Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.
OBJECTIVE
Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment.


METHODS
Alpha-gliadin affinity column was loaded with intestinal mucosal membrane lysates to identify the putative gliadin-binding moiety. In vitro experiments with chemokine receptor CXCR3 transfectants were performed to confirm binding of gliadin and/or 26 overlapping 20mer alpha-gliadin synthetic peptides to the receptor. CXCR3 protein and gene expression were studied in intestinal epithelial cell lines and human biopsy specimens. Gliadin-CXCR3 interaction was further analyzed by immunofluorescence microscopy, laser capture microscopy, real-time reverse-transcription polymerase chain reaction, and immunoprecipitation/Western blot analysis. Ex vivo experiments were performed using C57BL/6 wild-type and CXCR3(-/-) mouse small intestines to measure intestinal permeability and zonulin release.


RESULTS
Affinity column and colocalization experiments showed that gliadin binds to CXCR3 and that at least 2 alpha-gliadin 20mer synthetic peptides are involved in this binding. CXCR3 is expressed in mouse and human intestinal epithelia and lamina propria. Mucosal CXCR3 expression was elevated in active celiac disease but returned to baseline levels following implementation of a gluten-free diet. Gliadin induced physical association between CXCR3 and MyD88 in enterocytes. Gliadin increased zonulin release and intestinal permeability in wild-type but not CXCR3(-/-) mouse small intestine.


CONCLUSIONS
Gliadin binds to CXCR3 and leads to MyD88-dependent zonulin release and increased intestinal permeability.
Lammers
Karen M
KM
Mucosal Biology Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Lu
Ruliang
R
Brownley
Julie
J
Lu
Bao
B
Gerard
Craig
C
Thomas
Karen
K
Rallabhandi
Prasad
P
Shea-Donohue
Terez
T
Tamiz
Amir
A
Alkan
Sefik
S
Netzel-Arnett
Sarah
S
Antalis
Toni
T
Vogel
Stefanie N
SN
Fasano
Alessio
A
D000818:Animals; D001706:Biopsy; D018938:Caco-2 Cells; D002446:Celiac Disease / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D002772:Cholera Toxin / Q000378:metabolism*; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005903:Gliadin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006242:Haptoglobins; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D011498:Protein Precursors; D051381:Rats; D054367:Receptors, CXCR3 / Q000235:genetics / Q000378:metabolism*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects
C515187:CXCR3 protein, human; C515188:Cxcr3 protein, mouse; D006242:Haptoglobins; C507781:Myd88 protein, rat; D053594:Myeloid Differentiation Factor 88; D010446:Peptide Fragments; D011498:Protein Precursors; D054367:Receptors, CXCR3; C408043:zonulin; D005903:Gliadin; D002772:Cholera Toxin