatterton et al.⁽ Reference Chatterton, Nguyen and Bering ^{233 )} reviewed the anti-inflammatory mechanisms of milk proteins that assist in the prevention of a severe form of intestinal inflammation known as necrotising enterocolitis which is associated with a high mortality in neonates. In this review, the authors commented that although raw human milk contains many anti-inflammatory proteins (for example, immunoglubulins that chelate bacterial and viral proinflammatory antigens), bovine milk may have much fewer anti-inflammatory components due to digestive proteolysis and pasteurisation⁽ Reference Chatterton, Nguyen and Bering ^{233 )}. Various growth factors present in both human and bovine milk have been reported as having anti-inflammatory properties⁽ Reference Rutherfurd-Markwick and Moughan ^{15 ,} Reference Chatterton, Nguyen and Bering ^{233 )}. Transforming growth factor-β1 (TGF-β1) was reported by Letterio et al.⁽ Reference Letterio, Geiser and Kulkarni ^{234 )} to have anti-inflammatory properties, which were later attributed to the modulation of inflammatory responses⁽ Reference Boll, Markovich and Weber ^{179 )}. TGF-β regulates the differentiation of T helper 17 cells (Th17-cells) which maintain intestinal barrier integrity and produce the anti-inflammatory cytokine IL-10⁽ Reference Fei, Kanai and Nussberger ^{235 )}. Heparin-binding epidermal growth factor-like growth factor was found to attenuate bacterial binding to the intestinal mucosa⁽ Reference Tanida, Kataoka and Mizoshita ^{236 )}, repress the cytokine-induced activation of NF-κB and release of proinflammatory cytokines⁽ Reference Mehta and Besner ^{237 ,} Reference Mehta and Besner ^{238 )}.

The hypothesis that dietary proteins and their hydrolysates contain peptides that may affect mucin secretion has been studied by a number of researchers⁽ Reference Montagne, Toullec and Formal ^{239 –} Reference Han, Deglaire and Sengupta ^{242 )}. The casomorphins, a family of bioactive peptides derived from milk β-casein, are opioid agonists known to affect the secretion of mucin, a protective response that also stimulates the production of epidermal growth factor, that in turn promotes epithelial cell proliferation⁽ Reference Miner-Williams ^{243 )}. The effect of various opioid-acting casomorphins on mucin secretion has been reported by a number of researchers⁽ Reference Claustre, Toumi and Trompette ^{241 ,} Reference Trompette, Claustre and Caillon ^{244 )} who found that the intraluminal administration of β-casomorphin-7 provoked a 500% increase (over the controls) in the secretion of mucin. β-Casomorphin-7 seems unique in this respect as little or no increase in mucin secretion was observed from any of the other opioid peptides tested. Milk-borne opioid receptor ligands have been extensively reviewed by Clare & Swaisgood⁽ Reference Clare and Swaisgood ^{245 )}. Zoghbi et al.⁽ Reference Zoghbi, Trompette and Claustre ^{246 )} reported that β-casomorphin-7 increased the expression of rMuc2 and may contribute significantly to mucin production through a direct effect on intestinal goblet cells and the activation of μ-opioid receptors. Because intestinal mucins are an integral part of epithelial barrier function, dietary supplements containing β-casomorphin-7 are worthy of investigation for their potential to improve intestinal protection in IBD.

