The Barrier and Intestinal Permeability

The gastrointestinal tract is one of the most exposed parts of the human body to external elements covering 200m (squared).

Every day, thousands of microorganisms and compounds, which derive from digestion come into contact with it.

This is one of the reasons why a rather complex defence system capable of separating the intestinal contents from the host’s tissues is required in order to regulate the nutrient absorption. This enables an interaction between the resident gut microbiome and mucosal immune system inhibiting the translocation of pathogens into the tissues beneath: the intestinal barrier.

The gut barrier is a functional unit, organised as a multi-layer system, made up of two main components: a physical barrier surface, which prevents bacterial adhesion and regulates paracellular diffusion to the host tissues, and a deep functional barrier, able to distinguish between pathogens and commensal microorganisms, organising the immune tolerance to commensals and the immune response to pathogens.

Other mechanisms participate in the luminal integrity of the gut barrier.

For instance, the low pH of gastric juice is bactericidal and so it acts against the infective agents, whereas the pancreatic enzymes are likely to cause damage to the bacterial cell barrier.

The microbiota, found in the surface barrier fights against the pathogens in order to expand and gain energy resources. At the same time it forms all the necessary molecules protecting the mucosal integrity moulding the immunologic behaviour of the inner barrier.

The next level is composed of mucus layers which separate the endoluminal content from the inner layers and contain antimicrobial agents and lgA secretories.

Right beneath the mucus, there are monolayers of epithelial intestinal cells which form a barrier and contain the immune cells.

Their main function is to introduce the pathogenic antigens to the lower functional barrier in order to elaborate an adequate immune response.

The surface beneath is made up of a complex network of immune cells, strictly organised in a perfectly constructed system best known as “gut associated lymphoid tissue” (GALT). It is formed by isolated lymphoid follicles and aggregates and represents one of the main lymphatic organs containing up to 70% of all immune cells in the human body.

That is what triggers the response of all pathogenic micro-organisms and tolerates the commensal bacteria. It is carried out due to the physiologic interactions with the upper layer working with specific immune cells: the so called dendritic cells and the M-cells in Peyer’s Patches.

These cells are perfectly capable of capturing the microorganisms and macromolecules, thus introducing the antigenes to the T-lymphocytes,

which are responsible for generating an immune response through cytokines.

The integrity of these structures is necessary in order to maintain the normal intestinal permeability. The possible alteration of this balance could determine the transfer of luminal content into the tissues underneath and could therefore infiltrate within the bloodstream and activate an immune response, resulting in an inflammatory process.

This alteration in the permeability is what causes the pathogenesis of many gastrointestinal pathologies (among those better known, there are the enterocolitis, chronic inflammatory intestinal diseases (Ulcerative Colitis, Crohn’s disease)), there is also the Irritable Bowel Syndrome (IBS), Small Intestinal Bacterial Overgrowth (SIBO), the coeliac disease, the hepatic fibrosis, food intolerances, the development of atopy.

The capability to determine the level of intestinal permeability while keeping molecular mechanisms responsible for the integrity of the barrier into consideration, gives the possibility to research scientists to learn more about the different pathologies mentioned above. 

In vitro studies (Caco2 cells and HT29-MTX) and in vivo non invasive techniques  (sugar tests and radioisotope tests) represent very effective methods. The modulation of the different constituents of the barrier is of crucial importance for the regulation of the intestinal permeability.

 

Components of the Intestinal Barrier

The Gut Microbiota

Our gut, including funguses and bacteriophages, contains about 1kg of bacteria incorporating more than 3 millions genes having a mutualistic relation with their host.

 

They could perform different functions acting as a barrier, synthesis, immunostimulators, metabolism (the conversion of food/fuel to energy), tropism of mucus, metabolism of medications and toxins.

The microbiota helps improve digestion of all energetic substrates. It enriches the production of vitamins and hormones and protects the host from all harmful pathogens.

The microbiota is perfectly capable of consuming all nutrients necessary for the survival of the pathogenic bacteria and could produce molecules able to inhibit the growth of pathogenic flora.

