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Introduction

The survival of living organisms greatly depends on the ability of species and individuals to constantly provide a series of complex and dynamic repository responses to counteract internal and environmental threats. This functional equilibrium, named homeostasis, relies upon the adequate integration of every generated response to a threat. At the gastrointestinal level, the mucosal surfaces are the first location where immunogenic particles, environmental toxins and microorganism-derived antigens gain access to the immune system [1]. The luminal side of the mucosa of the ileum and jejunum is coated with hundreds of tiny finger-like structures called villi, which in turn are composed by myriads of microvilli, rendering a final physical contact area of about 400 m2. This enormous epithelial surface area favours nutrient absorption and water and electrolyte transport across. However, it also designed to select which luminal antigens should face the components of the mucosal-associated immune system. This selection process is aimed at preventing the generation of inadequate pro-inflammatory signals [2]. Mucosal processing of antigens and non-immunogenic molecules will at the end, determine whether tolerogenic or non-tolerogenic immune responses are raised to keep homeostasis [3].

The intestinal mucosal barrier consists of different consecutive layers, including the intestinal flora and external mucus, the columnar epithelium and extracellular matrix below, and the innermost lamina propria. Within the lamina propria we can find blood and lymph vessels, a plethora of resident immune cells (plasma cells, lymphocytes, macrophages, eosinophils, mast cells, dendritic cells, etc.), and a significant number of intrinsic and extrinsic nerve terminals (Fig. 4.1). All of these components may display effector and modulatory functions relevant to the control of inflammation, absorption and secretion, transport of macromolecules and metabolic processes [4]. Considerable evidence now supports the existence of multidirectional communication between the components of this local regulatory network [5, 6]. Communication is driven by the release of chemical mediators, such as neuropeptides, neurohormones, neurotransmitters, cytokines, chemokines, growth factors, and other regulatory molecules.

Fig. 4.1
figure 1

Intestinal barrier function. The intestinal barrier has evolved to guarantee homeostasis through the execution of basic weeping off functions, such as water secretion and intestinal peristaltism, and by the development of immunological surveillance. This barrier is composed by several levels of protection aimed at preventing and selecting toxin and antigen penetration across. The most external laters harbours mucus, enzymes, antimicrobial peptides and the intestinal microbiota. Just below, a single-cell layer of epithelial cells, sealed by intercellular junctions, regulates the transcellular and paracellular passage of substances. Intermingled goblet cells secrete mucins that dissolve in water to form mucus, a major contributor to the retention of secretions containing antibacterial peptides and digestive enzymes, and to keep epithelial hydration. The epithelium also displays microbial recognition receptors and is able to release immune mediators. Lamina propria leukocytes produce proteases and cytokines to modify epithelial secretory activity and permeability range of the epithelium. M cells are found in the follicle-associated epithelium of the Peyer’s patches and transport antigens from the luminal side to immune cells across the epithelial barrier. IgA is produced by plasma cells, and transported through, and secreted by, the epithelium to the luminal side. Both, the central and the enteric nervous system, interact with the immune system, the smooth muscle and the epithelium to regulate immune responses, absorption and secretion, motility, and also visceral sensitivity. Note: IEL intraepithelial lymphocyte

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The regulation of gut physiology is also achieved through the activity of both the enteric nervous system (ENS) and the central nervous system (CNS). ENS is an extensive neural network, also known as the second brain, containing approximately 100 million neurons embedded in the gastrointestinal lining, similar number to the spinal cord [7]. The ENS contains sensory neurons, inter-neurons, and motor neurons, which primarily control motility, absorption and secretion, but also visceral sensitivity. In addition, the ENS is wired with multiple terminals from ascending and descending CNS pathways that help to control gut function. To understand gut physiology and pathology, it is of particular importance to consider the role of the autonomic nervous system, and the hypothalamic pituitary-adrenal axis (HPA) because both systems also establish a vast and complex array of integrative and bidirectional interactions between the brain and the gut, the brain-gut axis.

The Intestinal Barrier

The intestinal barrier has evolved to guarantee homeostasis through the execution of basic weeping off functions, such as water secretion, to wash off harmful substances that may be present in the intestinal lumen, and by the development of a programme, that includes active immunological surveillance. One of the first steps to fight unwanted or harmful stimuli involves the release of mucus, defensins, secretory-immunoglobulin A, and other chemical mediators to the lumen [8]. In addition, the importance of maintaining epithelial permeability tight to prevent the passage of noxious substances, was emphasized in the early 1990s [9], and reiterated by many authors thereafter.

Structure and Function of Intestinal Barrier

Mucus

The entire intestinal mucosal surface is covered by a layer of mucus gel, thicker than 100 μm secreted by goblet cells (GCs). Mucus protects the epithelial lining from luminal sheer forces, adhesion and invasion by microorganisms, dietary, chemical and radiation toxins, and other antigens present in the intestinal lumen [10]. The mucus layer also contributes to the retention of mucosal secretions containing antibacterial peptides and digestive enzymes [11, 12] and keeps epithelial hydration. Mucus seems to participate in epithelial renewal, differentiation and integrity, and relates to other biological processes [13]. More recently, mucus has also been shown to enhance oral tolerance by imprinting dendritic cells with anti-inflammatory properties through the assembly of a galectin-3-dectin-1-FcγRIIB receptor complex that activated β-catenin, interfering with the expression of inflammatory, but not tolerogenic cytokines by dendritic cells [14].

Components of mucus include water, phospholipids, the negatively charged mucins (MUC), which provide a chemical barrier to protect the underlying epithelium, and a variety of trefoil factors and other antimicrobials such as secretory IgA [15], cathelicidins and defensins that provide the physical and immune protection against luminal agents [16]. Mucus secreted at the apical brush border binds the glycocalyx to form a viscoelastic gel with hydrophobic and surfactant properties, dependent on the presence of phospholipids at the most apical part. Hydrophobicity helps to fight enteric bacteria and to regulate gut permeability [17].

