The primary functions of the gastrointestinal tract traditionally include digestion and absorption of nutrients, and electrolytes and water homeostasis. Recent studies indicate that another key function of this organ is to regulate trafficking of environmental antigens across the host mucosal barrier. Together with the gut- associated lymphoid tissue and the neuroendocrine network, the intestinal epithelial barrier, with its intercellular tight junctions (TJ), controls the balance between tolerance and immunity to non-self antigens. During the continuous 15 years of funding of this grant, we have used a Vibrio cholerae-elaborated protein, zonula occludens toxin (Zot) as a tool to gain insights into the regulation of TJ function. Our studies led to the discovery of zonulin, a human eukaryotic Zot homologue, and to the definition of aspects of its physiological and pathological functions. To our knowledge, zonulin is the only reversible modulator of intercellular TJs described so far. Genome wide association studies have coupled genes controlling gut permeability to autoimmune and inflammatory diseases. Our studies have demonstrated that zonulin expression is increased in autoimmune disorders associated with gut barrier dysfunction, such as celiac disease and type 1 diabetes. Our data generated on intestinal cell lines, animal models, and human tissues indicate that Zot and zonulin both induce proteinase-activated receptor 2 (PAR2)-mediated transactivation of epidermal growth factor receptor (EGFR). Our overall hypothesis is that this PAR2-mediated transactivation of EGFR is coupled with TJ disassembly, and, ultimately, increased intestinal permeability. Further we hypothesize that the number of zonulin gene copies is associated with augmented zonulin expression and a constitutively dysfunctional, permissive gut mucosal barrier. To challenge these hypotheses, we propose three specific aims.
With Aim 1 we will expand our studies to establish the mechanisms through which zonulin because PAR2- mediated transactivation of EGF. We have demonstrated that this transactivation is followed by protein kinase C (PKC)a-mediated ZO-1 and myosin 1c serine/threonine phosphorylation, provoking ZO-1 disengagement from three of its binding partners, occludin, claudin-1, and transiently from myosin 1C, and induce actin polymerization, ultimately causing TJ disassembly.
With Aim 2 we will define the zonulin-triggered intracellular signaling events downstream to EGFR activation leading to increased intestinal permeability. We have recently established identity between zonulin and pre-haptoglobin (HP) 2, i.e., the uncleaved product of HP2. The HP2- 2 genotype is more frequently encountered in immune-mediated conditions, including celiac disease, Crohn's disease, and schizophrenia, and its expression is associated with more severe clinical manifestations. With our Aim 3 we will investigate the association of HP genotypes (HP1-1, HP1-2, and HP2-2) with serum zonulin expression and clinical severity in human disease states and transgenic murine models.

Public Health Relevance

Our in vivo data and clinical trials using a competitive zonulin synthetic peptide inhibitor (Larazotide acetate) demonstrate a direct link between zonulin release, increased intestinal permeability, and autoimmune pathogenesis. We predict that insights on zonulin mechanism of action and evaluation of its expression may help identify subjects at higher risk for autoimmune and inflammatory diseases. Our recently developed assays to measure serum zonulin levels and to perform high throughput HP genotyping can be used as potential biomarkers to stratify patients for specific targeted anti-zonulin treatments (i.., personalized medicine) that are currently tested in clinical trials.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK048373-16
Application #
8534755
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Carrington, Jill L
Project Start
1996-05-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
16
Fiscal Year
2013
Total Cost
$365,205
Indirect Cost
$155,317
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Shirey, K A; Lai, W; Patel, M C et al. (2016) Novel strategies for targeting innate immune responses to influenza. Mucosal Immunol 9:1173-82
Salerno-Goncalves, R; Safavie, F; Fasano, A et al. (2016) Free and complexed-secretory immunoglobulin A triggers distinct intestinal epithelial cell responses. Clin Exp Immunol 185:338-47
Guerrant, Richard L; Leite, Alvaro M; Pinkerton, Relana et al. (2016) Biomarkers of Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Brazil. PLoS One 11:e0158772
Salerno-Goncalves, Rosangela; Fasano, Alessio; Sztein, Marcelo B (2016) Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity. J Vis Exp :
Lammers, Karen M; Chieppa, Marcello; Liu, Lunhua et al. (2015) Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1. PLoS One 10:e0138338
Vajro, Pietro; Paolella, Giulia; Fasano, Alessio (2013) Microbiota and gut-liver axis: their influences on obesity and obesity-related liver disease. J Pediatr Gastroenterol Nutr 56:461-8
Rittirsch, Daniel; Flierl, Michael A; Nadeau, Brian A et al. (2013) Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system. Am J Physiol Lung Cell Mol Physiol 304:L863-72
Barry, Eileen M; Pasetti, Marcela F; Sztein, Marcelo B et al. (2013) Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol 10:245-55
Fasano, Alessio (2012) Intestinal permeability and its regulation by zonulin: diagnostic and therapeutic implications. Clin Gastroenterol Hepatol 10:1096-100
Fasano, Alessio (2012) Leaky gut and autoimmune diseases. Clin Rev Allergy Immunol 42:71-8

Showing the most recent 10 out of 53 publications