**1.5 Mechanisms of passage of different molecules through the intestinal epithelial barrier**

The intestinal epithelium is a single layer of cells that covers the intestinal mucosa, separating it from the lumen and has two critical functions: first, it acts as a barrier to prevent the passage of harmful intraluminal entities including antigens, foreign microorganisms and their toxins. Its second function is to act as a selective filter that allows the translocation of essential nutrients, electrolytes and water, from the intestinal lumen to the circulatory stream. Enterocytes have a high transport activity because they have ion channels, transporters and pumps in the apical and basolateral membranes. Net fluid absorption in the intestine is the result of the balance between absorption and secretion. This transport is carried out selectively via two routes: the paracellular route and the transcellular route. The paracellular pathway allows 85% of the total passive trans-epithelial flow of molecules through the space between two adjacent epithelial cells and is regulated by TJs, which have pores of different sizes, limiting and selecting the passage of the molecules. This pathway constitutes an effective barrier for the passage of luminal antigens and is decisive for establishing intestinal permeability [104].

Transcellular transport involves the transportation of solutes through the enterocyte membrane. There are several mechanisms that mediate the passage of molecules through the transcellular pathway. Small-sized lipophilic and hydrophilic compounds are spread, by passive transport, through the lipid double layer of the enterocyte membrane. Furthermore, epithelial permeability is conditioned by active *New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

transport, mediated by transporters and by various mechanisms of endocytosis, transcytosis and exocytosis for ions, amino acids or some antigens. Large molecules, such as proteins and bacterial products, are captured by cells through the mechanism of endocytosis and are actively transported through the cytoplasm, by transcytosis process, for further processing and presentations, as part of the intestinal immune response (**Figure 5**) [105].

### **1.6 Mechanisms of damage and rupture of the intestinal epithelial barrier**

The intestinal barrier is a dynamic system in which various factors intervene and the increase in the passage of substances due to the increased permeability does not necessarily imply its dysfunction. The progressive increase in intestinal permeability during the development of a pathological process implies an imbalance of the various factors that maintain the barrier function; the immune system being the main candidate to exert a greater effect on it, given the association between inflammation and barrier dysfunction in various digestive diseases. Under normal conditions, the increase in permeability is insufficient to cause a state of "intestinal disease" since the epithelial barrier has the ability to restore itself once the inducer stimulus has ceased. However, in certain pathological conditions, this self-regulating ability can be lost and this condition can facilitate an increase in permeability, facilitating chronic intestinal inflammation. Although the etiology of inflammatory bowel disease (IBD) is unknown, it has been observed that IBD patients have greater intestinal permeability than healthy subjects. It has been identified that this is due to the structural alterations of the TJ proteins, mainly due to the reduction of the expression of claudin-3, 4, 5 and 8 and of occludin, as well as an increased expression of claudin-2 and the phosphorylation of the myosin-light-chain (MLC); this phosphorylation is catalyzed by the specific myosin-light-chain kinase (MLCK), which is activated when

### **Figure 5.**

*Schematic representation of epithelia and transport pathways across a monolayer, and prototypic arrangement of junctions in polarized epithelial cells. The apical junction complex is formed by the tight junction, adherens junction and the most apically located desmosome. Gap junctions and additional desmosomes associate beneath the apical junction complex along the remainder of the lateral cell membranes. Hemidesmosomes interact with the basal lamina at the base of the cells. Intermediate filaments dock into desmosomes and hemidesmosomes whereas actin filaments attach to both tight and adherens junctions. Transcellular permeability is associated with the movement of solutes or water through intestinal epithelial cells. Paracellular permeability is associated with movement in the intercellular space between epithelial cells and is regulated by tight junctions located at the junction of the apical-lateral membranes.*

it binds to calcium and calmodulin, forming a complex (Ca ++−calmodulin-MLCK) which facilitates the contraction of the cytoskeleton and the opening of the junctions [106–108]. The exaggerated inflammatory response would presumably be the cause of these alterations, given the increase in IFN-γ and TNF-α in these patients [109] and the in vitro effect that these cytokines have on the epithelial barrier. In the final analysis, as mentioned above, the alterations of the intercellular junctional complex during enteropathy are linked to an altered mitochondrial function with an energy deficit of the epithelium.