MUC represent the most abundant component of the mucus gel. MUC are huge glycoproteins composed of a central protein backbone rich in serine, threonine and proline. These glycoproteins are highly glycosylated by attached oligosaccharides, which contain blood group structures and are initiated by N-acetyl-galactosamine that is O-linked onto serine or threonine at the protein core [18–20]. These O-linked oligosaccharides are responsible for MUC properties. Up to 20 different MUC genes have been identified to date (MUC1 to MUC20) [21], with site and cell-specific expression. Several secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6) function as extracellular viscous secretions whereas others appear as membrane-associated mucins (MUC1, MUC3 and MUC4) in the glycocalyx [22]. MUC1–4 represent the most abundant secreted mucins in the human intestine. The first identified human secretory mucin was MUC2 that is also the principal secreted MUC [23], and is normally restricted to GCs [24]. In mice, it has been shown that colonic mucus consists of two layers with similar protein composition, being MUC2 the major structural component. The inner layer is firmly attached to the epithelium and functions as a barrier to prevent bacterial invasion while the outer layer is a loose matrix usually colonized by bacteria [25]. Thickness of the inner mucus layer varies down along the intestine according to luminal concentration of bacteria, being thicker at the highly colonized colonic segment, and thinner at the less colonized small intestine [26]. Baseline secretion of MUC is a constitutive pathway where small vesicles transport MUC directly to the cell surface where immediate and full exocytosis of their contents takes place. The release and secretion of packaged MUC is a different pathway regulated by specific stimuli including microbes and their products, and neuroendocrine and inflammatory/immune mediators. Mucus production is tightly regulated by different protein families, such as MUC and protein O-fucosyltransferase 1 (POFUT1) family members. Dysfunction of mucus secretion can lead to the development of intestinal inflammation as shown by the susceptibility of MUC2 KO mice to develop spontaneous colitis, and by a more severe intestinal response to the administration of dextran sulphate sodium (DSS) [27]. These mice also display impaired host resistance to parasitic infection [28], and over-enhanced susceptibility to Salmonella enterica serovar typhimurium [29]. Decreased production and alteration of the O-glycosylation profile of MUC2 has been associated with increased inflammation in ulcerative colitis [30, 31]. Moreover, increased susceptibility to ulcerative colitis [32] and Crohn’s disease [33] has been linked to a rare variable number of tandem repeat alleles of the MUC3 gene. Mice defective in intestinal POFUT1 exhibit chronic intestinal inflammation in association with an alteration of mucus-associated flora, goblet cell hyperplasia and hypertrophy and elevated production of mucus [34].

Resistin-like molecule (RELM)-β is a cysteine-rich protein also present in the mucus layer and specifically produced by intestinal GCs. RELM-β upregulates MUC2 and M1/MUC5AC gene expression in the human colonic HT29 cell line. Pretreatment of murine colon with RELM-β significantly attenuates trinitrobenzene sulphonic acid (TNBS)-induced colitis [35] while RELM-β deficient mice show increased susceptibility to T-cell-dependent TNBS-induced colitis. Therefore, available evidence suggests that RELM-β plays an important role in colonic inflammation [36].

Trefoil factors, a group of small cysteine-rich peptides, are also essential protective components of the mucus layer and contribute to mucosal repair, particularly, the trefoil factor 3 synthesized and secreted by intestinal GCs [37, 38]. Trefoil factor 3 deficient mice are highly susceptible to DSS, chemotherapy and radiation-induced colitis [39, 40], and display prominent hypoxia-elicited increases in intestinal permeability [41].

Epithelial Lining

The intestinal epithelium is a single polarized continuous layer of columnar cells of only 20 μm thick that covers the intestinal surface and separates the intestinal lumen from the internal milieu. Although it functions primarily as a physical barrier, it also regulates the absorption of dietary nutrients, water and electrolytes. The passage of molecules from the intestinal lumen to the lamina propria takes place mainly through two different routes: (1) The paracellular pathway, which allows small molecules (<600 Da) diffuse through tight junctions (TJs) located between adjacent intestinal epithelial cells; and, (2) The transcellular pathway, which allows the passage of larger particles through the epithelial cells via endocytosis or exocytosis processes [42].

The intestinal epithelium contains several stem cell-derived cellular types, such as absorptive enterocytes, GCs, Paneth cells, enteroendocrine cells, and M cells, as shown in panel 1 of Fig. 4.2. This epithelial population renews every 3–5 days from pluripotential stem cells located in the intestinal crypts to ensure cellular integrity all along the intestinal epithelium. Pluripotential stem cells migrate to the tip of the villus where final differentiation takes place [43]. Signalling cascades such as the wnt and the Notch pathway are involved in epithelial proliferation and differentiation, essential processes to regulate homeostasis in the intestinal epithelium [44].

Fig. 4.2
figure 2

Ultrastructure of the intestinal mucosa. Transmission electron micrographs of the intestinal epithelium and the lamina propria of the human jejunum. The intestinal mucosa is responsible for nutrient absorption and water secretion, which require a selectively permeable barrier. Panel 1—Intestinal epithelium. The epithelium functions primarily as a physical barrier between the external environment and the internal milieu. It is composed by enterocytes, secretory cells and immune cells, all supported on the basal side by a basement membrane underneath which the lamina propria harbors blood and lymph vessels, resident immune cells and nerve terminals. GC goblet cell, IEL intraepithelial lymphocyte, EC enterochromaffin cell, Ep epithelial cell. Panel 2—Intercellular junctions. The epithelial cells are polarized cells bound together through specific junctions. The apical junctional complex delineates the apical and the basal regions of the epithelial cells. It limits the uptake of microbial and food-derived antigens and prevents the passage of cellular elements across. TJs are located at the most apical site of the epithelium followed by the subjacent adherens junction and the desmosomes. TJ tight junction, AJ adherens junction, D Desmosome. Panel 3—Lamina propria. Most of the immune elements of the intestinal barrier are located in the lamina propria, where they develop innate and adaptive responses in coordination with the nervous system and the epithelium. Eo eosinophil, NE nerve endings, PC plasma cell, MC mast cell, L lymphocyte, Ep epithelial cell

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Enterocytes

Enterocytes are key elements of the epithelial lining. Although, the most important endeavour of these cells is to maintain the integrity of the intestinal physical barrier, enterocytes reinforce barrier strength by also developing immunologic activity. Enterocytes express innate immune receptors [45], act as non-professional antigen presenting cells, and release several chemokines and cytokines such as fractalkine [46] or thymic stromal lymphopoietin [47] involved in leukocyte recruitment and in dendritic cell regulation.

Enterocytes are tightly bonded to each other through the apical junctional complex that separates the apical membrane from the basolateral membrane. This apical junctional complex is composed TJs, adherens junctions, and desmosomes, as shown in panel 2 of Fig. 4.2. The junctional complex limits the uptake of microbial and food derived antigens and prevents the passage of cellular elements across. TJs are located at the most apical site of the epithelium and composed of intracellular and surface-membrane proteins. Intracellular proteins are zonula occludens (ZO)-1, ZO-2 and ZO-3, as well as cingulin. Surface-membrane or transmembrane proteins include occludin, claudins, and junctional adhesion molecules (JAMs). TJs seal the intercellular space and regulate intestinal permeability. Adherens junctions are located below TJs and mainly composed by e-cadherin, catenin, and actin filaments. These protein complexes provide the necessary strength to hold the cells together.

Occludin was the first TJ transmembrane protein identified. It belongs to the TJ-associated MAL and related proteins for vesicle trafficking and membrane link (MARVEL) proteins, and contains a MARVEL domain. The function of occludin remains to be elucidated. On one hand, occludin deficient mice do not show alterations in TJ assembly and permeability [48], but, on the other hand, occludin seems to play a role in the regulation of integrity rather than in the de novo assembly of the TJs [21]. Furthermore, in vitro observations suggest that occludin localization to the TJ complex is regulated by phosphorylation [49]. Regulation of occludin phosphorylation implicates several kinases including protein kinases C, mitogen-activated protein kinases (MAPKs), Rho kinases, and the Src-Family kinases [50]. When occludin is highly phosphorylated on serine and threonine residues, it is selectively located at the TJ. In contrast, occludin dephosphorylation at those residues by protein phosphatases, results in redistribution of the protein to the cytoplasm [24].

