The gastrointestinal tract is the human habitat most exposed to the external environment with a global surface area of 200 m2.
Every day, thousands of microorganisms and compounds derived from digestion come into contact with it.
This condition requires a complex defense system that is able to separate the intestinal contents from the host tissues, regulate the absorption of nutrients and allow interaction between the resident microbial flora and the mucosal immune system, by inhibiting the translocation of pathogens into the underlying tissues: the intestinal barrier (1).
In particular, the intestinal barrier is a functional unit, organized as a multi-layered system, in which it is possible to recognize two main parts:
- superficial physical barrier, which prevents bacterial adhesion and regulates paracellular diffusion towards the underlying host tissues
- deeper functional barrier, which is able to discriminate between commensal and pathogenic microorganisms, organizing the immunological tolerance towards commensals and the immune response towards pathogens.
In addition, there are also other luminal intestinal mechanisms that participate in the integrity of the intestinal barrier.
The low pH of gastric juice is bactericidal.
The low pH of the juice This element, for example, acts against infectious agents. While pancreatic enzymes are capable of damaging the bacterial cell barrier, gastric is bactericidal.
The superficial barrier starts from the resident microbiota which competes with the pathogens to gain space and energy resources, processes the molecules necessary for mucosal integrity and modulates the immunological behavior of the deep barrier.
The next level is represented by the mucus layer, which separates the intraluminal contents from the innermost layers and contains antimicrobial products and secretory IgA. Underneath the mucus is a monolayer of intestinal epithelial cells. This forms a physical barrier and contains the immunological cells.
These have the task of presenting the pathogenic antigens to the lower functional barrier, with the aim of developing an adequate immune response (1-3).
The intestinal barrier.
The deep barrier is represented by a complex network of immunological cells, organized in a specialized and compartmentalized system known as "gut associated lymphoid tissue" or GALT.
The GALT consists of isolated and aggregated lymphoid follicles and is one of the major lymphoid organs, containing up to 70% of the body's immune cells.
It is responsible for the development of the response to pathogenic microorganisms and the immunological tolerance to commensal bacteria. An ability performed thanks to the physiological relationships with the superficial layer and the intimate contact with the external environment through specific immune cells.
Dendritic cells and M-cells within Peyer's patches.
These cells have the ability to acquire microorganisms and macromolecules and to present antigens to T lymphocytes, which produce cytokines by activating the immune response.
The integrity of the aforementioned structures is necessary for the maintenance of normal intestinal permeability. The alteration of this balance determines the passage of the luminal content towards the underlying tissues and therefore into the bloodstream, with a consequent activation of the immune response and the induction of an inflammatory state.
The alteration of permeability is in fact at the basis of the pathogenesis of many gastroenterological and non-gastroenterological diseases, including not only infectious enterocolitis and chronic inflammatory bowel diseases (IBD), which include rectocolitis ulcerative disease (UC) and Crohn's disease (CD), but also irritable bowel syndrome (IBS), small intestinal bacterial overgrowth syndrome (SIBO), celiac disease, liver fibrosis, food intolerances and even atopic manifestations (1)#.
Determining the degree of intestinal permeability and the molecular mechanisms underlying the integrity of the barrier could become an interesting parameter to consider in clinical practice to study and, above all, treat these pathologies.
Models in vitro (Caco2 and HT29-MTX cells) and techniques in vivo non-invasive (sugar tests and tests with radioisotopes) represent effective methods for this type of evaluation. Finally, the modulation of the various components of the barrier is of crucial importance for the control of intestinal permeability (3).
Components of the intestinal barrier.
- A) Gut microbiota
Our intestine, which in physiological conditions also includes fungi and bacteriophages, contains about 1 kg of bacteria which contain a genetic heritage of more than 3 million genes and which have a mutualistic relationship with their host.
In fact, they perform various functions: barrier, synthesis, immunostimulation, metabolism of nutrients, trophism of the mucosa, metabolism of drugs and toxins.
The microbiota collaborates in particular in the digestion of energy substrates, in the production of vitamins and hormones, and in the protection of the host from pathogenic species # ADDIN EN.CITE # ADDIN EN.CITE.DATA #(2, 4, 5). In fact, the microbiota is able to consume the nutrients necessary for the survival of pathogenic bacteria. It can also produce molecules that inhibit the growth of pathogenic flora.
