New Acquisitions Regarding Structure and Function of Intestinal Mucosal Barrier

Abstract

The purpose of this chapter is to illustrate the role of the intestinal barrier in keeping separate, but also communicating, the “world above” represented by the resident microbial flora (microbiota) and the “world below” (the immune system associated with the gastrointestinal tract or GALT). Description will be given for how it is possible that the intestinal microbiota, in the course of dysbiosis, can alter the junctional complex that unites the enterocytes, and how the probiotic bacteria (and their metabolites) to restore a homeostasis in the gastrointestinal tract. The fundamental role of enterocyte mitochondria will be highlighted, where being archaic methylotrophic bacteria have retained the ability to “interpret” the bacterial signals (eubiotic or dysbiotic) derived from the intestinal lumen. In this perspective, everything starts from an altered mitochondrial functioning, deriving from a condition of dysbiosis, which alters the tightness of the TJs, opening up to bacterial translocation and bacterial products. Probiotics and their metabolites act by restoring mitochondrial activity and function and the enteric barrier functionality. The author will exemplify this “story” with in vitro and in vivo tests, deriving from original studies on different animal models (mouse, dog, and cat) including humans (patients with IBD and with HIV-related enteropathy).

1. Introduction

The gastrointestinal system is, together with the skin and the respiratory system, the habitat most exposed to the external environment. Every day, thousands of microorganisms and compounds derived from digestion come into contact with it. This condition requires a complex defense system capable of separating the intestinal contents from the host tissues, regulating the absorption of nutrients and allowing the interaction between the resident microbial flora and the mucosal immune system, inhibiting the translocation of pathogens in the underlying tissues. All these functions are performed by the intestinal barrier.

The intestinal barrier is a functional unit, organized as a multi-layered system, in which it is possible to recognize two main parts: a physical surface barrier, which prevents bacterial adhesion and regulates the paracellular diffusion towards the underlying host tissues and a deeper functional barrier, which is able to discriminate between commensal and pathogenic microorganisms, organizing the immunological tolerance towards the commensal bacteria and the immune response towards the pathogens [1].

The intestinal epithelium is organized into a monolayer of cells with a thickness of only 20 μm and is composed of 5 different cell types: enterocytes (IECs), mucus-producing goblet cells (GCs), endocrine cells, “M” cells, “G” cells, and defensin-producing Paneth cells, all of which differentiate from intestinal epithelial Lgr5+ stem cells [2, 3, 4]. Lgr5+ cells are crypt base columnar (CBCSs) stem cells, a population of rapidly dividing cells at the crypt base expressing leucine-rich-repeat containing G-protein coupled receptor 5 (Lgr5), giving rise to all terminally differentiated intestinal epithelial cell (IEC) types [5] CBCSs divide into progenitor cells which move upward within the crypt into the transit amplifying zone [6]. It is here that the cells differentiate further and travel to the villus where their functions are required. At the villus tip, senescent IECs slough off through anoikis, a specific type of programmed cell death for anchorage-dependent cells, and make room for newly formed cells to take their place [6]. Paneth cells are the exception as these cells are long-lived secretory cells that migrate to the crypt base and reside between Lgr5^{+ CBCSs} where they produce and secrete antimicrobial peptides and stem cell factors such as epidermal growth factor (EGF), and other factors that sustain the stem cell niche [7].

IECs are the most represented cell type. They act as a physical barrier that inhibits the translocation of the luminal content into the innermost tissues; IECs form a seamless structure. In fact, they are connected by particular inter-cellular binding structures called adherent junctions (AJs) and tight junctions (TJs), characterized by trans-membrane proteins that interact with adjacent cells and with intracellular proteins, intimately connected with the enterocyte cytoskeleton. The fundamental elements on which the integrity of the “intestinal barrier” depends are, therefore, the IECs and the intercellular junctions.

1.1 Intestinal epithelial cells (IECs) and their metabolism

The main function of enterocytes is the absorption of nutrients, and this function is performed by the mature or “absorptive” IECs, which are differentiated from the intestinal stem cells, CBCSs, residing at the bottom of the crypt. Nutrients such as glucose and amino acids are transported and absorbed by various transporters embedded on the membranes of these enterocytes. Metabolism occurs in each cell along the crypt-villus axis (CVA). The intestinal epithelial cells are the most vigorous, self-renewing cells, regenerating from the crypt bottom to the villus tip in only 3–5 days. Intestinal epithelial cells continuously migrate and mature along the CVA; the energy metabolism in intestinal epithelial cells increases from the bottom of the crypt to the top of the villi. Moreover, the expression of proteins related to the metabolism of glucose, most amino acids, and fatty acids increases in intestinal epithelial cells during maturation along the CVA, while the expression of proteins related to glutamine metabolism decreases from crypt to villus tip. The expression of proteins involved in the citrate cycle is also increased in IECs during maturation along CVA [8].