The claudin family of transmembrane proteins consists of 24 members with a molecular weight ranging from 20 to 27 kDa. Each member shows a specific organ and tissue distribution. This protein family plays a central role in the regulation of barrier function. Some claudins make up pores that allow preferential passage of specific ions, while others reduce the transit of specific ions. The strength, size, and ion selectivity of TJs is determined by claudins, as reflected by massive trans-epidermal water loss and death of mice within one day of birth affecting claudin-1 deficient mice [51]. Moreover, segmental barrier properties along the crypt-villus axis and throughout the length of the intestine do correlate with the disposition of claudins [52, 53]. In the human intestine, both ileal and colonic mucosa express tightening claudins-1, -3, -4, -5 and -7 [54, 55]. However, the expression of the permeability mediator claudin-2 is restricted to the crypt, in the colon [30, 56], yet detected in the crypt and the villus, in the small bowel [31]. Differences in the expression and distribution of claudins may reflect adaptation to specific physiological functions carried out by the different segments down the intestinal tract.

A third group of transmembrane receptors found at TJs is the family of JAMs. JAMs have been implicated in the construction and assembly of TJs [57], and in the regulation of intestinal permeability and inflammation [58]. JAM-A deficient mice display increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis to DSS-induced colitis [59]. More recently, reduced intestinal JAM-A expression has been described in irritable bowel syndrome (IBS) patients, possibly contributing to intestinal barrier dysfunction in these patients [60]. JAMs are also present on blood cells, such as leukocytes, thereby contributing to the process of trans-endothelial migration [61].

The TJ transmembrane proteins, claudins, occludin, and JAMs are linked to the actomyosin fibers of the cytoskeleton by members of the ZO family [62]. This association to the peri-junctional actomyosin ring seems crucial for the dynamic regulation of permeability at paracellular spaces. Interestingly, only ZO-1 and ZO-2 are relevant for claudin recruitment, TJ formation and for epithelial barrier function [63].

Far from being static, TJs are quite mobile structures that readily adapt to changing conditions and challenging stimuli. Regulation of intestinal permeability involves different functional pathways. Fast changes in permeability occur usually via myosin light chain kinase (MLCK)-mediated cytoskeleton contraction, and by endocytosis of TJ proteins [64, 65]. In contrast, lasting permeability disturbances involve the transcriptional modulation of TJ proteins, epithelial cell apoptosis and ultrastructural alterations in the epithelium [66].

Phosphorylation of myosin II regulatory light chain (MLC) induces actomyosin cytoskeleton contraction and increased TJ junction permeability. Rho GTPases have been shown to regulate TJs through redistribution of ZO-1, and reorganization of JAM-1 away from the TJ membrane [67]. Up-regulation of zonulin expression increased intestinal permeability to bacterial and gliadin exposure. In fact, this zonulin-mediated intestinal barrier defect has been advocated to play a central role in the origin of celiac disease [68] and type 1 diabetes [69].

Secretory Cells

The intestinal epithelium also houses different types of specialized epithelial called secretory cells that contribute to the reinforcement of the intestinal epithelial barrier, mainly goblet cells, Paneth cells and enteroendocrine cells.

GCs are scattered through the epithelial lining. GCs that mainly secrete mucins, but also trefoil peptides, RELM-β and Fc-γ binding protein. GC distribution varies throughout the gastrointestinal tract, the number increasing from the duodenum to the distal colon. The number of GCs is probably regulated by the intestinal microbiota because germ-free mice have less and smaller GCs than regular mice [70].

Paneth cells are located at the base of the crypts of Lieberkühn. Similar to the other intestinal epithelial cell types, they evolve from stem cells at the bottom of the crypt. Contrary to other cell types, Paneth cells migrate downwards, to the bottom of the crypt, where they synthesize and secrete antimicrobial peptides and other proteins to the intestinal lumen. Among them, lysozyme, α-defensins, TNF-α, and secretory phospholipase A2 type IIA, contribute to maintain host-microbe homeostasis and to protect stem cells from pathogens [71, 72]. Certain defects in Paneth may be linked to the pathogenesis of Crohn’s disease [73, 74] and necrotizing enterocolitis [75, 76].

Gut enteroendocrine cells spread all along the intestinal epithelium where they function as highly specialized chemoreceptors sensing changes in luminal osmolarity, pH and nutrient composition. Although they represent less than 1 % of the entire gut epithelial population, enteroendocrine cells constitute the largest endocrine organ of the human body. Products released by enteroendocrine cells include hormones, such as ghrelin, somatostatin, cholecystokinin, gastric inhibitory polypeptide, glucagon-like peptides and peptide YY, and neurotransmitters such as serotonin [77]. Enteroendocrine cells inform the brain-gut axis mostly through the activation of neural pathways [78].

The Intestinal Immune System

Mucosa-associated lymphoid tissue is a diverse and diffuse defence system found at most mucosal surfaces of the body, such as the respiratory system and the eye conjunctiva. The immune response generated by this system provides generalized immunization at all mucosal surfaces [79]. About 70 % of whole body’s immune cells reside within the gastrointestinal tract shaping the gut-associated lymphoid tissue (GALT), which is conformed in two different compartments: the organized immune inductive sites, and the diffuse effector sites.

Diffuse GALT is composed of two lymphocyte populations distributed at both sides of the basal lamina. Intraepithelial lymphocytes are found between epithelial cells, above the basal lamina. Lamina propria lymphocytes reside in lamina propria along with many other types of immune cell, such as eosinophils, dendritic cells, mast cells, macrophages or plasma cells (panel 3 of Figs. 4.2 and 4.3). The majority of intraepithelial lymphocytes are CD8+ T cells that function as surface gatekeepers of the intestinal barrier because the constantly monitor and respond against luminal bacteria and other antigens. Lamina propria lymphocytes constitute a much more heterogeneous population, approximately 50 % of which correspond to plasma cells, 30 % to T lymphocytes, and the remaining 20 % to macrophages, dendritic cells, mast cells and eosinophils. Resident B lymphocytes complete their maturation into plasma cells, mostly producing IgA, but IgM and IgG. Activated T and B-lymphocytes express α4β7 integrin and mucosal endothelial cells of Peyer’s patches, mesenteric lymph nodes and lamina propria of the small and large intestine constitutively express the mucosal addressin cell adhesion molecule-1 that interacts with α4β7 integrin to recirculate lymphocytes between the blood and the gastrointestinal tract [80].