Studies have already shown that Bifidobacteria and Lactobacillus (as the Lactonbacillus acidophilus) produce acid bactericidal material, such as lactic acid, bacteriocins and Short-Chain Fatty Acids that could suppress the growth of Salmonella Typhimurium in animals.

A recent study has shown that a compound of Bacteroides thetaiotamicron and Eubacterium rectale could induce the production of mucosal glycans in the host.

Only these two bacterial strains can come into contact with them and all pathogenic organisms are basically kept off. That is what allows the intestinal barrier to perform functionally.

The enteric microflora is capable of producing nutrients for the mucosal cells: Bacteroides thetaiotamicron colonises the outer mucus layer and is capable of degrading the peptides and glycans (composing the mucus), in order to produce SCFA as the Butyrate.

The Short-Chain Fatty Acids (SCFA) trigger the production of mucus as it has been seen in vivo and in vitro. The SCFA help block the invasion and adhesion of Escherichia coli. At the same time they are responsible for the growth of the mucosal lgA and pathogen species.

The alteration of the microflora is one of the reasons why certain gastrointestinal pathologies arise such as Gastritis, Peptic ulcer disease, IBS, IBD, Stomach cancer (also known as Gastric cancer) and Colon cancer.

As far as IBD is concerned, the concentration of intestinal bacterial adhesion to enterocytes is even higher and grows progressively as the pathology takes over.

A reduction of bacterial diversity has been seen highlighting an increase of Enterobacteriaceae (Escherichia coli included). A stark reduction of Bacteroides and Clostridium has been detected. This specific condition leads to a reduction of enterocytes expanding the intestinal permeability.

 

  1. B) Intestinal Mucus

 

The mucus is the first barrier bacteria come across once they get into the alimentary tract. It acts as a shield for the epithelium keeping off all microorganisms and noxious antigenes. At the same time it helps lubricate the intestinal motility. It is made up of two separate layers: the inner layer, strictly adherent to the epithelial cells, its circular thickness is around 50um; the outer layer is definitely looser and less adherent, it is approximately 100um in depth.

 

The inner mucus layer is thick and does not let bacteria in rendering all epithelial surface germ-free. The mucus inner layer turns into the outer one, which is the actual habitat of commensal flora. The outer mucus takes up a lot of space, it is all due to the proteolyc activities managed by the host (at the same time provided by the protease and glycoside of commensal bacteria). Both mucus layers rely on the MUC2 Mucin creating a shapeless ceiling-like polymerous cover which is constantly secreted by calyciform cells.

The MUC2 mucin is a molecule which has been preserved over the millennia since the first metazoans appeared on Earth.

As soon as the secretion occurs, the MUC2 mucin places itself in a larger hydrated network and thus, forms a perfectly structured and organised mucus layer. The protein composition is very much alike in both mucus layers because it derives from the same cell source. The microbial flora resides in the outer mucus layers, whereas the inner layers is inaccessible to bacteria and acts as a protective barrier for the epithelial cell surface. This division in compartments is essential for permitting the intestinal homeostasis in a colic habitat highly colonised.

The importance of the mucus barrier has already been tested and demonstrated in animals, when traces of the MUC2 genes disappeared. In this specific case, bacteria are in contact with epithelial cells (considering the absence of mucus). The same happens in all intestinal crypts as in the epithelial cells. The increase of intestinal permeability (caused by the complete absence of mucus) determines an inflammatory response and, later on, it could even facilitate the development of colon cancer. Furthermore, the mucus does not only act as a barrier. The fact that it contains glycans connected to the MUC2 mucin, does not simply feed bacteria. It is important because it could select certain microbial species which are crucial for the integrity, homeostasis and gastrointestinal functionality.

 

  1. B) The epithelial cells

 

The intestinal epithelium is composed of a simple columnar epithelium. It is 20um in depth and contains five different types of cells: enterocytes (or intestinal absorptive cells), enteroendocrine cells, M cells, G cells and Paneth cells. Enterocytes are largely spread over. Their function most as a barrier inhibiting the translocation of luminal contents into the inner tissues. Enterocytes are basically connected by the so called tight junctions, mainly characterised by transmembrane proteins. These proteins interact with the neighbouring cells and intracellular proteins  connected to the cytoskeleton. All these components form a rather complex and uniform network indeed. There are two main junctions in the intestinal epithelium: the adherent junctions (AJs) and the tight junctions (TJs).