L-Glutamate is one of the most abundant amino acids in alimentary proteins, but its concentration in blood is among the lowest. This is largely because L-glutamate is extensively oxidized in small intestine epithelial cells during its transcellular journey from the lumen to the bloodstream and after its uptake from the bloodstream. This oxidative capacity coincides with a high energy demand of the epithelium, which is in rapid renewal and responsible for the nutrient absorption process. L-Glutamate is a precursor for glutathione and N-acetylglutamate in enterocytes. Glutathione is involved in the enterocyte redox state and in the detoxification process. N-acetylglutamate is an activator of carbamoyl phosphate synthetase 1, which is implicated in L-citrulline production by enterocytes. Furthermore, L-glutamate is a precursor in enterocytes for several other amino acids, including L-alanine, L-aspartate, L-ornithine, and L-proline. Thus, L-glutamate can serve both locally inside enterocytes and through the production of other amino acids in an inter-organ metabolic perspective. In colonocytes, L-glutamate also serves as a fuel but is provided from the bloodstream. Alimentary and endogenous proteins that escape digestion enter the large intestine and are broken down by colonic bacterial flora, which then release L-glutamate into the lumen. L-Glutamate can then serve in the colon lumen as a precursor for butyrate and acetate in bacteria. L-Glutamate, in addition to fiber and digestion-resistant starch, can thus serve as a luminally derived fuel precursor for colonocytes (Figure 1) [9].

Figure 1.

Metabolic role of glutamine at the cellular level. In the catabolic phase, glutamine is transformed into glutamate and ammonium ions, thanks to the mitochondrial enzyme glutaminase (GA), while in the anabolic phase, at the level of most tissues, glutamine can be synthesized starting from glutamate and ammonia, in the presence of ATP, thanks to the enzyme glutamine synthetase (GS). Ammonia can be converted into Carbamoyl-phosphate, while Glutamate can form a-Ketoglutarate but also Glucose in the liver and kidney, while it is the basis for the synthesis of Glutathione in most cells, and of GABA (Gamma aminobutyric acid) at the neuronal level.

Glutamine is the principal energy source for IECs, and during acute illnesses, patients experience nutritional depletion that is correlated to low plasma and low mucosal glutamine concentrations. Such deficiencies are common among hospitalized dogs and cats or human patients and are associated with an increased risk of developing infectious complications, organ failure, and death [10, 11]. A number of clinical studies reveal a significant benefit of glutamine use on mortality, length of hospital stay [12, 13], and infectious morbidity in critical illnesses, as well as in dog or cat parvovirus infection [11, 12, 14]. Patients receiving high-dose parenteral (rather than orally) glutamine presented the highest beneficial effects, and it is estimated that high doses of parenteral Gln (>0.50 g/kg/day) are the best treatment for humans and animals, demonstrating a greater potential to benefit [15]. However, the role of glutamine in the maintenance of normal gut and immune system function may be even more important for critically ill animals [16]. Glutamine is now considered by many investigators to be a conditionally essential nutrient during protein-calorie malnutrition, required in quantities that are greater than those that can be synthesized by the body. Based on this hypothesis and preclinical studies performed in dogs [17] the commercial veterinary critical care rations often recommended for cats and dogs with some severe enteropathies and cancer are routinely supplemented with glutamine. Glutamine supplementation has also been suggested as a way to promote more rapid resolution of acute side effects of the oral mucosa in dogs receiving oronasal radiotherapy and to maintain gut immunity and integrity in patients receiving radiotherapy or chemotherapy [18].