Fig. 4.3
figure 3

Resident immunocytes in the intestinal mucosa. The majority of the immune cells within the body reside in the gastrointestinal tract (Gut-associated lymphoid tissue, GALT), and are distributed in two different compartments: the organized inductive sites, and the diffuse effector sites. The diffuse GALT is composed of intraepithelial lymphocytes, between the epithelial cells, and the lamina propria lymphocytes, which reside below the basal lamina, along with other immune cells. The figure shows intestinal micrographs (×400 magnification) processed for H&E staining to identify mucosal eosinophils (1), and immunohistochemistry for T-lymphocytes (2, CD3+), B-lymphocytes (3, CD20+), macrophages (4, CD68+), plasma cells (5, CD138+), and mast cells (6, CD117+)

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Inductive sites of the GALT include organized lymphoid structures in the small intestine such as Peyer’s patches, mesenteric lymph nodes, isolated lymphoid follicles, and lymphocytes and antigen-presenting cells. Peyer’s patches are macroscopic lymphoid aggregates found at the submucosal levels in the antimesenteric border of the intestine. The follicle-associated epithelium covering Peyer’s patches contains M cells, another special cell type that plays a role in monitoring the gut lumen and maintaining intestinal barrier function. M cells display several unique properties including apical microfolds instead of microvilli, no mucus layer, and a reduced glycocalyx, which facilitate the capture of luminal antigens and microorganisms and their transport to contact underlying immune cells [81]. Peyer’s patches also contain antigen-presenting cells, mainly dendritic cells, but also macrophages. These antigen-presenting cells capture luminal antigens (taken up by M cells in the Peyer’s patch dome), to further process and present them to immunocompetent cells in association with the major histocompatibility complex.

Innate immunity is present in both animals and plants [82]. It serves the host defence via immediate, but non-specific, responses to a wide variety of pathogens. The main components of innate immune response include pattern recognition receptors (PRRs), and antimicrobial peptides.

PPRs are a class protein that responds to small molecular sequences consistently found on pathogens, named pathogen-associated molecular patterns (PAMP). PRRs include Toll-like receptors (TLRs) and Nod-like receptors (NLRs).

The TLR family consists of at least 13 transmembrane receptors containing a large leucine-rich repeats extracellular domain that recognizes different bacterial, viral, parasite or self-derived ligands, such as lipopolysaccharide, peptidoglycan, muramyl dipeptide, lipoteichoic acids, and bacterial DNA. After activation upon PAMP recognition, TLRs initiate downstream signalling cascades, leading to transcriptional responses and to the initiation of both innate immune responses (macrophage activation and induction of antimicrobial peptides for various cell types) and the adaptive immune response (induction of T cell responses and maturation of dendritic cells) [83]. In many tissues, mast cells, dendritic cells, monocytes/macrophages and B cells express TLRs [84]. Healthy intestinal epithelial cells express relatively low levels of TLRs, such as TLR-4, perhaps explaining why lipopolysaccharide does not induce a potent inflammatory response in normal intestine [85]. By contrast, and consistent with the idea that chronic intestinal inflammation may be the result of uncontrolled responses to components of the intestinal bacterial flora, the intestinal epithelium of patients with inflammatory bowel disease (IBD) shows increased expression of TLR-4 [86]. The cellular localization of TLRs is also influenced by the polarized epithelial cell organization. TLR5 is expressed on the basolateral surface of intestinal epithelia only, where if becomes stimulated by luminal flagellin exposure when disruption of the epithelial barrier. Therefore, its localization prevents inappropriate stimulation by flagellin, but allows recognition of invasive pathogens [87]. Similarly, TLR9 activation through apical and basolateral surface domains induces distinct transcriptional responses. Whereas basolateral TLR9 strongly stimulates proinflammatory chemokine secretion, through NF-kappaB activation, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation conferring mucosal tolerance towards microbial exposure [88].

NLR constitutes a large family of 23 intracellular PRRs, being nucleotide-binding oligomerization domain (NOD)1, NOD2 and NALP3 the most extensively described. NOD1 and NOD2 recognize intracellular bacterial cell products, and NALP3 responds to multiple stimuli to form a multi-protein complex termed the NALP3 inflammasome, which promotes the release of the IL-1 family of cytokines. Most NLRs share a similar structure consisting of a centrally located NOD, a C-terminal leucine-rich repeat that detects PAMPs, and a variable N-terminal domain that is critical for downstream signalling through the recruitment of adaptors or effector molecules [89]. NOD1 recognizes γ-d-glutamyl-meso-diaminopimelic acid, which is found in the peptidoglycan structures of all gram-negative as well as in several gram-positive bacteria [90]. In contrast, NOD2 recognizes muramyl dipeptide, which is found in nearly all gram-positive and gram-negative organisms [91]. Upon ligand recognition, NOD1 and NOD2 induce the activation of NF-kappaB and MAPKs pathways leading to the activation of both innate and adaptive immune responses. In contrast, other NLRs such as Ipaf and cryopyrin respond to microbial components through the assembly of multiprotein complexes termed “inflammasomes” that promote caspase-1 activation to generate the proinflammatory cytokines IL-1β and IL-18 [92]. NOD1 is expressed by intestinal epithelial cells [93] while NOD2 expression is predominantly found in monocytes and Paneth cells [73]. Both NOD1 and NOD2 have been shown to modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis [94]. In addition, NOD2-deficient mice display an increased load of commensal resident bacteria, and a diminished ability to prevent intestinal colonization by pathogenic bacteria [95]. NOD-2 mutations have been identified in Crohn’s disease patients and could be related to an impaired release of antimicrobial peptides from Paneth cells [96].

Antimicrobial peptides are endogenous antibiotics that are constitutively expressed in intestinal epithelial cells, yet may be also inducible in immune cells and Paneth cells [97]. They include compounds such as lactoferrin, hepcidin, bactericidal/permeability increasing protein, lysozyme and overall, defensins and cathelicidins.

Defensins are a family of small cationic peptides (29–45 amino acids) that exhibit a wide and potent antimicrobial activity spectrum against gram-negative, and gram-positive bacteria, fungal and yeast, parasites, viruses, and even tumor cells [98]. Defensins have been identified in both prokaryotes and eukaryotes. Although structurally different, most defensins display cationic and amphiphilic properties which confer them the capacity to permeabilize the bacterial cell membrane. In mammals, these peptides are expressed in mucosal epithelial cells and phagocytes, but also are released into the intestinal lumen, several grams daily, by Paneth cells [99]. Defensins act as effector and regulatory molecules of the innate immune response. In addition, defensins also enhance adaptive response acting on phagocytic cells and mast cells to induce the release of inflammatory mediators and to regulate the complement system. Defensins also interact with dendritic cells and T cells to increase antigen-specific immune response [100].

These peptides are classified as α and β-defensins according to their disulphide bond pairing pattern. The human α-defensins 1–4, conventionally referred as to neutrophil defensin (human neutrophil peptide, HNP), although defensins HNP1-3 are also expressed in epithelial cells of inflammed mucosa [101]. In contrast, human α-defensin 5 and 6 (HD5 and HD6) are only expressed in Paneth cells of the small intestine [102]. HD5 has been shown to induce IL-8 expression on intestinal epithelial cells [103], and to protect mice from DSS colitis and Salmonella infection [104]. More recently, HD6 has been shown to form fibrils and nanonets that surround and entangle bacteria to protect the small intestine against invasion by diverse enteric pathogens [105].