IECs culture and enteroid models have provided important mechanistic insight, suggesting that decreased mitochondrial function in epithelial cells drives a loss in barrier integrity and subsequent bacterial invasion of the underlying intestinal tissue. Loss of barrier function can manifest from epithelial cell death or leakiness of paracellular epithelial cell-cell junctions. DSS-induced colitis is associated with epithelial barrier dysfunction and mechanistic studies using Caco-2 cell monolayers demonstrated that mitochondrial reactive oxygen species (mtROS) play a key role in the loss of barrier integrity during DSS via stimulating the redistribution of Occludin and ZO-1 from intercellular junctions into intracellular compartments, causing leakiness of the tight junctions without altering cell viability [110]. Many forms of ROS have been implicated in disrupting tight junctions through the rearrangement of the actin cytoskeleton to decrease its interaction with tight junction proteins Occludin and ZO-1 and interaction with myosin heavy chain [111]. Additionally, hydrogen peroxide alters phosphorylation of Occludin, disrupting the tight junction, and phosphorylation of β-catenin, disrupting the adherens junction due to the redistribution of E-cadherin preventing interaction with β-catenin [111]. Indeed, dysfunctional mitochondria and accumulation of mtROS during deficiency of the autophagy mechanism induced epithelial barrier defects and the transcellular passage of bacteria that perpetuated intestinal inflammation [112].

In healthy dogs, similarly to the results of Ohta et al. [113], we describe a characteristical pattern of expression of AJ proteins along the small and large intestine [106]. Occludin-specific labeling is uniformly expressed throughout the epithelium of the small and large intestine, with the most intense labeling at the epithelial cell AJC, with fainter labeling observed along the basolateral membranes. Concerning the overall intensity of E-cadherin expression, we observe a decrease from the luminal epithelium to the distal crypts. At the luminal epithelium, E-cadherin labeling is uniform along the length of the intercellular junction, while the expression becomes polarized toward the AJC in the distal glands/crypts. At cellular levels, E-cadherinspecific labeling is restricted to the AJC and basolateral membranes of intestinal epithelial cells. Moreover, there is little evidence of specific labeling outside the epithelium. Claudin-2 readily detectable in the duodenal epithelium and glands and in the colonic crypt epithelium, decreasing in intensity from the distal to the proximal crypt, and remaining minimally detectable at the luminal surface of the colon. Interestingly, the expression pattern of AJC proteins in healthy dogs of our study, is very similar to the AJC proteins distribution, associated with clinical improvement, in IBD suffering dogs, after an oral probiotic treatment of 60 days [106] instead, a different pattern of AJC protein expression was observed in a homogeneous group of IBD affected dogs, apparently improved after a canonical association of metronidazole and prednisone therapy. In this classically treated group, claudin-2 expression was severely increased in the large intestine, particularly at the level of the proximal crypt and luminal epithelium. On the contrary, in the same group of dogs occludin was significantly lower, with a weak to absent expression in the luminal epithelium

### *New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

and in the small intestinal glands. No discernible difference in the distribution or staining intensity of E-cadherin was observed between normal and all IBD affected dogs. This greater deviation from the physiological conditions in the expression of Occludin in the small intestine and Claudin-2 in the colon of IBD suffering dogs, treated with a classical therapeutic protocol, resembles that previously described in samples from the colon of dogs with colitis [114]. In our experience, the effects of a multi strain, live and highly concentrated probiotic association, restored the epithelial barrier integrity, also from a morphological point of view, increasing the number and average size of IECs mitochondria [92]. In our studies, this restoration suggests a potential anti-inflammatory effect of probiotics, on the moment that in treated dogs, decreased mucosal CD3+ T-lymphocytes, and increased FoxP3+ and TGF-β+ positive cells were observed 30 days after the end of probiotic administration. More specifically, the probiotic treated dogs showed increases in CD3+/FoxP3+ cells in the intestinal mucosa, while dogs treated with prednisone and metronidazole displayed an overall decrease in all inflammatory cell populations that was accompanied by a decrease of FoxP3+ lymphocytes and TGF-β expressing cells.

The combination of different factors, genetic, environmental and defects in the barrier function, it is what ultimately predisposes the patient to an abnormal immune response and a greater susceptibility to developing intestinal inflammation. In fact, the appearance of IBD has been linked to the presence of mutated proteins such as X-box binding protein 1 (XBP1) or mutations in the NOD-2 gene related to lower IL-10 production or inadequate immune tolerance to antigens and luminal microbial products [115, 116].

