Assessment of immunological methods for quantification of human zonulin and the effects of intestinal bacteria on zonulin secretion in vitro

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http://urn.fi/URN:NBN:fi:hulib-201906283093
Title: Assessment of immunological methods for quantification of human zonulin and the effects of intestinal bacteria on zonulin secretion in vitro
Author: Kanerva, Sonja
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2019
URI: http://urn.fi/URN:NBN:fi:hulib-201906283093
http://hdl.handle.net/10138/303606
Thesis level: master's thesis
Abstract: Increased intestinal permeability and its role in autoimmune, metabolic and chronic intestinal diseases is under extensive research as the “leaky gut” is considered as a potential target for preventive and therapeutic strategies in wide range of diseases. Zonulin, an eukaryotic analogue of Vibrio cholerae derived Zonula occludens toxin, which induces tight junction disassembly, has recently become a popular serum-based biomarker of intestinal permeability in biomedical research, even though the link between serum zonulin levels and functional measures of intestinal permeability has never been validated properly in humans. In addition, surprisingly little is known about the location and regulation of zonulin expression in the humans despite the protein was discovered almost two decades ago. Zonulin, also known as pre-haptoglobin-2, is an uncleaved precursor form of haptoglobin that is abundantly expressed in the liver. Zonulin, in turn, based on studies on rats, rabbits and monkeys, is expressed in the small intestine and stimulated by exposure to bacteria and gliadin, but no other stimulators have been described so far. It is also unclear, if different bacteria can induce different responses in zonulin secretion as only the effect of gram-negative enterobacteria has been documented so far. The aim of this study was to evaluate the effect of selected intestinal bacteria and of two known upregulators of haptoglobin, interleukin-6 (IL-6) and bacterial lipopolysaccharide (LPS), on zonulin secretion in vitro. The impact of two gram-positive probiotic bacteria (Lactobacillus rhamnosus GG & Bifidobacterium bifidum) and of two commensal gram-negative bacterial strains (Escherichia coli DH5α & Escherichia coli RY13) were tested for zonulin secretion in HT-29 intestinal epithelial cells, in addition to IL-6. Two separate lineages of immortalized human hepatocytes were tested for zonulin secretion by stimulation of LPS and IL-6. In addition, different immunological methods were assessed for quantification of zonulin, as the potential cross-reactivity of our primary analysis method, a commercial zonulin ELISA kit from Immundiagnostik AG that is also the main method used in the published zonulin studies, became more evident at the beginning of this thesis project. The main findings of this study were that the widely used commercial zonulin ELISA from Immundiagnostik AG is not specific for zonulin, but instead cross-reacts at least with complement C3, in line with the results published by other group during this work. Our further experiments comparing the signals of the above-mentioned zonulin ELISA and complement C3 ELISA for serum samples showed that there was only weak correlation between the obtained signals, suggesting that the zonulin antibody does not directly bind to complement C3. By using dot blot, western blot and immunoprecipitation, we found that the cross-reaction only occurred in native conditions. Based on zonulin ELISA measurements of the cell culture media from the in vitro experiments, very low signal was obtained for both intestinal and hepatic cells. Among the tested bacteria, only exposure to Lactobacillus rhamnosus GG led to a significant increase in the release of target protein. In hepatic cells, LPS had no effect, while IL-6 led to a significant increase of zonulin ELISA signal in one of the tested hepatic lines. However, it is currently difficult to differentiate if the low detected “zonulin” levels in this study are due to low level of secretion, or rather due to the lack of a proper method to detect zonulin. Taken together, these observations suggest that the most commonly used zonulin ELISA and other related, commercially available antibody-based methods for zonulin detection should be utilized with caution, as these antibodies cross-react with other protein(s). Hence, the serum “zonulin” cannot be considered as a biomarker of intestinal permeability until the captured protein(s) are identified, and similarly the anticipated effects of intestinal bacteria on zonulin expression cannot be reliably investigated with the currently available antibodies.
Subject: Zonulin
pre-haptoglobin
intestinal permeability
intestinal bacteria
zonulin ELISA
complement C3
Discipline: yleinen mikrobiologia
General Microbiology
allmän mikrobiologi


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