• Genetic and Functional Characterization of Zonulin as Prehaptoglobin-2 and its Role in Inflammation and Autoimmunity

      Sturgeon, Craig; Fasano, Alessio; Shea-Donohue, Terez (2017)
      Increased small intestinal permeability has recently been described as an integral element, along with genetic makeup and environmental triggers, in the pathogenesis of chronic inflammatory diseases (CIDs). We identified zonulin as a master regular of intercellular tight junctions, and both our group and others have linked the zonulin pathway to the development of several CIDs. We further characterized zonulin as the precursor of haptoglobin (Hp)-2. In this dissertation, my aim is to study the role of zonulin-mediated small intestinal permeability in the pathogenesis of CIDs by using a zonulin transgenic mouse model as well as zonulin genotyping and disease modeling in several CIDs. Zonulin transgenic Hp2 mice (Ztm) were subjected to dextran-sodium-sulfate (DSS) treatment, and their morbidity and mortality compared to C57Bl/6 (WT) mice. We observed increased morbidity (more severe body weight loss and colonic inflammation) and mortality at 11 days post DSS treatment in zonulin transgenic Hp2 mice compared to WT. Ztm had increased small intestinal permeability at baseline compared to WT, which was exacerbated by DSS treatment and associated with upregulation of the zonulin gene in the small intestine. Treatment with the zonulin inhibitor AT1001 prevented the DSS-induced increased small intestinal permeability and completely reversed mortality rates. We went on to study the risk Hp genotypes in disease development by using a combination of HLA and Hp genotypes to predict disease outcome. We observed increased HP2 allele frequency in several CIDs studied. Additionally, the HP2 allele (both in homozygosity and heterozygosity) seems to increase the risk of early age of onset of Type 1 diabetes (T1D). Using the combination of HLA and Hp genotypes, we were able to create accurate positive predictive models for celiac disease (CD) and T1D. Taken together, these data show that the zonulin gene can be mechanistically linked to increased small intestinal permeability that leads to a break of tolerance with subsequent development of CIDs. Additionally, the use of the Hp genotype, either alone or in addition to other genetic markers, may be a useful tool to create predictive models for disease development for precision and preventive medicine approaches.
    • Physiological and pathological role of zonulin in the regulation of intercellular tight junctions

      Wang, Wenle; Fasano, Alessio; Bucci, Enrico, M.D., Ph.D. (2001)
      The paracellular route is the dominant pathway through which passive solutes flow across both the endothelial and epithelial barriers, and its functional status is regulated, in part, at the level of intercellular tight junctions. Tight junctions readily adapt to a variety of developmental, physiological, and pathological circumstances. The physiological regulation remains undefined. Zonula occludens toxin (Zot), a protein elaborated by Vibrio cholerae, reversibly regulates tight junction permeability. Zot interacts with a specific surface receptor(s) with subsequent protein kinase C alpha-dependent polymerization of actin microfilaments strategically localized to regulate the paracellular pathway. Based on this observation, we postulated that Zot might mimic an endogenous modulator(s) of tight junctions and that Zot and its putative eukaryotic analogue could be structurally and immunologically related. Accordingly, the specific anti-Zot antibody and the Using chamber assay were used to screen for the human intestinal Zot analogue(s). Non-primate intestinal tissues were used as an indicator system to identify and purify this analogue. A single protein was picked up and named zonulin. Purified zonulin reduced the electrical resistance of small intestine in a reversible fashion similar to that of Zot. All characteristics of zonulin that have been tested are comparable with Zot's characteristics. We conclude that zonulin and Zot share the same mechanism in regulating tight junctions. To date, there is no clear explanation for the disturbed physiological regulation of the intestinal permeability secondary to proximal bacterial contamination. Zonulin can, however, protect the small intestine from bacterial colonization. When rabbit ileum was exposed to Salmonella typhimurium and E. coli 6-1, resistance decrement was induced. These changes in Rt correlated with increases in zonulin secretion into the luminal (but not the serosal) side of the mucosa. This result suggests that when there is bacterial colonization in the small intestine, the tissue will secrete zonulin that is either stored or newly synthesized. Pre-treatment with FZI/0, a zonulin receptor binding inhibitor that prevents the permeating effect of zonulin, completely blocked the E. coli 6-1-induced Rt changes in both jejunum and ileum and the paracellular passage of inulin without affecting zonulin luminal secretion. Taken together, our data demonstrate the some tissues contain zonulin which can regulate tight junctions by using the same mechanism as Zot. They share the same receptors and the same signal transduction pathway, and they share a conservative domain responsible for binding to receptors. Zonulin is induced when there are bacteria or other specific proteins in the small intestine. In addition, overexpression of zonulin has been found in celiac disease. (Abstract shortened by UMI.)