TNF-α and IFN-γ have been extensively studied for their effects on the tight junction barrier in the gut. The effect of TNF-α on the intestinal barrier has been associated with IBD [117]; graft-versus-host disease [118], and celiac disease (CD) [119]. In patients with Crohn's disease (CrD) anti-TNF-α treatment is able to correct barrier disruption seen in the colon [117].

The mechanism of TNF-α barrier disruption has been shown to be mediated by MLCK. MLCK activation alone has been shown to decrease tight junction permeability both in vitro and in vivo [120, 121]. IFN-γ increases intestinal permeability through changes in expression and localization of tight junction proteins as well as rearrangement of the cytoskeleton [122].

Toll-like receptors (TLRs) are a class of transmembrane PRRs that are important for microbial recognition and control of immune responses. TLR2 is one member of the TLR family, which recognizes conserved patterns on both Gram-negative and Gram-positive bacteria. TLR2 is expressed on many cell types through the intestine including epithelial cells [123]. Stimulation of TLR2 in vitro increased trans epithelial electrical resistance through protein kinase C (PKC = a group of enzymes activated by signals such as increases in the concentration of diacylglycerol or calcium ions, and involved in several signal transduction cascades) activation and translocation of ZO-1 to the tight junction complex [123]. Proteinase activated receptors (PARs) are a family of g-protein-couple-receptors that are activated by proteolytic cleavage of their N-terminus revealing a tether ligand. PAR2 is found on both the apical and baso-lateral sides of enterocytes [124]. Stimulation of basolateral PAR2 results in increased permeability through redistribution of ZO-1, occludins, and F-actin [125]. Stimulation of PAR1 has also been shown to increase intestinal permeability [126].

In humans, a large number of chronic inflammatory diseases (CID) have been described to have alterations in intestinal permeability, including IBD [127], IBS [128], type-1-diabetes (T1D) [129], etc. Under normal physiological conditions,

the majority (∼90%) of antigens that pass through the intestinal epithelium travel through the transcellular pathway. The transcellular pathway is regulated and leads to lysosomal degradation of antigens into small non-immunogenic peptides. The remaining ∼10% of proteins cross the epithelium through the paracellular pathway as full intact proteins or partially digested peptides as tightly regulated antigen trafficking through intestinal tight junction modulation, which leads to antigenic tolerance [130].

Zonula occludens toxins (Zot), is an enterotoxin which is able to reversibly open intracellular tight junctions [131]. Zot causes polymerization of actin of targeted cells leading to disassembly of tight junction complexes through a protein kinase C (PKC) dependent mechanism [132]. Immunofluorescent studies have shown that Zot is able to interact with epithelial cells along the GI tract with the highest binding in the jejunum and distal ileum and also decreasing along the villous to crypt axis [133]. Anti-Zot antibodies led to the identification of a ∼47 kDa human analog to Zot, named zonulin [134]. Ex vivo studies show endogenous human zonulin is able to increase permeability in both the jejunum and ileum [135].

Studies on human sera from CD patients, who have increased zonulin levels [134] as determined by ELISA measurement using polyclonal zonulin cross reacting anti-Zot antibodies [136], revealed that zonulin is pre-haptoglobin (Hp)-2, the pro-protein of Hp2 before enzymatic cleavage into its mature form. After this discovery, an analogue of human zonulin (Hp2) has been evidenced in dogs. Dog and human Hp2 are proteins with a 98% similarity.

It was therefore hypothesized that zonulin may disassemble TJ through epidermal growth factor (EGF) activation, since it has been described that EGF can modulate the actin cytoskeleton, similar to the effects seen with zonulin [134, 135]. In vitro studies in Caco-2 cells showed zonulin caused EGF receptor (EGFR) phosphorylation and subsequent increases in permeability, which were blocked by an EGFR inhibitor. To confirm the effect was due to zonulin and not mature Hp2, trypsin digested zonulin was tested and showed no EGFR activation. Additionally, it was shown that EGFR activation was dependent on PAR2 as demonstrated both in Caco2 cells in which the receptor was silenced, and in PAR2−/− mice [136]. Zonulin contains a PAR2 activating peptide-like sequence in its β-chain, and it had been reported previously that PAR2 is able to transactivate EGFR [137].

