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

modulating transcription factors related to lipid and carbohydrate metabolism, including farnesoid X receptor (*FXR*) and G-coupled membrane protein 5 (*TGR5*) [84]. *FXR* is a target of NAD-dependent protein deacetylase *silent regulator 1 (SIRT1)* [85] and regulates the steroid response element binding protein-1c, carbohydrate response element binding protein, and Peroxisome proliferator-activated receptor alpha (*PPAR-*α), which stimulates fatty acid uptake and oxidation [74]. There is increasing evidence that secondary bile acid metabolism might also directly modify *SIRT1* expression as well as mitochondrial biogenesis, inflammation, and intestinal barrier function in different types of cells (**Figure 3**) [86, 87].

Together, these results consistently show that mitochondria undergo chemical communication with bacteria, a process modulating metabolic and senescent states of eukaryotic cells. The impact of microbiota on mitochondrial functions has been further supported by studies intending to manipulate gut microbiota through the use of probiotics. One example is the probiotic *Escherichia coli* Nissle 1917 (EcN) with proven effectiveness in the treatment of inflammatory intestinal disorders and acute diarrhea. Outer membrane vesicles (OMVs) released by the probiotic EcN and the commensal ECOR63 are taken up by intestinal epithelial cells, and modulate the epithelial barrier integrity through several mechanisms, mediated by the restoring of the mitochondria [88]. Administration of the probiotic *Lactobacillus rhamnosus* CNCMI–4317 induced a series of modulating factors that modified the oxidative phosphorylation (OXPHOS) capacity of mitochondria [89]. Certain intestinal bacteria such as *Eubacterium hallii* and *Anaerostipes caccae* have the capacity to transform the byproduct of anaerobic glycolysis lactate into SCFA during glucose depletion thus

### **Figure 3.**

*Effect of mitochondrial morpho-functional alterations at the basis of the "leaky gut" as observed in the course of IBD. Dysbiosis, bacterial toxins and free radicals linked to a reduced intake of glutamine, involve the activation of signals of the extrinsic and intrinsic pathways of apoptosis, which pass through the structural and functional alterations of the mitochondria (i.e., increased permeability, translocation of HSPs and of the APAF-Cytochrome C complex, loss of Ca ++ etc.). A reduction in mitochondria, resulting in a reduction in ATP, causes a decreased activity of ETC complexes, accumulation of mtROS, accumulation of misfolded or unfolded proteins in the matrix, and ultrastructural changes such as cresting. Subsequent loss of epithelial barrier integrity, epithelial cell apoptosis, and bacterial invasion have been demonstrated following mitochondrial dysfunction in the epithelium. mtDNA is released in the serum of IBD patients and acts as a DAMP for the activation of immune cells. Furthermore, damaged mitochondria can signal the activation of the inflammasome, leading to the production of pro-inflammatory cytokines and increasing leukocyte infiltration of the intestinal mucosa.*

creating an alternative energy source for the host, while bypassing OXPHOS [90, 91]. Finally, probiotic mixture Slab51™ administration for a period of two or six months, restores mitochondria inducing HSP60 and 70 mitochondrial internalization and increasing number and size of mitochondria in intestinal cells of IBD, and suffering dogs, and HIV chronically affected patients [39, 92, 93].

Unlike the beneficial effects commensal bacteria and certain probiotics have on energy metabolism, pathogens such as *Salmonella* and *E. coli* [94] can produce negative effects on the host mitochondria energy metabolism by degrading sulfur amino acids to produce hydrogen sulfide (H2S) in the large intestines. H2S is an important mediator of many physiological and pathological processes. High amounts of H2S can inhibit a key component of the mitochondrial respiratory chain by penetrating cell membranes and inhibiting COX activity and energy production [56]. Pathobionts can also produce NO, which may affect host mitochondrial activity and favor bacterial infection [95]. Beaumont et al. [96] concluded that exposure of high levels of H2S to HT-29 human cells showed not only reduced mitochondrial oxygen consumption but also an increase in the expression of inflammatory genes such as IL-6, which was increased following a high protein diet. Mottawea et al. [56] recently demonstrated that a proliferation of pathobionts, many of which are known to be potent H2S producers, down regulated mitochondrial proteins. Additionally, H2S induces genotoxic damage to the epithelium, inhibits metabolism of SCFAs, and induces breaks in the mucus barrier, allowing exposure of luminal contents to the underlying tissue [7, 97].

### **1.4 The system of intercellular junctions**

In the intestinal epithelium there are two main types of junctions: adherent junctions (AJs) and tight junctions (TJs). Both types are formed from the proteins of the classes of cadherins, claudins and occludins, present in different concentrations and control the paracellular permeability through the intercellular spaces. In epithelial barriers, TJs and AJs are well defined and distributed: the TJs are present in the apical part, while the AJs are located in the basolateral part, below the TJs (**Figure 4**). Both are connected to the actin cell cytoskeleton.

The tight junctions seal the paracellular space and for their assembly they need adherent joints. As can be seen in **Figure 4**, they are multi-protein complexes made up of integral membrane proteins (claudins, occludins and junctional adhesion molecules), peripheral membrane proteins (zonula occludens) and regulatory molecules such as kinases.

Claudins (18–27 kDa) are proteins with 2 extracellular loops and a C-terminal cytoplasmic domain. They constitute a large gene family in which 24 isoforms have been identified that determine the selectivity of the paracellular pathway in terms of tissue, charge and size. They are expressed in a tissue-specific way and a mutation or deletion of one of the members of this family can have significant effects on the function of the epithelial barrier [98, 99].