Studies have shown that Bifidobacteria and Lactobacilli such as Lactonbacillus acidophilus produce acidic bactericidal substances, such as lactic acid, bacteriocins and short-chain fatty acids, which suppress the growth of Salmonella Typhimurium in animal models. A recent study demonstrated that a compound formed by Bacteroides thetaiotamicron and Eubacterium rectale is capable of inducing the production of mucosal glycans by the host.
These have the particularity of being used exclusively by these two bacterial strains and not by pathogenic organisms, which therefore are not enabled to proliferate # ADDIN EN.CITE # ADDIN EN.CITE.DATA #(6-9). These properties are essential for maintaining adequate functionality and efficiency of the intestinal barrier.
The enteric microflora is also capable of producing nutrients for the mucosal cells: the Bacteroides thetaiotamicron colonizes the outermost mucus layer and is able to degrade the peptides and glycans, which make up the mucus, so to produce SCFA, such as butyrate.
SCFAs are involved in the stimulation of mucus production in in vivo and in vitro models:
- in blocking the invasion and adherence of Escherichia coli
- and increased levels of total and pathogen-specific mucosal IgA # ADDIN EN.CITE # ADDIN EN.CITE.DATA #(3, 10-15).
The alteration of the microflora is the basis of many gastrointestinal pathologies such as gastritis, peptic ulcer, IBS, IBD and also the gastric and colon cancer.
In IBD, for example, the concentration of intestinal bacteria adhering to enterocytes is higher than in healthy controls and increases progressively with the severity of the disease. Furthermore, a reduction in bacterial diversity was found with an increase in Enterobacteriaceae, including Escherichia coli, and a marked reduction in Bacteroides and Clostridium.
This condition leads to a reduction in the growth and maturation of enterocytes with a consequent increase in intestinal permeability (16-20).
- B) Intestinal mucus
Mucus is the first physical barrier that bacteria encounter within the digestive tract. It acts as a shield for the epithelium, protecting it from harmful microorganisms and antigens, but also as a lubricating agent for intestinal motility. It consists of two layers: an inner layer, firmly adherent to the epithelial cells and about 50μm thick; and a looser, less adherent outer layer that is approximately 100μm thick as measured in laboratory animals (21)#.
The inner mucous layer is dense and does not allow bacteria to penetrate, therefore making the epithelial cell surface free from bacteria. The internal mucus layer is transformed into the outermost one, which is the habitat of the commensal flora. The external mucus is distributed over a very large volume thanks to the proteolytic activities of the host, but also provided by the proteases and glycosidases of the commensal bacteria.
Both mucus layers are organized around the highly glycosylated mucin MUC2, which forms an amorphous polymer-like coating and which is secreted by goblet cells. The mucin MUC2 is a molecule that has been preserved along the evolution starting from the first metazoans (22).
After secretion, the mucin MUC2 organizes itself into a hydrated and expanded network and thus forms, together with other secreted proteins, a well-organized mucous layer (23). The protein composition is similar in the two mucous layers as it derives from a common cellular source. The physiological microbial flora resides in the outermost mucus layer, while the innermost layer is impervious to bacteria and acts as a protective barrier for the epithelial cell surface.
This compartmentalization is critical for intestinal homeostasis within a highly colonized colonic environment #ADDIN EN.CITE #ADDIN EN.CITE.DATA #(24, 25). The importance of the mucosal barrier was further demonstrated in animals whose MUC2 genes were deleted.
The bacteria, due to the absence of mucus, are in direct contact with the epithelial cells, but also in the depths of the intestinal crypts, as well as inside the epithelial cells. The increase in intestinal permeability, caused by the loss of the mucus barrier, therefore determines the triggering of an inflammatory reaction and, subsequently, can favor the development of colon cancer. Furthermore, mucus not only performs a barrier function.
Its content in glycans, linked to the mucin MUC2, not only serves as a nourishment for the bacteria, but also as a site of binding and, probably, also of selection of specific microbial species, which are essential for maintaining the integrity, homeostasis and intestinal function (26-28).
- c) Epithelial cells
The intestinal epithelium is organized into a monolayer of cells, with a thickness of only 20 μm and composed of 5 different cell types: enterocytes, endocrine cells, M cells, G cells and Paneth cells. Enterocytes are the most represented cell type. They act as a physical barrier that inhibits the translocation of luminal contents into the innermost tissues. The enterocytes form a seamless structure. In fact, they are connected by particular intercellular binding structures called tight junctions, characterized by transmembrane proteins, which interact with adjacent cells and with intracellular proteins connected with the cytoskeleton (29)#. Overall these components form a complex and homogeneous network. There are two main types of junctions in the intestinal epithelium:
- Adherent Junctions (AJs)
- the tight junctions (TJs).