Recently, glutamine parenteral supplementation evidenced restoration of interdigestive migrating contraction in an experimental canine model of postoperative ileus [19]; in this research is hypothesized that the benefit derives from glutamine’s ability to maintain glutathione concentration and thereby counteract the deleterious effects from surgical injury, inflammation, and oxidative stress. Similarly, parenteral administration of L-alanyl-L-glutamine [20] in dogs prevented the immune suppression induced by high-dose methylprednisolone sodium succinate, and experimental studies in the current literature indicate that glutamine use may prevent the occurrence of lung injury, tissue metabolic dysfunction, and reduce mortality after injury [21]. Glutamine’s beneficial effects on critical illnesses or during IBD, may result from two principal ways: (a) the direct effect on IECs metabolism that helps to maintain the integrity of the epithelial barrier, preventing bacteria translocation; and (b) enhanced heat shock proteins (HSP) expression [22, 23] by enterocytes, and leucocytes [10, 24]. Heat shock proteins are a group of proteins essential to cellular survival under stressful conditions. The stress-inducible HSP60, HSP70 and HSP72 are inducible forms of the stress protein, which may confer cellular protection [10]. The cellular functions of intracellular HSP70 and HSP72 are responsible for limiting protein aggregation, facilitating protein refolding, and chaperoning proteins; an intra-mitochondrial concentration of these proteins is associated with an increase of mitochondria wellness, metabolic activity and ATP production for IECs (see below). IECs-specific deletion of the mitochondrial chaperone protein heat-shock protein 60 (HSP60) led to mitochondrial dysfunction, impairment of cell proliferation and loss of stemness of intestinal stem cells [25]. Additionally, mitochondrial dysfunction impaired the ability of the CBCSs to produce ATP, leading to altered CBCSs self-renewal and differentiation. Furthermore, L-glutamine potentiation of HSP72 is associated with increased gut epithelial resistance to apoptotic injury, and reduced HSP72 may be associated with increased caspase activity in glutamine-deficient [26]. In fact, glutamine induces autophagy under stressed conditions, and prevents apoptosis under heat stress through its regulation of the mTOR and p38 MAP kinase pathways [27]. Glutathione (GSH) metabolism is also closely related to the apoptotic processes of epithelial and immune cells. The increase of intracellular GSH is sufficient to reduce Fas-triggered increase in apoptotic cells. Over expression of Bcl-2, an anti-apoptotic protein, causes redistribution of glutathione to the nucleus, thereby altering nuclear redox and blocking caspase activity [28, 29].

Also, the amount and type of dietary fiber influence the end-products of fermentation and thus fuel availability to intestinal tissue in a specie depending manner. The metabolic fuel usage was studied in intestinal cells isolated from dogs consuming a commercial diet to examine preferential fuel usage and the effect of diet on canine enterocytes and canine colonocytes, respectively, indicating that glutamate/glutamine is preferentially used by enterocytes, while butyrate (found in food and produced as an intestinal fermentation by-product of dietary fiber by gut bacteria) followed by glutamine is preferentially used by isolated canine colonocytes [30].

IBD has been suggested to involve a state of energy-deficiency, whereby oxidative metabolism is altered within IECs [31, 32]. Butyrate undergoes catabolic degradation through β-oxidation in the mitochondrial matrix of colonocytes, providing over 70% of the energy demand of the colonic epithelium [33]. Butyrate metabolism was demonstrated to be impaired in an animal model of colitis [34], and numerous studies have reported impaired metabolism in the intestinal mucosa of patients with IBD [35, 36]. Similarly, intestinal mucosal inflammation results when butyrate oxidation is inhibited in experimental animals [33]. Santhanam et al. [37] showed that the mitochondrial acetoacetyl CoA thiolase, which catalyzes the critical last step in butyrate oxidation, was significantly impaired in the colonic mucosa of patients with ulcerative colitis. Furthermore, they conclude that an increase in mitochondrial ROS may trigger this enzymatic defect [37]. Thus, defective β-oxidation in the mitochondria has deleterious effects beyond energy requirements. Likewise, a dysfunctional gut microbiome or a poor diet may also result in a decrease of butyrate metabolism in the colonic epithelium. Enhanced production of butyrate may potentially benefit the colonic epithelial cells by stimulating an enhancement in cellular homeostasis, including antioxidant and anti-inflammatory roles as well as protective gut-barrier functions.

1.2 Role of mitochondria in IECs homeostasis and barrier integrity

The integrity of the intestinal epithelium, tight junction maintenance, and β-oxidation are key cellular processes within the intestinal epithelium that are not only dependent upon properly functioning mitochondria but are also known to be altered in animal models of intestinal inflammation and in humans with IBD.

Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using murine IECs, Berger et al. [25] demonstrated that loss of mitochondrial chaperone HSP60, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction [25].