The signaling pathways triggered by Zot and zonulin leading to tight junction disassembly have been extensively studied and resulted being similar, passing by PAR2 binding, and increasing permeability through displacement of ZO-1 and occludin from the cell junctions [138]. The displacement of ZO-1 and occludin was shown to be secondary to PCKα-dependent phosphorylation of ZO-1, causing decreased tight junction protein-protein interactions, and of myosin-1C that, together with the cytoskeletal rearrangement, temporarily removes ZO-1 and occludin from the junctional complex. While ZO-1 displacement per se is not sufficient to cause a barrier defect [139], the combination with other intracellular signaling events affecting TJ, including occludin displacement, actin polymerization, and myosin-1C phosphorylation [132] may contribute to a more profound rearrangement of the junctional complex that ultimately causes transient TJ disassembly (**Figure 6**).

High alteration in intestinal barrier permeability was observed also during IBS syndrome in man and, recently, in dogs [140]. In both species, IBS is associated with low grade inflammatory infiltration, often rich in mast cells, in both the small and large bowel. The close association of mast cells with major intestinal functions, such

*New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

### **Figure 6.**

*Schematic representation of the gliadin and bacteria-induced release mechanism of zonulin, with the consequent increase in intestinal mucosal permeability, alteration of the barrier and increase in paracellular permeability. In phase 1, some specific peptides, such as gliadin, or deriving from other food sources or from bacteria, induce the release of zonulin mediated by the activation of the C-X-C Motif Chemokine Receptor 3 (CXCR-3 receptor or IFN-gamma induced G protein-coupled chemokines receptor 3—CD183) and dependent on MyD88 (or Myeloid differentiation primary response 88—a innate immune signal transduction adaptor) (phase 2). Zonulin transactivates EGFR (Epidermal Growth factor Receptor) via the PAR2 receptor leads to disassembly of the PCK-α-dependent (Protein kinase alfa) tight junction (phase 3). There is therefore an increase in intestinal permeability due to the opening of the intercellular junctions and the paracellular passage of "non–self-" antigens (phase 4) which diffuse into the lamina propria where they are able to interact with the immune system.*

as epithelial secretion and permeability, neuroimmune interactions, visceral sensation, and peristalsis, makes it necessary to focus attention on the key roles of mast cells in the pathogenesis of IBS. Numerous evidence showed a positive relationship between the number of mucosal MCs and intestinal permeability [141], and the MC-derived tryptase was well identified as a key factor disrupts the intestinal barrier [142]. MC tryptase cleaves PAR2 on colonocytes to increase paracellular permeability by acting, as previously described, on the intercellular apical junction complex, which mainly consists of the tight junctions such as claudins, occludin, zonula occludens, junctional adhesion molecule, and the adherens junction such as E-cadherin [143]. Furthermore, PAR2 may induce the activation of extracellular signal-related kinase 1/2 (ERK1/2) and phosphorylation of MLCK, which regulates reorganization of F-actin and cytoskeleton and redistribution of tight junction, to increase epithelial permeability [105]. Other MC mediators such as interferon-γ, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-4, IL-13, and prostaglandin E2 also have destructive effects on both trans- and paracellular permeability.

The most important triggers of zonulin release that have been described are bacteria, gliadin, and intestinal mast cells (MCs) tryptase. Enterotoxins and several enteric pathogens such as *E. coli*, and *Salmonella typhi* have been shown to cause a release of zonulin from the intestine when applied to the apical surface of IECs. Following the release of zonulin, that may be find and quantified in intestinal lumen (in fecal material) or in plasma, intestine showed increased permeability and disassembly of ZO-1 from the tight junction complex, permitting antigen and bacteria translocation and/ or inflammatory cells passage throughout the epithelial layer. As we described in the previous paragraphs, conditions of dysbiosis, IECs absorption of bacterial/alimentary toxins, and other substances can induce IEC mitochondrial dysfunction with an

