The role of Zonulin-mediated gut permeability in IBD

Zonulin has emerged as a popular marker to assess the integrity of the intestinal mucosal barrier. Discovered by Dr Alessio Fasano, Zonulin (haptoglobin 2 precursor) is a protein that increases the permeability of tight junctions between cells of the wall of the digestive tract.

This discovery has bypassed challenge testing with lactulose and mannitol which can be challenging for patients who are diabetic or intolerant to the challenge solutions.

Zonulin was first discovered as an endogenous human analogue of the bacterial enterotoxin, zonula occludens toxin (Zot), which is produced by the intestinal bacterium Vibrio cholera.[1]

Zonulin is secreted mainly from the liver, but also from enterocytes, adipose tissue, brain, heart, immune cells, lungs, kidney, and skin.

The surge in chronic inflammatory diseases (CIDs) including infectious, allergic, neuroinflammatory, neurodegenerative, autoimmune diseases, and cancer has been extensively reported by the Human Genome Project. This surge of GI-diseases has been generated from the hygiene hypothesis and a Western diet and lifestyle.

The intestinal epithelium regulates fluid exchange and absorb nutrients. The intestinal epithelium together with gut-associated lymphoid tissue and the neuroendocrine network work at regulating transport of environmental or dietary antigens from the intestinal lumen into the sub-mucosa.[2]

Dysregulation of the mucosal barrier leads to “leakiness” of antigens and other macromolecules into the host to initiate systemic inflammation and immune activation which in turn influences tolerance and immunity.

Intercellular tight junctions (TJs) tightly regulate paracellular luminal antigen trafficking across the epithelium. TJs are extremely dynamic structures that operate in several key functions of the intestinal epithelium under both physiological and pathological circumstances. TJs were previously believed to be impermeable and static, forming a sealing barrier. This paradigm was subverted in 1993 by the discovery of Dr Fassano’s work on Zonulin.

Dr Fassano’s discoveries clarified that a sophisticated meshwork of proteins forms the tight junctions. The discovery of a toxin, called the “zonula occludens toxin” or Zot (zonula occludens is Latin for “tight junction”), provided a valuable tool for clarifying the control process. [3]

Zonula occludens was first discovered in 1993, as the first component of the TJ complex is made up of more than 150 proteins, including occludin, claudins, junctional adhesion molecules, tricellulin, and angulins. [4]

A major breakthrough in Dr Fasano’s work in the discovery of Zonulin as the only physiologic intestinal permeability modulator has been reported as a biomarker of gut permeability.

Elevations in Zonulin with subsequent “gut Leakiness” has been associated with irritable bowel syndrome (IBD), and gastrointestinal disorders such as coeliac disease and irritable bowel syndrome (IBS). [5]

Zonulin levels may rise in obesity, high blood pressure, high blood sugar, high triglycerides, high cholesterol, and autoimmunity, so do rule out co-morbidities with a leaky gut patient.[6]

Active ulcerative colitis (UC) is associated with a decrease in claudin-1, claudin-4, claudin-7, and occludin and an increase in claudin-2. Chron’s disease (CD), on the other hand, is associated with decreased claudin-3, claudin-5, and claudin-8, as well as increased expression of claudin-2.[7]

As a result of upregulation of TJ proteins, barrier dysfunction in CID’s is related to inflammatory responses and TJ alteration.[8]

How Zonulin impacts IBS, IBD and Non-coeliac Gluten sensitivity

Among IBD patients, zonulin was significantly higher in CD patients compared to UC patients and was associated with IBD severity. An increase in the serum concentration of zonulin was associated with an increase in the functional activity of circulating neutrophils and an increase in the number of CD3+CD8+ cells, NK cells, and a decrease in the number of CD19+ cells.[9]

In a more recent study, serum zonulin concentration was found to be higher in both diseases, and an inverse correlation was observed between serum zonulin concentration and disease duration. [10]

Certain gut microbes, in particular pathogens, might induce the release of zonulin from the gut suggesting a mechanistic link between alterations in the gut microbiota and gut barrier function.

Patients with diarrhea-associated irritable bowel syndrome showed increased zonulin levels and involvement of the protease-activated receptor 2 (PAR2) the zonulin target receptor.[11]

The effect of Gluten on Zonulin

When gluten peptides bind onto the chemokine receptor CXCR3, zonulin is released and subsequently increases intestinal permeability. The inhibition of zonulin has led to a novel treatment option for CD.

This discovery has led to propose that it is the enhanced intestinal permeability in CD patients that allows gluten, the environmental factor, to seep out of the gut and to interact freely with genetically sensitised elements of the immune system. That understanding, in turn, suggests that removing any one factor of the autoimmunity-causing trinity – the environmental trigger, the heightened immunity or the intestinal permeability – should be enough to stop the disease process.