Both types are formed from the proteins cadherins, claudins and zonulins, present in different concentrations. The regulation of the connection strength of these junctions, and therefore of the permeability of the epithelial barrier, occurs through the phosphorylation of myosin and the contraction of the actin-myosin complexes.
Their alteration increases mucosal permeability, causing the passage of intraluminal molecules into the deeper layers, determining the activation of the adaptive immune response and leading to the inflammatory state # ADDIN EN.CITE # ADDIN EN.CITE.DATA #(3, 30- 33). A condition seen especially during infectious enterocolitis and IBD. In the first condition, the E. coli enterohaemorrhagic (EHEC) and E. Enteropathogenic coli (EPEC) have the ability to adhere to intestinal epithelial cells and disrupt the integrity of the barrier via disruption of the tight junctions.
In IBD, on the other hand, a dysfunction of the AJ proteins has been described, due to a reduction in E-cadherin. This alteration causes a weakening of the intercellular adhesion and the triggering of the inflammatory response. Recent studies have shown that high concentrations of IFN-γ and TNF-α, typical of UC and CD, can cause a reorganization of numerous proteins that constitute the tight junctions such as zonulin-1, JAM-A, occludin, claudin-1 and claudin-4. Also in this case this leads to an increase in intestinal permeability. This increase is also seen in IBS, especially in the diarrheal-predominant subgroup of IBS # ADDIN EN.CITE # ADDIN EN.CITE.DATA #(34, 35).
Consequences of damage to the intestinal barrier: associated pathologies.
The ability of the various components of the intestinal barrier to guarantee physiological permeability even in the presence of pathogenic noxae from the external environment is essential for maintaining health. In fact, as the protection guaranteed by the intestinal barrier fails, the immune cells come into direct contact with the antigens present in the intestinal lumen, with consequent alteration of the normal physiological functions of the barrier, including the immunological response to the various pathogens (bacteria, viruses, fungi and parasites), recognition of "self" antigens, tolerance of commensal flora and desensitization to food antigens.
Numerous scientific evidences show a relevant association between gastro- and extra-intestinal pathologies and the alteration of intestinal permeability. These include autoimmune disorders, including celiac disease, IBD, type I diabetes mellitus, multiple sclerosis and rheumatoid arthritis; but also cirrhosis of the liver, acute pancreatitis, infectious gastroenteritis and small intestinal bacterial overgrowth syndrome (SIBO). Even pathologies apparently not directly connected to the functionality of the intestinal barrier, such as heart failure or autism, can lead to an increase in intestinal permeability.
In some cases, such as in IBD and celiac disease, the increase in permeability could represent the primum movens of the unleashing of the pathology. In others, such as in liver cirrhosis, it would determine the passage of microbial antigens in the enteroportal circulation with worsening of liver fibrosis and portal hypertension and consequent further increase in permeability (36). However, whether the alteration of the barrier function is an epiphenomenon, an early manifestation, or an essential step in the pathogenesis of these diseases remains to be ascertained, while it has been demonstrated that an increase in intestinal permeability contributes to the exacerbation and chronicisation of these pathologies.
Often the same drugs that lead to their resolution, even temporarily, determine a recovery of physiological permeability and therefore of adequate intestinal homeostasis (1)#. The IBD are an example in which the increase in intestinal permeability plays a central role in the pathogenesis of the disease. In this case, the altered intestinal permeability precedes the development of the pathology, whose exact pathogenesis remains unknown but certainly involves genetic, immunological and environmental factors that initiate the autoimmune process.
In patients with clinically asymptomatic Crohn's disease, an increase in intestinal epithelial permeability precedes the disease flare by about 1 year. This suggests that a defect in intestinal permeability is an early event and predictive of the clinical exacerbation of the disease (37). While the primary intestinal barrier defect appears to be involved in the early stages of the pathogenesis of IBD, the production of cytokines, including IFN-γ and TNF-α, secondary to the inflammatory process, would serve to perpetuate the increase in intestinal permeability following the reorganization of the proteins that form the intercellular junctions.
In this way, a vicious circle would be created in which the primary dysfunction of the barrier would cause an initial passage of luminal content, which in turn would trigger an immune response with the maintenance of an altered permeability and the chronicity of the disease (38). In this context, therefore, new therapeutic strategies focused on the recovery of the physiological functionality of the intestinal barrier can offer an innovative approach for the improvement of the clinical picture of these highly debilitating chronic pathologies.