During IBD, a destruction of the intestinal epithelial barrier, an increased gut permeability, and an influx of immune cells through the intestinal mucosa are observed. Given that, most cellular functions as well as maintenance of the epithelial barrier are energy-dependent, it is logical to assume that mitochondrial dysfunction may play a key role in both the onset and recurrence of disease. Indeed, several studies have demonstrated evidence of mitochondrial stress and impaired functions, such as oxidative stress and impaired ATP production, within the intestinal epithelium of patients with IBD and mice undergoing experimental colitis [38].

Recently, we have observed that mitochondria dysfunction has a central role in human detrimental intestinal barrier effects of chronic HIV infection [39].

Mitochondria are membrane-bound organelles that maintain cellular energy production through oxidative phosphorylation [40], and contain a circular small genome that encodes only 13 proteins [41]. Despite the limited coding-capacity of the mtDNA, mitochondria regulate vital cellular functions aside from energy production, such as the generation of ROS and reactive nitrogen species (RNS), the induction of programmed cell death, and the transduction of stress and metabolic signals [42]. The current literature would support a key correlation between mitochondrial function and intestinal barrier dysfunction/inflammation. Nonetheless, it is important to understand how any alteration in the multifaceted functionality of the mitochondrion may contribute to the initiation and propagation of an inflammatory insult (Figure 2).

Figure 2.

A condition of eubiosis involves the correct synthesis/absorption of glutamine and glutathione by the enterocytes. Furthermore, the presence of “healthy” bacterial species producing NEFAs in the correct proportion, with an excess of butyrate, preserves the mitochondria from oxidative damage from ROS. A condition of dysbiosis increases mitochondrial damage, critically reducing the number of mitochondria but above all modifying their morphology and permeability. A critical reduction in mitochondria leads to a decrease in the production of ATP by the enterocyte (due to a reduction in the Krebs cycle and beta-oxidation). A reduction in energy leads to a lower “hold” of the intercellular junctional complexes and an increase in bacterial translocation through the intestinal epithelium, which becomes more permeable. At the submucosal level, this condition increases inflammation and the recall of leukocytes, further worsening the condition of the mucosal barrier.

Supporting the importance of mitochondrial form and function, enterocytes isolated from patients with IBD have been reported to exhibit swollen mitochondria with irregular cristae [43, 44]. Abnormal mitochondrial structure is also seen in IECs from mice subjected to experimental models of colitis [45]. Similar observations are made on canine IECs during IBD or lymphangiectasia [46].

These morphological changes are suggestive of cellular stress and bioenergetic failure. Indeed, patients with IBD have reduced ATP levels within the intestine [33, 47]. As would be expected, morphological changes in mitochondria have been shown to result in deficiencies in the β-oxidation of short-chain fatty acids (SCFA) [48]. The intestinal mucosa of IBD patients has been demonstrated to be in a state of energy deficiency characterized by low ATP levels and low energy charge potential [33, 49], calling into question the functionality of this organelle during disease. To further prove this, in a recent study, it was demonstrated that mtDNA released into the serum in IBD patients was recognized as a damage-associated molecular pattern (DAMP) potentially by toll-like receptor 9 (TRL9), and could provide a biomarker of inflammation [50].

Thus, defects in intestinal epithelial homeostasis result in an inadequate intestinal barrier defense, which may allow luminal antigens and/or microbes to interact with or violate the intestinal epithelium and consequently cause inflammation [51]. However, the role of mitochondrial dysfunction during IECs differentiation needs to be further evaluated in order to understand the role it may play in the development of intestinal inflammation. Recently, Bär et al. [52] demonstrated that altered mitochondrial oxidative phosphorylation activity influences intestinal inflammation in mice models of experimental colitis. The study suggests that increased regeneration of the intestinal epithelium (by means of increased mitochondrial function) is a key factor in combating intestinal inflammation. Mucosal healing also results in improved mitochondrial structure in the IECs of patients with ulcerative colitis [53].

1.3 Mitochondria cross-talking with intestinal microbiota maintaining intestinal barrier integrity

Maintenance of TJ integrity is an energy-dependent process, and it is not surprising that disruption of the barrier by toxins, pathogens, or noxious stimuli can be initiated by damaged mitochondria [39, 54, 55].

Mitochondria in animals, as well as chloroplasts in plant cells, are old- primitive bacteria that have lost the ability to live a “free” life by entering into a complex system of cooperation, the eukaryotic cell, and leaving some fundamental functions to the nucleus and other cellular organelles. The fact that mitochondria are ancestral bacteria makes them particularly sensitive to metabolic “motifs” produced by other bacteria. New research shows bidirectional communication exists between the gut microbiota and mitochondria [56, 57].