Both contain cadherins, claudins and zonulins. The permeability of the epithelial barrier takes place thanks to the myosin’s phosphorylation and the contraction of complexes Actin-Myosin. In case there is an alteration, the mucosal permeability expands letting endoluminal molecules into the inner layers thus activating the adaptive immune defence leading to an inflammation. It could easily occur during an infectious enterocolitis and IBS (Irritable Bowel Syndrome).

The Enterohemorrhagic E. coli (EHEC) and Enteropathogenic Escherichia coli (EPEC) are capable of sticking to the intestinal epithelium breaking down the integrity of the barrier through the alteration of the tight junctions. For what concerns the IBD, it has been seen that a dysfunction of the AJ-proteins occurs due to the reduction in E-cadherin. This alteration determines an enfeeblement of intercellular adhesion thus activating an inflammatory response. Recent studies have clearly shown that a higher rate of IFN-y and TNF-a, typical of UC and CD, could cause a rearrangement of a large number of proteins (those composing the tight junctions such as zonulin-1, JAM-A, occludine, claudine-1 and claudine-4). In this case it is possible to observe an increase of the intestinal permeability. The same increase is easily observed in the IBS as well (especially in the IBS’s diarrhoeic subgroup.

Consequences of the Damage to the Intestinal Barrier:

Pathologies

The unique ability of the intestinal barrier to guarantee the permeability keeping off all noxious pathogens coming from the outside is essential for the preservation of the state of health of the human body.

In case the body runs short of the guaranteed protection of the intestinal barrier, the immune cells come easily in contact with the antigenes present in the lumen thus altering the normal physiological functions of the barrier, including the immune response to all pathogenic agents (bacteria, viruses, funguses and parasites), the recognition of the “self” antigenes, the tolerance of commensal flora, the desensitisation to food antigenes.

A large number of scientific studies have shown there is a connection between the gastro / extra intestinal pathologies and the alteration of intestinal permeability.

There are autoimmune pathologies such as the celiac disease, the IBD, Diabete Mellitus (type 1), multiple sclerosis, rheumatoid arthritis, hepatic cirrhosis, acute pancreatitis, the infectious gastroenteritis, the Small Intestine Bacterial Overgrowth (SIBO).

Cardiac decompensation or autism could determine an increase of intestinal permeability. As in the case of the IBD and the celiac disease, the increase of permeability itself could spark off the pathology. In the case of the hepatic cirrhosis it could lead to the entrance of microbial antigens into enteroportal circulation thus damaging the hepatic fibrosis and portal hypertension allowing an increase in the permeability.

It is not that clear whether the alteration of the barrier’s function is an essential part of the pathogenesis of all these diseases previously mentioned above. Still, it has been shown that an increase of intestinal permeability contributes to the exacerbation of these pathologies leading to a chronic state. Medicines could regain the physiological permeability bringing back the intestinal homeostasis.

The IBD’s, for instance, have shown that the increase of the intestinal permeability has a key role in the pathogenesis of the disease. In this case, the modified intestinal permeability precedes the development of the disease whose exact pathogenesis remains clearly unknown but it may include different genetical factors both immunologic and environmental which could start off the autoimmune process. In the asymptomatic patient suffering from Crohn’s disease, the increase of intestinal epithelium permeability occurs about 1 year before the relapse.

 

Whereas the first defects of the intestinal barrier seems to be involved since the very first stages of the IBS’s pathogenesis, the production of cytokines (IFN-y and TNF-a), secondary to the inflammatory process, could perpetuate an increase of intestinal permeability following a rearrangement of the proteins composing the intercellular junctions.

That is how the primary dysfunction could let the lumen content in activating an immune response maintaining an alteration in the permeability. It could lead to a chronic state of the disease afterwards.

New therapeutic strategies concentrating on the recovery of the physiologic functionality of the intestinal barrier could provide an innovative approach in order to improve the case history of all these chronic diseases.

 

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