Human β-defensin-1 is constitutively expressed in the small intestine and the colon. In contrast, Human β-defensins-2-4 expression is inducible [106] in inflammatory conditions such as IBD [107, 108] or infection by enteroinvasive bacteria [109].

The other major class of antimicrobial peptides is the cathelicidin group. In mammals, about 35 members have been identified, but only one in humans: hCAP18/LL37 [110]. Although regarded as neutrophil specific, hCAP18/LL37is also expressed in other leukocytes, keratinocytes and epithelial cells of the respiratory, genitourinary and gastrointestinal tract [111], and in human breast milk [112].

Expression of hCAP18/LL37 in human colonic epithelial cells has been related to cell differentiation [113]. Infection of intestinal epithelial cells by Shigella spp. inhibits the expression of hCAP18/LL37 [114], while bacterial components such as sodium butyrate [115] or TLR-ligands such as bacterial DNA [116] induce its expression.

Acquired immunity is restricted to vertebrates and constitutes a second line of defence against pathogens. It is driven by B and T lymphocytes through specific receptors and confers protection against re-exposure to the same antigen. Antigen binding to these receptors results in clonal expansion of these cells and the initiation of a directed immune response. Functionally speaking, within the adaptive immunity, we can distinguish inductive and effector compartments. Antigen presentation and naive T and B-lymphocytes activation occurs in the inductive compartment. In the effector compartment sensitized cells against different antigens extravasate and differentiate to carry out the destruction of pathogens. IgA secretion has been shown to be regulated through TLR-signalling [117] but also by changes in the composition of intestinal Microbiota [118].

Intestinal Barrier Dysfunction

Stress, Hormones and Neurotransmitters

Stress represents a threat to the internal homeostasis. In response to stress, a coordinated response is initiated to maintain stability through the autonomic, endocrine, and immune systems. The main systems activated during the stress response are the sympatho-adrenomedullary, a component of the sympathetic division of the autonomic nervous system, and the HPA axis. The autonomic nervous system provides, through its sympathetic and parasympathetic arms, the fastest response to stressor exposure, leading to rapid alterations in physiological state through neural innervation of end organs. Stress activation of the HPA axis stimulates the parvocellular neurons in the paraventricular nucleus of the hypothalamus to secrete corticotropin-releasing-factor (CRF), which in turn travels to the anterior pituitary to promote the synthesis of corticotropin (ACTH) [119]. ACTH, when released into the systemic circulation, activates the adrenal cortex to induce cortisol and corticosterone secretion that circulate through the bloodstream to reach every tissue [120]. Adaptation to stress through the activation of the sympatho-adrenomedullary system and the HPA axis to maintain homeostasis is called “allostasis”. However, excessive stress exposure impairs this adaptive response, eventually predisposing these subjects to the development of disease or to exacerbation of previous existing ones [121], specially in stress-sensitive disorders, like IBS.

At the experimental level, different type of stresses, acute and chronic, physical or psychological, have been shown to influence properties of the intestinal barrier function, including as ion and water secretion, intestinal permeability, mucus secretion, and also intestinal flora. Ion and water secretion allows the intestine to wash away noxious substances present in the intestinal lumen, preventing adhesion to the mucosal surfaces and penetration to the lamina propria. The jejunum of rats submitted to restraint stress or cold restraint stress was found to show an increase its baseline short-circuit current, indicative of enhanced anion secretion [122]. Later, it was observed that peripheral CRF and repetitive exposure to water avoidance stress reproduced stress-induced rat jejunal and colonic epithelial barrier dysfunction via cholinergic and adrenergic nerves and mast cells [123, 124]. More recently, it has been shown that chronic psychosocial stress also activates mucosal mast cells and increases baseline short-circuit current in both the jejunum and the colon [125]. In humans, studies using jejunal segmental perfusion techniques reveal that acute physical or psychological stress either reduce net water absorption or increase secretion in healthy subjects and in patients with food allergy [126, 127] through the parasympathetic nervous system and mast cell activation [128]. More recently, we have extended these observations to show that in healthy female volunteers that intestinal water secretion during cold pain stress was significantly reduced in those with moderate background stress compared to those with low stress [129]. This observation could indicate a loss of regulatory mechanisms in subjects suffering from continuous life stress.

Both paracellular and transcellular permeability to small and large molecules increased in response to acute and chronic stress in the rodent jejunum and colon [130–133]. Several mechanisms, including mast cells, CRF [134], MLCK, and cytokines like interferon gamma, and interleukin-4 [135] have been implicated. In humans, it is known that surgery, trauma, and gastrointestinal infections [136] increase intestinal permeability. CRF has been shown to enhance transcellular uptake of macromolecules in human colonic mucosa via CRF-R1 and CRF-R2 receptors, located on subepithelial mast cells [137]. Unpublished observations from our group indicate that intravenous CRF increased intestinal permeability in healthy subjects and in IBS patients [138]. Acute psychological stress also increases small intestinal permeability in humans and peripheral CRF reproduces the effect of stress and mast cell stabilization blocks the effect of both stress and CRF, suggesting the involvement of mast cells [139]. Cold pain stress also increased intestinal permeability in female healthy subjects, although this response was larger in women with moderate background stress. Increased intestinal permeability has been found in diarrhoea prone IBS patients [140]. These findings provide new insight into the complex interplay between the central nervous system and gastrointestinal function in man.

Acute stress causes mucin release in the rat colon, along with enhanced secretion of rat mast cell protease II and prostaglandin 2. These changes were reproduced by intravenous or intracerebral injection of CRF in non-stressed rats, and were inhibited by the administration of a CRF antagonist or a mast cell stabilizer [141]. In addition, stress-induced release of mucin was abolished in mast-cell deficient mice, highlighting a key role of mast cells in stress-mediated mucin release [142]. In contrast, rats submitted to chronic stress displayed mucus depletion along with increased bacterial adhesion and penetration into enterocytes [143].

Stress can also induce microbiological changes in the intestinal flora. Maternal separation in infant rhesus monkeys decreased faecal bacteria, especially Lactobacilli, and increased their susceptibility to opportunistic bacterial infections [144]. Similarly, prenatal stress reduced the overall numbers of Bifidobacteria and Lactobacilli in the newborn infants [145]. Interestingly probiotic treatment ameliorates stress-induced changes in the gastrointestinal tract [146] and attenuates the observed Lactobacilli reduction in maternally-deprived rat pups [147]. In addition to these microbiological changes, dexamethasone administration in rats enhanced bacterial adherence to the mucosa, decreased secretory-immunoglobulin A secretion, and increased intestinal permeability [148]. More recently, Söderholm et al. showed that chronic psychological stress in rats, leads to an increased antigen and bacterial uptake in follicle associated epithelium from Peyer’s patches [149] as well as in the villous ileal and colonic epithelium. Emotional stress during take-off in cosmonauts induced changes in faecal Bifidobacteria and Lactobacillus, as well as an increase in Escherichia coli, whereas a substantial increase in Enterobacteria and Clostridia was found after the flight [150]. These stress-induced changes in the faecal flora have been related to catecholamine release into the intestinal lumen and/or into the systemic circulation, as the addition of various catecholamines to cultures of gram negative bacteria resulted in dramatic increases in growth of E. coli, Yersinia enterocolitica and Pseudomonas aeruginosa [151].