Certain insults, such as NSAID exposure, are known to disrupt the structure and function of the mitochondria, and at least transiently, increase gut permeability [58, 59, 60]. Additionally, it has been reported that some patients with Crohn’s disease develop immune reactivity against components of their gut microbiome [61]. Consistent with these reports, Nazli et al. [44] demonstrated that treating a cell monolayer with dinitrophenol (an oxidative phosphorylation uncoupler) resulted in cellular internalization of a non-invasive strain of Escherichia coli. From this, the authors hypothesized that under metabolic stress resulting from mitochondrial dysfunction, the enteric epithelium loses its ability to distinguish between commensals and pathogens, and as a result, begins internalizing commensal organisms, which can lead to an exacerbated intestinal inflammatory response [44]. Studies do suggest that both mitochondrial dysfunction [62] and increased gut permeability [63] affect the overall competence of the intestinal epithelial barrier, but the stimuli that initiate either process are not known. Nonetheless, these studies reinforce the implication of epithelial mitochondrial dysfunction as a predisposing factor for an increase in gut epithelial permeability and a loss of gut barrier function, resulting in intestinal inflammation. The intestinal lumen and epithelium are continuously exposed to noxious stimuli, such as ingested nutrients, local microbes or infections, gastric acid production, and periods of ischemia/reperfusion that have the potential to stimulate the generation of oxygen and nitrogen radicals [64, 65, 66]. Additionally, the infiltration of leukocytes, monocytes, and neutrophils during inflammation can further enhance intestinal ROS production through both respiratory burst enzymes and prostaglandin and leukotriene metabolism [67]. Several studies have demonstrated increased ROS/RNS levels within the intestinal epithelium of animals and patients with spontaneous and experimentally induced IBD [68, 69, 70].

Typical gut bacterial families found in healthy dogs and cats include Bacteroidaceae, Clostridiaceae, Prevotellaceae, Eubacteriaceae, Ruminococcaceae, Bifidobacteriaceae, Lactobacillaceae, Enterobacteriaceae, Saccharomycetaceae, and Methanobacteriaceae [71].

The gut microbiota are key to host metabolism as they aid in the digestion and absorption of food, neutralize drugs and carcinogens, synthesize choline [72], secondary bile acids [73, 74], folate, vitamin K2 and short chain fatty acids (SCFA). Additionally, the gut microbiota protects the host against pathogenic infection, stimulating and maturing the immune system [75] and epithelial cells [76] and regulating oxidative stress [77].

Bacterial metabolites, including short-chain fatty acids (SCFAs) and hydrogen sulfide (H₂S), serve as messengers to enteric/colonic epithelial and immune cells, impacting their metabolism, epigenetic modifications, and gene expression. SCFAs are currently the most studied bacterial metabolites and are beneficial to intestinal and colon homeostasis. The three major SCFAs, acetate, propionate, and butyrate, are produced in the colon by bacterial fermentation of carbohydrates and are an important source of energy for colon epithelial cells. SCFAs are ligands for free fatty acid receptors 2 and 3, which modulate glucose metabolism and mitochondrial fatty acid β-oxidation (FAO). Additionally, SCFAs regulate PGC1α, a transcriptional coactivator that is a central inducer of mitochondrial biogenesis in cells [78]. These responses to SCFAs result, at the organelle level, in increased glucose uptake, FAO, oxidative phosphorylation, and mitochondrial biogenesis. In terms of intestinal homeostasis, these responses to SCFAs in colon epithelial cells facilitate the development of a tolerant mucosal immune system, promote epithelial barrier integrity, promote “physiologic hypoxia”, and suppress colitis [7]. In addition, steady-state inflammasome machinery activation in the colon is mediated by SCFAs, which produces basal IL-18 levels, regulates the microbiome composition, and dampens overt inflammatory responses.

