Intestinal barrier, disruption

A proper definition (and related ontology) of the intestinal barrier and permeability would benefit the understanding of this biological event central in many diseases. However, it is generally accepted that the intestinal barrier is a multilayer system encompassing :

- a chemical barrier able to detoxify bacterial endotoxins,

- a mucus layer providing a physical barrier against bacteria,

- an one-cell-thick epithelial layer which physical barrier function is ensured by epithelial cell integrity and by tight junction proteins (occludins, claudins and zonulins), adherence junctions and desmosomes ^2,4,5

- the cellular immune system present in the lamina propria underlying the epithelial cell layer

- the antibacterial proteins secreted by the specialized intestinal epithelial cells or the Paneth cells.

Together with the chemical barrier of the mucosal layer and the cellular immune system, the intestinal epithelial cell layer has actually two barrier functions:^1–3

1. It acts as a physical barrier against external factors (pathogens, toxins),
2. It acts as a selective barrier by regulating the absorption of essential dietary nutrients and  ions, meaning their transport from the lumen into the blood.

Intestinal permeability⁶ describes the movement of molecules across the intestinal barrier from the lumen to the blood (Figure 1), and as such, is the measurable feature of the intestinal barrier.

Figure 1. Created with Biorender.com

Molecules can cross the epithelium via paracellular or transcellular route. Transcellular permeability encompass passive diffusion from the apical to the basal side (from the lumen to the blood), vesicle-mediated transcytosis and uptake mediated by a membrane receptor. Paracellular permeability is regulated by the tight junctions between adjacent cells and by the integrity of the epithelium.

Alteration or disruption of one or more layers of the intestinal barrier leads to increased intestinal permeability, also called intestinal hyperpermeability or “leaky gut”, enhancing the transport of pathogens, toxins (such as lipopolysaccharides), undigested nutrients and the translocation of bacteria of the gut microbiota from the intestinal lumen into the systemic circulation³.

The definition of intestinal permeability being relatively broad includes altered paracellular route, regulated by TJ proteins, transcellular routes involving membrane transporters and channels, and endocytic mechanisms. Paracellular intestinal permeability can be assessed in vivo via different molecules and via putatiive blood biomarkers and ex vivo in Ussing chambers combining electrophysiology and probes of different molecular sizes. The latter is still the gold standard technique for assessing the epithelial barrier function, whereas in vivo techniques are also broadly used despite limitations (doi: 10.3389/fnut.2021.717925).

In humans.

Virtually all in vivo methods to assess paracellular intestinal permeability rely on the urinary excretion of orally ingested probes. Several markers, including different sizes of PEG, ⁵¹CrEDTA, and especially sugars have been used, each with advantages and disadvantages (doi: 10.3389/fnut.2021.717925). Intestinal Permeability Assessment (IPA) directly measures the ability of two non-metabolized sugar molecules (lactulose and mannitol) to permeate the small intestinal barrier by paracellular passage (sign of perturbed TJ-lactulose) or by transcellular passage (giving information of the whole epithelial absorptive area-mannitol), respectively. The patient drinks a premeasured amount of those sugars and 6h after, the ratio of Lactulose/Mannitol levels is measured in the urine ¹¹.

Levels in plasma/serum or in feces of:

• Markers of epithelial cell damage, such as intestinal fatty acid binding protein (FABP)
• Markers of tight junction alterations, such as zonulin levels (doi: 10.1080/21688370.2016.1251384)
• Microbial translocation, such as peptidoglycans and lipopolysaccharides (LPS) and gut microbiota alteration.

In vitro systems¹²

Transepithelial electrical resistance (TEER) or the Lucifer Yellow (LY) leakage assay are techniques to measure barrier integrity and permeability of a cell layer¹³. Caco-2 cells are human epithelial colorectal adenocarcinoma cells with a structure and function similar to the differentiated small intestinal epithelial cells (e.g. exhibit microvilli). Caco-2 cells can be plated in wells as monolayers^14,11. Other cell lines can be used, such as intestinal epithelial cells (IEC) or primary epithelial cells from human intestinal biopsies¹². Co-culturing of enterocyte-like cells with immune cells in three-dimensional structure and within a microfluidic gut-on-chip has been shown to reflect better the physiology of the gut epithelium. Epi-Intestinal^TM is an example of 3D human primary cell-based organotypic small intestinal model which allows evaluation of TEER and LY leakage assay (doi: 10.1007/s11095-018-2362-0).

In vivo system

In mice, one way to study intestinal paracellular permeability is by measuring the ability of fluorescein isothiocyanate (FITC)-dextran to cross from the lumen into the blood. After gavaging mice with FITC-dextran, the concentrations are measured in collected serum samples (doi: 10.3791/57032).

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12.          Fedi, A. et al. In vitro models replicating the human intestinal epithelium for absorption and metabolism studies: A systematic review. J. Control. Release 335, 247–268 (2021).

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14.          Li, B. R. et al. In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability. J. Vis. Exp. 1–6 (2018). doi:10.3791/57032

15.          Ayehunie, S. et al. Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption Seyoum. Pharm. Res. 35, 72 (2019).