Secretory mucin MUC2 is the predominant structural component of the mucus layer and is abundantly expressed by goblet cells in the colon⁽ Reference van Klinken, Einerhand and van der Wal ^{247 ,} Reference Van Klinken, Van der Wal and Einerhand ^{248 )}. MUC2 synthesis is decreased in both human and animal models of IBD⁽ Reference Tytgat, Opdam and Einerhand ^{249 ,} Reference Tytgat, vanderWal and Einerhand ^{250 )}, and the expression of MUC2 is considered as a phenotypic marker, which can be inversely correlated with the severity of inflammation⁽ Reference Shaoul, Okada and Cutz ^{251 )}. Quantitative changes in mucin secretion occur in IBD that include structural changes in the glycoprotein core together with the sulfation and sialylation of the oligosaccharide residues. Such changes are associated with a dysfunctional mucous barrier⁽ Reference Boltin, Perets and Vilkin ^{252 )}. In UC, defensin synthesis and activity are not disturbed, even in inflamed mucosa; however, deficiencies in the mucus layer of UC patients are indicative of defects in goblet cell differentiation. With a diminished mucus layer the secreted defensins in physiologically normal concentrations are not retained and this allows bacteria to pass through the epithelium and induce inflammation⁽ Reference Gersemann, Wehkamp and Stange ^{6 )}. There is evidence that matrix metalloproteinases (MMP) are the predominant extracellular proteinases within the mucosa disorders such as IBD and peptic ulcer disease⁽ Reference Xie and Costello ^{253 –} Reference Kirkegaard, Hansen and Bruun ^{255 )}. Both serum and tissue levels of MMP-9, localised in the colonic mucosa⁽ Reference Castaneda, Walia and Vijay-Kumar ^{256 )}, are known to correlate with disease activity in UC⁽ Reference Baugh, Perry and Hollander ^{254 ,} Reference Baugh, Evans and Hollander ^{257 ,} Reference Kossakowska, Medlicott and Edwards ^{258 )}. Garg et al. ⁽ Reference Garg, Ravi and Patel ^{259 )} demonstrated that MMP-9 modulates MUC2 expression by regulating goblet cell differentiation. Overexpression of MMP-9 inhibits goblet cell differentiation with a concomitant decrease in MUC2 mucin⁽ Reference Shields, Christy and Yang ^{260 ,} Reference Ng, Waring and Ristevski ^{261 )}. The aberrant expression of MMP-9, observed in inflammatory conditions, leads to the impaired differentiation and a consequent decrease in goblet cell function, known to be associated with increased susceptibility to bacterial infection/inflammation⁽ Reference Garg, Ravi and Patel ^{259 )}. A target for future IBD therapies may be to strengthen the mucous barrier through the up-regulation/down-regulation of MUC genes, the manipulation of post-transcriptional processing, or targeting the mucin molecule itself⁽ Reference Boltin, Perets and Vilkin ^{252 )}.

Epithelial barrier dysfunction, peptides and coeliac disease

CeD is a chronic immune-mediated disorder that primarily affects the mucosa of the small intestine. The condition is a food antigen-triggered autoimmune disorder that involves an immune response (both innate and adaptive) following exposure to dietary gluten-containing foods in genetically predisposed individuals. The pathogenesis of CeD involves a triad of predisposing genes, gluten and other environmental factors. The inception of CeD follows the deamination of gliadins, monomeric proteins contained in gluten, by tissue transglutaminase. Gliadin immunogenic fragments, resistant to endopeptidases, then bind to the chemokine CXCR3 present on the luminal surface of the enterocytes. Zonulin, one of the transmembrane proteins that regulate epithelial barrier permeability, may then be released from the TJ as a result of gliadin binding to CXCR3⁽ Reference Lammers, Lu and Brownley ^{262 )}. The MyD88 adapter protein-dependent release of zonulin, highly expressed in CeD, results in the disassembly of the TJ and a subsequent increase in intestinal permeability⁽ Reference Fasano, Not and Wang ^{263 )} and activation of antigen-presenting cells (for example, macrophages, dendritic cells and B cells). These cells in turn display the gliadin peptides and interact with gluten-specific CD4⁺ T cells in the lamina propria. CD4⁺ T cells release inflammatory cytokines (for example, IFN-γ and IL-15) that facilitate the transformation of intraepithelial lymphocytes into cytotoxic CD8⁺ T cells that kill the intestinal epithelial cells. The cumulative effect of this inflammatory cascade is the manifestation of villous atrophy and crypt hyperplasia⁽ Reference Green, Lebwohl and Greywoode ^{264 )}. Inflammation of the intestinal epithelia consequent to the gliadin assault on the enterocytes may then exacerbate intestinal barrier dysfunction leading to the increased passage of antigens involved in the pathogenesis of CeD. In genetically predisposed individuals (those carrying the HLA-DQ2 or less commonly HLA-DQ8 haplotype) environmental factors, such as rotavirus infection, toxins or Fe-deficiency anaemia, are thought to initiate the development of CeD⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. It is interesting to note that constitutive abnormalities in intestinal permeability are not the hallmarks of food allergy; increased epithelial permeability is more the consequence of immunological changes producing villous atrophy, rather than the cause of food sensitisation⁽ Reference Heyman ^{265 )}. For example, significant increases in the trans-epithelial transport of horseradish peroxidase (about 44 kDa) are observed in children with active cows’ milk allergy, which return to normal levels following a cows’ milk-free diet⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. However, increases in epithelial permeability are the cause of a self-perpetuating cycle that maintains allergic inflammation⁽ Reference Heyman, Abed and Lebreton ^{266 )}.