The data obtained in some in vitro studies indicate that the claudins -1, -3, -4, -5, -8, -11, -14, and -19 play a determining role in the selectivity of the paracellular barrier. The permeability of the ions through the TJs is regulated by the claudins -4, -8 and -14 which are involved in the cationic barrier, while other claudins such as -2, -7 and -13 form the paracellular pores for cations and anions. In the gastrointestinal tract claudins -2, -3, -4, -7, -8, -12, and -15 are expressed, but the levels of expression and their subcellular localization are different in the different intestinal segments [98].

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

### **Figure 4.**

*Molecular structure of tight junctions. When the intestinal barrier is intact, the paracellular space between two enterocytes is sealed by TJs which are made up of a series of transmembrane proteins that include occludin, claudins, and the junctional adhesion molecule-1 (JAM-1). Thanks to TJs, the intestinal barrier is perfectly able to keep the luminal environment separate from the underlying immune system. Claudins adhere to each other in a homotypic as well as a heterotypic manner. ZO-1, -2, and -3 bind the cytoplasmic tail of occludin and link the TJ to the actin cytoskeleton. Proteins of the ZO family can shuttle to the nucleus to influence transcriptional processes in cellular proliferation and differentiation. The ZO-proteins have also been shown to interact with claudins and provide molecular scaffolds for TJ assembly. In the composition of TJ we also find cingulin, a protein of 140 kDa, which is associated with the cell cytoskeleton of actomyosin. Tyrosine phosphorylated Par3 / 6 regulates tight junction assembly and promotes cell polarity via intracellular signaling. Localization of TJ-associated 7H6 antigen along the cell border of vascular endothelial cells has been shown to be related to paracellular barrier function. The ZO-1 and ZO-2 scaffold proteins form dimers and bind to claudins, thereby contributing to the targeting and polymerization of claudins at tight junctions. Dimerization involves the SH3/GUK domain of ZO-1 / ZO-2. Also, ZO-1 and ZO-2 interact with the underlying actin cytoskeleton and act as a scaffold at tight junctions. The apical polarity protein complexes, including the Crumbs and Par complexes, localize to tight junctions.*

Occludins (65 kDa) are proteins with 4 transmembrane domains and 2 extracellular loops and exist in 2 isoforms. The C-terminal domain, located in the cytoplasm, binds directly to ZO-1 (zonula occludens) which in turn binds the apical part of the actin. This portion of occludin is rich in sites of phosphorylation (thyroxine, serine and threonine) which can be modified by kinases and phosphatases. The non-phosphorylated occludin is distributed in the basolateral membrane and in the cytoplasmic vesicles, while the phosphorylated occludin is localized in the TJ and determines a reduced paracellular permeability [100]. Alterations (chronic inflammations or hyperplasias) have been observed in occludin deficient mice in all those districts characterized by the presence of TJs, suggesting more complex functions to be attributed to occludin, whose role is not yet fully known [101]. The interaction of occludins, claudins, JAMs, and tricellulin between cells and with ZOs maintains the integrity of the tight junction and controls the passage of molecules through the paracellular space.

Junctional adhesion molecules (JAM) (32 kDa, 3 isoforms) contain a transmembrane segment and an extracellular domain. They are proteins involved in the adhesion between the barrier cells and between the barrier and the blood cells and can form homophilic and heterophilic interactions with different ligands including integrins. They can also interact with partners such as ZO-1 and the protease-activated receptor PAR-3 [98].

Peripheral membrane proteins associated with zonula occludens (ZO) are crucial for the assembly and maintenance of TJs because they have multiple domains for interaction with other proteins, including integral membrane proteins and actin. On the

intracellular side of the membrane, the carboxy-terminal ends of claudin, occludin and actin interact with the proteins ZO-1 (220 kDa), ZO-2 (160 kDa) and ZO-3 (130 kDa). These proteins belong to the membrane-associated guanylate kinase (MAGuK) superfamily and have an enzymatically inactive guanylate kinase domain. The TJ multiprotein complex, hitherto described, is linked to the actin cytoskeleton through the ZO proteins that bind to the integral membrane proteins with the N-terminal domain and to the actin cytoskeleton with the C-terminal domain. The protein that plays the central role is ZO-1 which directly and indirectly connects the integral membrane proteins (occludin and claudin) to the other cytoplasmic proteins of the TJs and to the actin cytoskeleton. It has been shown that, like occludins, ZO-2 and ZO-3 cannot interact directly with actin filaments since their C-terminal domains show similarities only towards ZO-1. Therefore, the binding to the actin cytoskeleton is limited to ZO-1 which has the potential to organize the structural components and to modulate the paracellular pathway [102].

There are many other proteins involved in TJ: tricellulin, the coxsackie and adenovirus receptor (CAR), the selective adhesion molecule for endothelial cells (ESAM), JAM4, AF-6/afadine, PAR3, MUPP-1, cingulin, PILT (protein subsequently incorporated into TJ) and JEAP (junction-enriched and -associated protein). All this gives the idea of the complex organization of TJs [98].

Until a few years ago, tight junctions and adherent junctions were seen as discrete and independent complexes. However, new evidence has emerged that highlights their interdependence. From these studies, it is clear that there are both physical and biochemical connections between adherent and tight junctions. The ZO-1 complex physically connects the two junctional complexes through its interactions with the binding proteins of actin, α-catenin and afadin. These interactions promote the maturation of the AJs and the subsequent assembly of the TJs [103].