Non-celiac gluten sensitivity patients have shown to have increased levels of Zonulin triggered by gluten as in CD but without autoimmune enteropathy.[12]

Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten-containing grains in genetically susceptible individuals and can be reversed when gluten is eliminated from the diet. Indigestible fragments of gluten are able to bind CXCR3 and release zonulin.[13]

Studies have shown that elevated serum zonulin levels increased in obese versus non-obese subjects, and there is evidence of a correlation between total bacteria and serum zonulin levels, suggesting that the gut microbiota may cause increased zonulin levels, and subsequent abnormal gut permeability to endotoxin and ultimately micro-inflammation has been reported in obesity.[14]

Therapeutic strategies to drive down Zonulin

A qualitative and quantitative imbalance in the composition of the intestinal microbiota or dysbiosis contributes to intestinal barrier dysfunction and leaky gut syndrome.

Therapeutic considerations in normalising Zonulin in leaky gut syndrome should influence the modulation of intestinal immunity, regulation of the intestinal epithelial barrier, amelioration of mucosal abnormalities, and growth of epithelial cells.

List of therapies include:

• diet modification avoiding fats, sugars, additives, and ultra-processed foods, low FODMAPs
• probiotics/prebiotics
• amino acids such as arginine and glutamine
• polyphenols
• vitamins
• fibres

Diet plays a major role on zonulin. Limit foods that trigger Zonulin like refined carbohydrates (white bread and pasta), glutinous grains (barley, rye, and oats), white sugar (candy, baked goods, cereal), dairy, legumes, nuts and seeds.

A Low FODMAP diet is recommended. FODMAP is a collective term that consists of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols.

Slow or no absorption of FODMAPs results in an increase in osmotic action, resulting in increased luminal water content and subsequent distention of the small intestine, leading to IBS symptom induction.[15]

Glutamine is a crucial amino acid capable of regulating the expression of tight junction proteins, allowing the membrane of intestinal cells to remain impermeable. Glutamine has value in leaky gut, diarrhea-predominant intestinal inflammation.[16]

Targeted probiotics exhibit anti-inflammatory properties against TNF-α or IL-6. They can also strengthen the mucosal barrier and reduce intestinal permeability, upregulating TJS proteins. Probiotic supplementation reduces zonulin, an indicator of leaky gut. [17]

Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus Plantarum, Bifidobacterium infantis, Bifidobacterium animalis lactis BB-12, and Escherichia coli Nissle 1917 have shown its beneficial role in epithelial intestinal modulations and possible mechanisms of actions in the intestinal barrier that eventually can potentially provide health benefits for leaky gut syndrome-related diseases.[18]

Targeted probiotics may surpass certain lactic acid bacteria that activate Toll-like receptor 2 (TLR2) signalling pathway. TLR2 is localized in cell membranes of the intestinal epithelium. It induces epithelial resistance in the activated form.[19]

Vitamins A and D play critical functions in regulating gastrointestinal homeostasis. They can inhibit T-cell IFN-γ production and inhibit Th17 cells in vitro.[20]

Butyrate is the most critical substrate the colonocyte prefers, providing between 60 and 70% of the energy requirements. SCFAs are critical in metabolism, immunity, and intestinal barrier functions. The deficiency of SCFAS and dietary fibres can compromise epithelial and mucus barrier functions by increasing gut permeability. Partially hydrolysed guar gum, serum bovine derived immunolins and 2-Fucollysate found in human milk oligosaccharides have been shown to be butyrogenic.

Polyphenols play a critical role in providing antioxidant support in the inflammatory process.

Quercitin upregulates occludin, claudin-1, claudin-3, claudin-4, and claudin-7 in Caco2 cells.[21]

Epigallocatechin gallate upregulates IFN-γ in T84 cells.[22]

Berberine upregulates claudin-1 and claudin-2 in HT29/B6 cells.[23]

Curcumin upregulates ZO-1, and claudin-1 in CaCo2 cells.[24]

Medicinal phototherapeutic herbs such as:

Camellia sinensis improves the intestinal barrier, alleviating dysbacteriosis.

Hibuscus Sabdariffa suppresses the expression of inflammatory cytokines, including IL-6 and TNF-α in dextran sodium sulphate-induced colitis.[25]

Glycyrrhiza glabra maintains the integrity of the intestinal epithelium via HuR in IEC-6 cells.[26]

Althea officinallis decreased TNF-a and IL-1b and promotion of mucin, NO, PG-E2, and PG-12[27]

Zingiber officinale modulates nuclear factor-κB activity and interleukin-1β.[28]

Plantago lanceolate inhibits the enzymatic activity of inducible nitric oxide synthase expressed in both macrophages and neutrophils and 5 lipoxygenase [29]

Medicinal mushrooms are a viable source of prebiotics as they contain different polysaccharides, such as chitin, chitosan, hemicellulose, xylans, mannans, galactans, and α- and β-glucans. Mushroom polysaccharides affect SCFAs production mainly butyrate, propionate, and acetate.[30]

Conclusion

Due to its recognised role in reversible tight junction disassembly, the level of zonulin has emerged as an increasingly popular biological marker of mucosal barrier integrity.

Testing for Zonulin is therefore recommended in assessing intestinal mucosal barrier integrity in clinical conditions where a “leaky gut” is suspected.

References

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