increase of intracellular mitochondrial reactive oxygen species. MtROS, mostly from complex III, provides a pathway through which PAR1 and PAR2 are activated. Other sources of ROS do not participate in this induction. While PAR1 signaling ultimately involves NF-kappaB activation, inducing nuclear transcription for many proinflammatory molecules, PAR2 induces the activation of ERK1/2 and phosphorylation of MLCK, which regulates reorganization of F-actin and cytoskeleton and redistribution of tight junction, particularly of ZO-1 and occluding that break the integrity of the TJ complex [144], increasing the epithelial permeability [145]. This pathogenic mechanism is proposed for IBD pathogenesis. Gliadin is the other trigger that has been described to release zonulin; only when applied to the IECs apical surface, gliadin causes a release of zonulin and a subsequent increase in permeability, in both cell culture models and *ex vivo* studies of intestinal tissue. Lammers et al. described that specific non-digestible gliadin peptides are able to bind the CXCR3 receptor on the apical surface of enterocytes with subsequent MyD88-dependent zonulin release [146, 147]. The CXCR3 receptor is also overexpressed on the apical IECs surface of biopsies from celiac disease suffering patients (CD), which may explain the increased levels of zonulin detected in intestinal explants obtained from CD patients when exposed to gliadin [148].

CD suffering patients have a reorganization of actin filaments and an altered expression of occludin, claudin-3 and claudin-4, as well as ZO-1 and the adhesion protein E-cadherin [149, 150]. Generally, under physiological circumstances, there is a tight control of mucosal antigen trafficking (antigen sampling) that, in concert with specific immune cells and chemokine and cytokine mediators, leads to anergy and, therefore, to mucosal tolerance. In the pathological conditions above expressed, the inappropriate production of an increased amount of zonulin causes a functional loss of barrier function, with subsequent inappropriate and uncontrolled antigen trafficking instigating an innate immune response by the submucosal immune compartment, with production of pro-inflammatory cytokines, including IFN-γ and TNF-α that cause further opening of the paracellular pathway to the passage of antigens, creating a vicious cycle.

In conclusion, the loss of gut barrier function, through increased zonulin release from of both epithelial and endothelial barriers, as an essential step to initiate the intestinal inflammatory process. In many human and canine chronic intestinal diseases, whole bacteria or bacteria toxins, as well as gliadin or MCs tryptase are the triggers of zonulin release, leading to gut barrier dysfunction. Similar results, with increase plasma and fecal levels of Zonulin, plasma LPS and cleaved C18 cytokeratin [93] were recently described in sera of dogs with lymphangiectasia, and in cats with enteritis associated T cell lymphoma type II (EATCL II) [151] by the author [46].

An imbalanced microbiome or its inappropriate distribution along the gastrointestinal tract causes dysbiosis, mitochondrial dysfunction with an increase of intracellular mitochondrial reactive oxygen species (MtROS), and the induction of the release of zonulin leading to the passage of luminal contents across the epithelial barrier, causing the release of pro-inflammatory cytokines. The presence of cytokines eventually sustains the ulterior increased permeability, causing a massive influx of dietary and microbial antigens, leading to the activation of T-cells. Depending on the genetic background of the host, these T-cells can remain within the GI tract, causing chronic inflammation restricted to the intestinal mucosa (IBD, IBS, CD), or migrate to several different organs to cause a systemic chronic disease. Generally the main alterations in the expression of TJ proteins are the decrease in ZO *New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier DOI: http://dx.doi.org/10.5772/intechopen.105463*

### **Figure 7.**

*Pathogenic mechanism of chronic intestinal diseases (CID), linked to the loss of impermeability and selectivity of the intestinal barrier induced by the action of TJs-released zonulin. In phase 1 it is observed that, thanks to the barrier effect, the condition of eubiosis, and the physiological traffic through the barrier of non–selfantigens, which are suitably presented to the leukocyte cells of the lamina propria (Th3, Tregs, etc.), there is the establishment of "oral tolerance" with the homeostasis of the mucosa. In phase 2 it is observed how environmental stimuli cause an imbalance of the microbiota, triggering the release of zonulin, loss of paracellular permeability, and an increase in the flow of antigens from the intestinal lumen to the lamina propria. In phase 4, the antigens in the lamina propria activate the immune system in a "pro-inflammatory" manner by causing the release of IFN-γ and TNF-α. This inflammation further exacerbates the increase in intestinal permeability and immune response, worsening and chronicizing the inflammation. This vicious circle, even more serious in genetically predisposed individuals, causes the interruption of oral tolerance to food antigens and causes the aggravation of chronic enteropathies.*

and occludin, as well as an increase in claudin-2 and myosin light chain MLC phosphorylation (**Figure 7**) [152].