Butyrate, a by-product of the microbial fermentation of SCFAs, is one of the key molecules of mitochondria/gut microbiota cross-talk; butyrate may influence mitochondrial-endoplasmic reticulum (ER) contact signaling pathways. A body of recent evidence reveals that the microbiome impacts the host by communicating with its intracellular relatives, the mitochondria. This perspective mode of chemical communication between bacteria and mitochondria may help us understand complex and dynamic environment-microbiome-host interactions regarding their vital impacts on health and diseases. Communications between bacteria and mitochondrial are mediated by chemical signals from intestinal bacteria. In one case, a cluster of bacterial metabolites including betaine, methionine, and homocysteine initiate a signaling cascade that triggers the nuclear receptor 5A nuclear receptor and activates hedgehog signaling to regulate mitochondrial fission-fusion balance in intestinal cells [79]. This bacteria-mitochondria communication ultimately regulates fat storage homeostasis in the host [80]. Additionally, a slime polysaccharide named colanic acid, a major biofilm component of E. coli, secreted from intestinal bacteria, after entering the host cytoplasm via endocytosis, increases the fragmentation of intestinal mitochondria in a dependent fashion to the Dynamin Related protein-1 (Drp-1), a cytosolic guanosine triphosphate (GTPase) protein-key player of mitochondria fission, as well as enhances Mitochondrial Unfolded-Protein Response (UPR^mt) in response to mitochondrial stress. These signaling effects of bacterial colanic acid on mitochondrial dynamics and UPR^mt consequently lead to lifespan extension and protection against age-associated pathologies, like germline tumor progression and toxic amyloid-beta accumulation, in the host [81]. Besides SCFA, secondary bile acids produced by the gut microbiota also play an important role in regulating mitochondrial energy metabolism. Anaerobic bacteria of the genera Bacteroides, Eubacterium, and Clostridium hiranonis degrade 5–10% of the primary bile acids, forming secondary bile acids [71, 82, 83] Secondary bile acids interact with mitochondria by 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].

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.

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 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.

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.

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].

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].

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 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].

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.

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 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, 107, 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-cadherin-specific 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 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).

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.

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

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–self-antigens, 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.

2. Conclusions

The gastrointestinal system is, together with the skin and the respiratory system, the habitat most exposed to the external environment, microorganisms and compounds derived from digestion. This condition requires a complex defense system capable of separating the intestinal contents from the host tissues, regulating the absorption of nutrients and allowing interaction between the resident microbial flora and the mucosal immune system, inhibiting the translocation of pathogens into the underlying tissues. All these functions are performed by the intestinal barrier, a functional unit, organized as a multi-layered system: The barrier is more superficially composed of a physical surface barrier, which prevents bacterial adhesion and regulates the paracellular diffusion towards the underlying host tissues. More in depth, we find a deeper functional barrier, which is able to discriminate between commensal and pathogenic microorganisms, organizing the immunological tolerance towards the commensal bacteria and the immune response towards the pathogens. The fundamental elements on which the integrity and functionality of the “intestinal barrier” depends are therefore the IECs and the intercellular junctions. Glutamine plays a fundamental role in the metabolism of IECs. A condition of eubiosis involves the correct synthesis/absorption of glutamine and glutathione by the IECs. Furthermore, the presence of “healthy” bacterial species producing NEFAs in the correct proportion, with an excess of butyrate, preserves the IEC’s mitochondria from ROS oxidative damage. A condition of dysbiosis increases mitochondrial damage, critically reducing the number of mitochondria but, above all modifying their morphology and permeability. A critical reduction in mitochondria leads to a decrease in the production of ATP by the IECs. A reduction in energy leads to a lower “hold” of the intercellular junctional complexes and an increase in bacterial translocation through the intestinal epithelium, which becomes more permeable. At the submucosal level, this condition increases inflammation and the recall of leukocytes, further worsening the condition of the mucosal barrier. Pathogenic mechanism of chronic intestinal diseases (CID), linked to the loss of impermeability and selectivity of the intestinal barrier, are induced by the action of TJs-released zonulin. Zonulin is a protein that modulates the permeability of TJs between cells of the intestinal barrier. Zonulin has been implicated in the pathogenesis of important GI diseases (i.e. coeliac disease and diabetes), and some glycoproteins, such as the gluten protein gliadin, activate zonulin signaling, increasing intestinal barrier permeability of macromolecules and contributing to “leaky gut” conditions. Thanks to the barrier effect, the condition of eubiosis, and the physiological traffic through the barrier of non-self-antigens, 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 GI mucosa. When 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, antigens 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.

Acknowledgments

The author would like to thank Dr. Lucia Biagini for her helpful advice on various technical issues examined in this paper, and for her help in the graphic realization of the figures that exemplify the concepts expressed in the paragraphs of this chapter. The author also wishes to thank Dr. Livio Galosi, for his usual support activities in the revision of the proofs and in the editing of this and other papers written by the author. We also thank Mrs. Subeide Mari for her technical assistance for all the experimental parts of the numerous papers that have been written by the author over the years on the topics covered in this chapter.

Conflict of interest

The author declares no conflict of interest.