Mast cells are known to modulate stress-mediated responses of the epithelial barrier function, to orchestrate the mucosal immune function and to participate in the defence against bacteria [152, 153]. To exert these functions, enteric mast cells are strategically located within the gastrointestinal tract, developing an optimal sensory and effector interaction within the local regulatory neuroendocrine networks. Upon activation, mast cells act as effector cells, through the selective (piecemeal degranulation) (Fig. 4.4) or massive release (anaphylactic degranulation) of preformed or newly produced biological mediators. More relevant to stress-mediated inflammation is their ability to communicate, bidirectionally, with both the enteric, autonomic and central nervous systems. Anatomical contacts between mast cells and enteric nerve fibres have been demonstrated in the human gastrointestinal mucosa and these contacts increase, when inflammation is present [154]. An increase in the nerve-to-mast cell proximity in the colonic mucosa of IBS patients has been positively correlated with the severity and frequency of abdominal pain [155]. This mast cell-enteric nerve interaction provides a physical substrate for bidirectional communication between the CNS and the gut, by which stress might influence gastrointestinal physiology. This is reflected in vivo by the release of mast cell products into the lumen of the human small intestine after cold stress, which is accompanied by increased epithelial secretion [128]. Mast cell mediators released after degranulation can sensitize mesenteric afferents and nociceptive receptors [156]. Among the potential mast cell mediators involved, both histamine and serotonin induce intestinal secretion of water, electrolytes and mucus. In addition, mast cells from IBS patients release more histamine and tryptase than intestinal mast cells from normal subjects [157] a fact that has been linked to the generation of visceral hypersensitivity, through the activation of proteinase-activated receptors type 2. These receptors can modulate enteric neurotransmission, secretion, motility, epithelial permeability, and visceral sensitivity, and are also known to regulate intestinal inflammation [158]. However, altered expression of histamine H1 and H2 receptor subtypes has recently been reported in mucosal biopsies from distal gut of IBS patients, suggesting that these receptors could also play a role in these processes [159].

Fig. 4.4
figure 4

Intestinal mast cells. Enteric mast cells are known to modulate the epithelial barrier function, to orchestrate the mucosal immunity and to participate in the defence against bacteria. They are strategically located within the gastrointestinal tract, developing sensory and effector interactions within the local regulatory neuroendocrine networks. Upon activation, mast cells act as effector cells, through the selective (piecemeal degranulation) or massive release (anaphylactic degranulation) of preformed or newly produced mediators. The figure shows transmission electron micrographs of ultrastructural characteristics of mucosal mast cells: a resting mast cell in health (H), with granules filled (white arrows) and no signs of degranulation; piecemeal degranulation in a mast cell from a patient with irritable bowel syndrome (IBS), identified by partial or total emptiness of granules content (black arrows) and intact granules (white arrow); and anaphylactic degranulation in a mast cell from a food allergy patient (FA), identified by fusion of granule membranes devoid of content (black arrow). Barr indicates 2 μm

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CRF and related peptides are the most important neuroendocrine factors mediating the effects of stress, both at the central and peripheral level. CRF urocortin (Ucn) 1, Ucn 2 and Ucn 3 exert their effects after binding to G protein-coupled receptor subtypes, CRF-R1 and CRF-R2, signalling through cAMP [160]. After physical or psychological stress, neural or immune release of CRF and urocortins mediate autonomic, hormonal, and behavioural responses to stress and stimulate the ENS to modulate gastrointestinal motility and secretion [161–163]. Increased CRF and urocortin expression has been demonstrated in the colonic mucosa of IBD patients [164, 165].

Vasoactive intestinal peptide is also involved in the regulation of chloride secretion, mucin release, paracellular permeability and epithelial cell proliferation [166, 167]. Psychological stress increases vasoactive intestinal peptide levels in the small intestine of mice [168] and vasoactive intestinal peptide has been implicated in the regulation of the intestinal barrier function, through its direct effect on tight junction-associated protein, ZO-1, in epithelial cells [169].

Substance P participates in gut inflammation by interacting mainly with the neurokinin-1 receptor, expressed on nerves, epithelial, endothelial and smooth muscle cells, and immune cells, such as mast cells, macrophages, and T cells [170]. This neuropeptide has been found to stimulate macrophage and eosinophil secretion of pro-inflammatory cytokines, to increase NK cell activity and migration, and to activate the release of chemokines from leukocytes. It also induces the release of vasoactive mediators from mast cells, contributing to chloride secretion, intestinal permeability, vascular leakiness and oedema at sites of inflammation, modulating diarrhoea, inflammation, and motility [171]. Substance P mediates stress-induced CRF expression in mice eosinophils, and eosinophil-derived CRF is responsible for mast cell activation and consequently, epithelial barrier dysfunction [172].

Nerve growth factor (NGF) has been involved in the development of stress-induced barrier dysfunction [173] and hyperalgesia during inflammation [174, 175]. These effects seem to be mediated by CRF and mast cells [176, 177]. Maternal deprivation has been shown to induce hyperalgesia to rectal distension and to enhance colon permeability in association with elevated NGF expression [173]. A subsequent study from the same group showed that CRF, acting through its receptor CRF-R1, stimulated NGF release from mast cells, which in turn increased gut paracellular permeability [178]. More recently, norepinephrine has been shown to induce visceral sensitivity to colorectal distension by increasing the expression of NGF in the rat colon wall [179]. These findings support the importance of NGF in stress-induced visceral hypersensitivity, but also in stress-induced barrier dysfunction.

Sex steroids also play a role in modulating intestinal barrier, although conflicting results have been described. Estrogen can bind to two different receptors named estrogen receptor-α and β. Estrogen receptor-α mediates estrogen signalling in the development of secondary sex characteristics, and the regulation of the menstrual cycle and sperm maturation [180]. In contrast, estrogen receptor-β is mainly expressed in epithelial cells and is the most abundant estrogen receptor in the colon [181]. Both progesterone and estradiol have been shown to reduce chloride secretion in intestinal epithelial cells [182, 183], whereas estradiol has also been found to reinforce epithelial permeability [184], and to up-regulate JAM-A and occludin expression [185].

Other hormones have been involved in the regulation of intestinal barrier function (Table 4.1).