A number of non-dietary therapies are currently under investigation that target specific aspects of CeD pathogenesis including intraluminal agents, immunomodulators and vaccination⁽ Reference Perez, de Villasante and Ruiz ^{267 ,} Reference Crowe ^{268 )}. Larazotide acetate (AT-1001), a peptide derived from cholera toxin, is thought to regulate intestinal paracellular permeability by inhibiting the disassembly of intestinal TJ. However, current clinical trials with larazotide have not demonstrated any decrease in intestinal permeability in CeD patients taking the drug, although decreased tissue transglutaminase IgA levels and improved clinical symptoms were observed⁽ Reference Leffler, Kelly and Abdallah ^{269 ,} Reference Kelly, Green and Murray ^{270 )}. Other proposed treatments include: reduced gluten exposure by genetic modification of the cereal grains containing gluten⁽ Reference Spaenij-Dekking, Kooy-Winkelaar and Van Veelen ^{271 ,} Reference Carroccio, Di Prima and Noto ^{272 )}; or using co-polymeric binders of gluten⁽ Reference Pinier, Fuhrmann and Galipeau ^{273 )}; pre-digestion of gluten before intestinal epithelial cell uptake using prolyl-endopeptidases⁽ Reference Mitea, Havenaar and Drijfhout ^{274 ,} Reference Tack, Van de Water and Bruins ^{275 )}; transglutaminase inhibitors or the blockade of HLA-DQ2/DQ8⁽ Reference Schuppan, Junker and Barisani ^{276 –} Reference Xia, Siegel and Bergseng ^{278 )}; and immune tolerance induction⁽ Reference Keech, Dromey and Chen ^{279 ,} Reference Brown, Daveson and Marjason ^{280 )}.

Peptide permeability: is it the cause or consequence of intestinal disorders?

That intestinal barrier dysfunction is the primary cause of intestinal disorders, such as food allergy, CeD and IBD, involving the transformation of antigenic tolerance into antigenic sensitisation following the excessive absorption of antigens, is a longstanding hypothesis to explain the pathogenesis of these diseases⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. However, in food allergy, constitutive abnormality in intestinal permeability is unrecorded, and permeability to horseradish peroxidase in infants with active bovine milk allergy returns to normal following treatment with a milk-free diet⁽ Reference Heyman, Grasset and Ducroc ^{281 )}. Infection might be the triggering factor in CeD where inflammation increases intestinal permeability to gliadin peptides and thus immune responses in susceptible individuals. Rotavirus infection is associated with a higher risk of CeD in early childhood⁽ Reference Stene, Honeyman and Hoffenberg ^{282 )}. Increased permeability to intact gliadin peptides observed in active CeD⁽ Reference Matysiak-Budnik, Candalh and Dugave ^{283 )} ceases in most coeliac patients once treated with a gluten-free diet.

The hypothesis that a primary defect is the cause of intestinal barrier dysfunction in patients with IBD arises from observations of hyperpermeability in CD patients before phenotypic symptoms arise and an increased permeability in the healthy relatives of IBD patients⁽ Reference Hollander, Vadheim and Brettholz ^{22 )}. A genetic susceptibility to CD has been indicated by genome-wide association studies where a number of distinct genomic loci are involved in the maintenance of epithelial barrier integrity⁽ Reference Xavier and Podolsky ^{8 ,} Reference Van Limbergen, Wilson and Satsangi ^{284 )}. Variants of the myosin IXB (myo 9B) gene, that codes for the myosin IX motor protein, have been associated with IBD⁽ Reference Latiano, Palmieri and Valvano ^{285 ,} Reference Van Bodegraven, Curley and Hunt ^{286 )} and CeD⁽ Reference Monsuur, de Bakker and Alizadeh ^{287 )}, and may suggest a primary defect in intestinal permeability in these diseases⁽ Reference Menard, Cerf-Bensussan and Heyman ^{28 )}. Foster & Zheng⁽ Reference Foster and Zheng ^{199 )} demonstrated that the inflammatory cytokine IFN-γ increases intestinal PepT1 expression, which in turn mediates the absorption of the proinflammatory bacterial peptide fMLP⁽ Reference Foster, Landowski and Welage ^{288 )}. They demonstrated that cephalexin inhibits fMLP transport across cultured intestinal monolayers, which partially attenuates polymorphonuclear leucocyte-induced intestinal hyperpermeability. From their data it may be concluded that the use of pharmacological PepT1 substrates such as cephalexin may represent a novel means of preserving intestinal barrier integrity⁽ Reference Foster and Zheng ^{199 )}.