Volume 123, Issue 5, Pages 1607-1615 (November 2002) Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure  Rahzi El Asmar, Pinaki Panigrahi, Penelope Bamford, Irene Berti, Tarcisio Not, Giovanni V. Coppa, Carlo Catassi, Alessio Fasano  Gastroenterology  Volume 123, Issue 5, Pages 1607-1615 (November 2002) DOI: 10.1053/gast.2002.36578 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) TEER changes of unstripped rabbit small intestine mounted in the micro-snapwell system and exposed for 3 hours to nonpathogenic Escherichia coli 6.1, pathogenic E. coli 21-1, or Salmonella typhimurium. Uninfected controls are shown for comparison. The average baseline TEER was 120 Ω/cm2; n = 4. (B) Zonulin concentration in the media collected from the lower chamber (serosal side, open bars) or upper chamber (mucosal side, closed bars) of rabbit small-intestinal tissues mounted in the micro-snapwell system and incubated for 3 hours with E. coli 6.1, pathogenic E. coli 21-1, or S. typhimurium added to the mucosal aspect of the intestine. Uninfected tissues are shown for comparison; n = 4. Gastroenterology 2002 123, 1607-1615DOI: (10.1053/gast.2002.36578) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Kinetics of zonulin release from rabbit small-intestine organ cultures either uninfected (▴) or inoculated with the nonpathogenic bacteria E. coli 6-1 (○) or DH5α (●). All means were significantly higher than in uninfected control tissues after 24 (P < 0.05) and 48 (P < 0.01) hours; n = 3–5 determinations. Gastroenterology 2002 123, 1607-1615DOI: (10.1053/gast.2002.36578) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunoblotting of rabbit small-intestine organ cultures exposed to E. coli 6.1. Culture aliquots containing increasing zonulin concentrations as established by ELISA were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with zonulin-specific anti-Zot polyclonal antibodies. Zonulin concentrations (ng/mg protein): A = 0 (medium alone); B = 13.4; C = 20.5; D = 31.1; E = 36.9. A 47-kilodalton band corresponding to the predicted zonulin molecular weight19 was visualized, along with 2 other bands of approximately 46 and 20 kilodaltons. The intensity of both the 47- and 46-kilodalton bands paralleled the increasing zonulin concentration from B to E. No significant change of the 20-kilodalton band was noted in B, C, and D, whereas the band was not visible in E. Purified zonulin is shown for comparison (F). Gastroenterology 2002 123, 1607-1615DOI: (10.1053/gast.2002.36578) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) Inulin paracellular passage (from mucosa to serosa) through monkey small intestine mounted in the micro-snapwell system. A significant increase (P ranging between 0.038 and 0.01) of the paracellular marker was observed in tissues exposed to E. coli 6.1 (▨) as compared with uninfected tissues (□). This increment was already significant after 30 minutes of incubation and remained sustained up to 6 hours after inoculation. Pretreatment with the zonulin receptor synthetic peptide binding inhibitor FZI/0 (1 μg/mL) completely prevented the inulin passage (■), whereas the scrambled peptide FZI/1 (1 μg/mL) (●) did not. These results suggest that the permeating effect induced by the exposure of monkey small intestines to bacteria is zonulin dependent; n = 8 tissues. (B) Kinetics of zonulin release from monkey small intestines mounted in the micro-snapwell system. Zonulin concentration was determined in the same culture media obtained during the experiment described in (A). No significant changes in zonulin concentration were detected over time in uninfected tissues (♦). Culture media of monkey small intestines exposed to E. coli 6.1 (●) showed a progressive increase in zonulin concentration that became significant after 30 minutes of incubation (P < 0.05), confirming that the bacteria-induced increased paracellular permeability was zonulin dependent. Zonulin release by bacteria-exposed small-intestinal tissues was not affected by preincubation with FZI/0 (○), suggesting that the inhibitory effect observed in (A) was due to FZI/0's competitive binding to the zonulin receptor18 rather than its effect on zonulin release; n = 8 tissues. Gastroenterology 2002 123, 1607-1615DOI: (10.1053/gast.2002.36578) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 TEER changes of stripped monkey small intestines mounted in Ussing chambers and exposed to zonulin-containing tissue culture media. Zonulin (final concentration 1 ng/mL) induced a significant (P between 0.05 and 0.001) decrease in TEER (●) starting as early as 20 minutes after incubation and reaching a plateau at 80 minutes after incubation. Pretreatment with the zonulin receptor binding inhibitor FZI/0 (○) completely abolished the permeating effect that became indistinguishable from the negative control (♦). The average baseline TEER was 42.5 Ω/cm2; n = 4 observations. Gastroenterology 2002 123, 1607-1615DOI: (10.1053/gast.2002.36578) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of bacteria infection on the junctional complex protein ZO1 localization in Caco2 cells. Human intestinal Caco2 cells were exposed to E. coli 6-1, fixed either 30 minutes or 4 hours after incubation, and immunostained with anti-ZO1 antibodies. Uninfected monolayers showed the typical ZO1 localization at the cells' periphery both 30 minutes (A) and 4 hours (D) after incubation. Monolayers infected with E. coli 6-1 showed ZO1 redistribution, with reduced or lost ZO1 staining at the edge of the cell paralleled by the appearance of immunoparticles within the cell cytoplasm (B and E). Pretreatment with the zonulin receptor binding inhibitor FZI/0 (1 μg/mL) completely prevented the ZO1 redistribution (C and F), suggesting that ZO1 disengagement from the junctional complex is zonulin dependent. Gastroenterology 2002 123, 1607-1615DOI: (10.1053/gast.2002.36578) Copyright © 2002 American Gastroenterological Association Terms and Conditions