Table 4.1 Hormones and intestinal barrier
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Infections

Intestinal pathogens have developed specific strategies to gain access to the lamina propria. Strategies include direct TJ disruption, the production of toxins that induce fluid and electrolyte secretion, and the activation of the inflammatory cascade [186]. Vibrio cholerae can directly alter TJs through its cytotoxin hemagglutinin protease, a metalloproteinase that disrupts occludin-ZO-1 interactions leading to TJ and cytoskeleton anchorage destabilization [187]. In addition, other toxins have been involved in TJ disruption by V. cholerae such as the RTX toxin, that crosslinks actin inducing cell rounding and increased permeability [188], or the ZO toxin, that fragments ZO-1 and occludin and disrupts the actin cytoskeleton [189, 190]. Clostridium difficile infection produces two distinct exotoxins, Toxin A and B (TcdA and TcdB), that through RhoA GTPases inactivation cause actin filament disaggregation and cell rounding, resulting in increased paracellular permeability [191, 192]. Recent findings suggest that toxin A could even disrupt directly TJ proteins [193]. Clostridium perfringens enterotoxin utilizes claudin-3 and 4 as receptors [194] to bind the enterocyte surface where it forms small protein complexes in the plasma membrane that interact with other proteins forming a large complex, that at the end triggers massive permeability changes [195]. Enteropathogenic E. coli infection directly disrupts TJ through occludin dephosphorylation and dissociation from TJs to the cytoplasma [196] and MLC phosphorylation [197] enhancing intestinal permeability.

Intestinal Microbiota

Intestinal microbiota has been shown to influence intestinal barrier function and the brain-gut axis [198, 199]. Intestinal microflora displays several important functions to maintain gut homeostasis, such as nutrient digestion, vitamin and hormone production and most importantly, protection from microbial colonization, achieved through competition for intestinal nutrients and for attachment sites [200]. Probiotics are live microorganisms which, when consumed in adequate amounts, confer a health benefit on the host. Increasing evidence suggests that probiotics implement intestinal epithelial homeostasis and enhance barrier tightness and integrity. In contrast with pathogens, probiotics have been shown to increase occludin expression [201], and to enhance ZO-2 expression in parallel to its redistribution towards the cell boundaries via silencing of PKCζ [202] thereby leading to TJ stabilization and the restoration of the epithelial barrier. Specific Lactobacilus salivarus strains prevent hydrogen peroxide-induced reduction in transepithelial resistance when added to Caco-2 cell monolayers [203]. Similarly, Lactobacillus rhamnosus GG improves intestinal barrier function in the immature murine gut through the induction of claudin 3 expression [204], the regulation of apoptosis and the promotion of cytoprotective responses [205]. Interestingly, probiotics have also demonstrated beneficial effects in other tissues such as the skin barrier [206] or the respiratory tract [207, 208].

There is a significant body of evidence indicating that probiotics can also prevent intestinal barrier damage in conditions such as IBD or experimental stress. In rats, DSS-induced colitis was ameliorated by Lactobacillus reuteri decreasing the bacterial translocation from the intestine to mesenteric lymph nodes [209]. E. coli Nissle 1917 has been shown to confer protection against murine DSS colitis-associated increase in mucosal permeability through up-regulation of ZO-1 expression [210]. Moreover, a probiotic mixture of Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus plantarum and Bifidobacterium breve helped to maintain the integrity of colonic mucosal barrier in the DSS model by down-regulating macrophage nitric oxide production and by enhancing mucus production [211]. In this model, the administration of a probiotic mixture prevented not only the decrease in TJ proteins expression, but also the increase of epithelial apoptotic ratio induced by acute colitis [212]. Furthermore, in patients with severe pouchitis, probiotics were able to restore the mucosal barrier, as they decreased E. coli K12 passage through the intestinal epithelium in Ussing chambers [213].

Probiotics also play a role in stress-induced intestinal damage and psychiatric comorbidity. Lactobacillus farciminis has been shown to suppress stress-induced hyperpermeability and endotoxemia, and to prevent HPA axis response and neuroinflammation in rats submitted to partial restraint stress [214]. Probiotic administration to mice submitted to food and mobility restriction increased IgA producing cells, CD4+ cells in the lamina propria of the small intestine, and secretory IgA in the lumen and also reduced the levels of IFN-γ [215]. Bifidobacterium lactis CNCM I-2494 has been shown to suppress gut hypersensitivity and colonic barrier disruption induced by partial restraint stress in rats [216, 217]. In the last years, attention has been also pointed to the potential role of microbiota in the pathophysiology of psychiatric disorders such as depression and anxiety [218] and neurodevelopmental disorders such as autism. Interestingly, treatment with the human commensal Bacteroides fragilis restores gut permeability, alters microbial composition, and ameliorates defects in communicative, stereotypic, anxiety-like and sensorimotor behaviors in a mouse model of the autism spectrum disorder [219]. Since psychiatric comorbidities are highly common in functional gastrointestinal disorders, the emerging role of microbiota and probiotics in the regulation of intestinal and brain barrier function and its implication in behavioral changes in the host certainly will boost investigations in this field in the years ahead.

Inflammatory Mediators

Several inflammatory mediators have been involved in intestinal barrier regulation. In vitro experiments with epithelial cell monolayers demonstrated that interferon-γ and TNF-α induce epithelial barrier dysfunction through MLCK up-regulation and MLC phosphorylation [220, 221], although they can also disrupt intestinal permeability through down-regulation of occludin transcription [222] and up-regulation of the channel-forming TJ protein claudin-2 expression. In addition, TNF-α but also IL-1β have been shown to inhibit electrogenic sodium absorption in the rat distal colon [223], and mice injected with TNF-α present diarrhoea as a consequence of Na+/H+ exchange inhibition [224].

Similarly, IL-13 and IL-4 increased paracellular permeability in a dose- and time-dependent fashion and IL-4, but not IL-13, stimulated chloride secretion in T84 cells [225] through a PI3K pathway [226]. In contrast, IL-10 has been identified as a protector cytokine in barrier function as the addition of this cytokine to T84 cells prevents interferon-γ-induced disruption of T84 monolayer barrier integrity and limits chloride secretion [227]. Moreover, IL-10 deficient mice display increased intestinal permeability [228] and most importantly, develop spontaneous colitis [229], suggesting that increased permeability predisposes to intestinal inflammation.

Although, beyond the limits of this chapter. It is to know that many other cytokines have been involved in barrier function such as IL-17A, IL-17F, IL-22, and IL-26, interferon-α, interferon-β, transforming growth factor-α, and -β [230, 231].

Nutritional Factors

Some dietary compounds are able to induce intestinal barrier dysfunction in susceptible individuals such as in celiac disease and food allergy. The gliadin fraction of wheat gluten is the environmental triggering of celiac disease. In genetically predisposed subjects gluten exposure may lead to increased intestinal permeability and inflammation. Recent evidence has shown that the increase in intestinal permeability occurs through the activation of the zonulin pathway in a MyD88-dependent fashion [232]. The protein zonulin is the target of the Zot toxin of the V. cholerae and has been show to play a pivotal role in TJ regulation in different autoimmune disorders such as type 1 diabetes and celiac disease [233]. Food allergies are adverse reactions against food antigens that are IgE and mast cell mediated. Altered intestinal permeability has also been involved in the pathophysiology of food allergy, as these patients display an enhancement of intestinal permeability even in the absence of food allergens [234]. Moreover, patients under tacrolimus treatment have been shown to develop new-onset food allergies that could be related to tacrolimus-induced increase in intestinal permeability [235].

In contrast with these observations, several diet products such as glutamine or butyrate have been shown to exert a protective effect on the intestinal barrier. Butyrate, a short chain fatty acid produced by intestinal microbial fermentation of dietary fibres, maintains intestinal barrier function through an increase in mucus production [236] and an enhancement in TJ protein expression [237]. Glutamine has also been shown to protect intestinal barrier function through the regulation of TJ proteins such as claudin-1, and occludin [238].

Drugs and Toxins

Ethanol has been shown to promote separation of ZO-1 proteins in Caco-2 monolayers and disassembly and displacement of perijunctional actin and myosin filaments from the perijunctional areas and MLCK activation [239]. Recent findings point to one of its metabolites, acetaldehyde, as the main toxic product for intestinal barrier because it raises tyrosine phosphorylation of ZO-1, e-cadherin, and β-catenin [240]. Further investigations revealed that the deleterious effects of ethanol require the presence of resident microflora, to oxidize ethanol into acetaldehyde in situ, and downstream mast cell activation [241], and that the ethanol-mediated increase in intestinal permeability is modulated through iNOS-mediated activation of RhoA [242] and IL-22 [243].

NSAIDs can increase intestinal permeability. Several factors play a role in NSAIDs-induced intestinal barrier dysfunction. In vitro experiments with gastric epithelial monolayers showed that barrier dysfunction was associated with decreased expression of claudin 7 and involved phosphorylation of p38 MAPK [244]. NSAIDs also affect intestinal barrier through inhibition of intestinal epithelial restitution by decreasing calpain activity and membrane-associated expression of calpain-2 [245], and also through the increase of intestinal NO synthase [246].

Other drugs causing intestinal barrier dysfunction appear in Table 4.2. It is of particular interest the development of new drugs, such as larazotide, that may decrease intestinal permeability in celiac disease by acting on TJs.

Table 4.2 Drugs and intestinal barrier
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Other Disorders Associated with Barrier Dysfunction

Many other conditions such as chronic kidney disease [247], type 1 diabetes [248], primary biliary cirrhosis and primary sclerosing cholangitis [249], liver cirrhosis [250], alcoholic liver disease [251], autoimmune thyroiditis [252] and IgA nephropathy [253] have been associated with TJ dysfunction. In addition, some life threatening conditions have been related to intestinal barrier dysfunction and translocation of bacteria or/and endotoxin from gastrointestinal tract. In this line, hemorrhagic shock has been associated with increased intestinal permeability and bacterial translocation [254] through mucus damage and the generation of free radical species [255]. Estrogens exert a protective role against hemorrhagic shock-induced gut and lung injury by the activation of estrogen receptor-α, β or both [256] receptors. Similarly, gut inflammation and loss of gut barrier function has been related to splachnic ischemia-reperfusion through HIF-1 activation [257]. Multiple injured patients also show an increased intestinal permeability that correlates with IL-6 levels [258]. Severe burn injury also results in the loss of intestinal barrier function involving MLCK-dependent MLC phosphorylation signalling pathway [259] and p38 MAPK activation [260] in a TLR-4-dependent process [261].

Intestinal Barrier and Disorders of the Brain-Gut Axis

The pathophysiology of several gastrointestinal disorders involves intestinal barrier dysfunction and dysregulation of brain-gut interactions, particularly functional gastrointestinal disorders including IBS and functional dyspepsia. In recent years, due to new imaging techniques, such as positron emission tomography, it has been possible to characterize the role of the CNS in modulating gut motility and visceral pain in patients with functional gastrointestinal diseases. There is significant overlap between the brain regions responsible for modulating visceral sensitivity and regions involved in emotion processing in these patients. IBS patients display a higher activation of the anterior cingulate cortex in response to rectal distension [262] that correlates with the presence of psychosocial disorders when compared to healthy subjects [263].

At the peripheral level, mucosal inflammation, increased intestinal permeability and visceral hypersensitivity are findings associated with clinical manifestations of IBS. Mast cells play a key role in IBS pathophysiology because they modulate intestinal permeability, and target visceral afferents involved in abdominal pain [155]. Stress has been associated with the development, exacerbation and perpetuation of IBS through the brain-gut-axis. Early life stress plays a major role in the vulnerability of individuals to develop IBS in adult life [264–266]. Post-traumatic stress syndrome or sexual abuse are also important risk factors in the development of IBS and functional gastrointestinal disorders [267] and both acute psychological and physical stress have been associated with enhancement of visceral sensitivity [268] and small-intestine motility in IBS [269].

Functional dyspepsia is characterized by postprandial fullness and early satiation or by epigastric pain or burning in the absence of an organic cause Functional dyspepsia has been show to share some of the pathophysiological features of IBS. Particularly, patients with functional dyspepsia display low-grade inflammation in the duodenal mucosa, characterized by an increased infiltration of mucosal mast cells and eosinophils, and increased duodenal permeability [270]. Acute gastroenteritis has been shown to be a risk factor for functional dyspepsia development [271], as well as the presence of psychosocial comorbidities such as anxiety and depression [272], and life stress [273].

Stress, acting through the brain-gut axis, also modulates intestinal inflammatory conditions such as IBD. Social Gibbon monkeys submitted to social upheaval develop spontaneous colon inflammation [274]. Intracolonic infusion of TNBS induced a significantly higher inflammatory reaction in maternally deprived rats than in control animals [275]. Collins et al. [276] found that rats recovering from TNBS-induced colitis and submitted to mild restraint stress displayed a significant increase in myeloperoxidase activity. Moreover, overt inflammation was induced when animals were exposed to stress in combination with a small dose of TNBS, suggesting an additive effect [277]. In keeping with these findings, a significant association between stress and relapse in IBD has been reported, especially in patients with ulcerative colitis [278, 279]. Although the mechanism underlying the association between stress and IBD remains unclear, disturbances of brain-gut axis, peripheral neuroendocrine-immune interactions and altered intestinal barrier function [280–284] have been demonstrated in IBD patients [285].

Finally, a heterogeneous group of conditions associated with chronic manifestations affecting the CNS and the gut may possibly reflect the existence of primary or secondary alterations of brain-gut axis, intestinal microbiota and barrier function. This is the case of diabetes and the metabolic syndrome [286], liver encephalopathy [287], neuropsychiatric disorders [288], autism [289], chronic fatigue [290] or fibromyalgia [291], although the ultimate pathophysiological mechanisms are not well known.